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I 73-2008 GR0SSK0PF, J a c k C a r l, 191+6- CHARACTERIZATION OF IMMOBILIZED PRONASE BY SELECTED SUBSTRATES. The O hio S t a t e U n i v e r s i t y, P h.d., 1972 A g r i c u l t u r e, g e n e r a l University Microfilms. A XEROX C om pany, A nn Arbor. Michigan 1 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

CHARACTERIZATION OF IMMOBILIZED PRONASE BY SELECTED SUBSTRATES DISSERTATION P r e s e n te d i n P a r t i a l F u l f i l l m e n t o f t h e R e q u ire m e n ts f o r t h e D eg ree D o c to r o f P h ilo s o p h y in t h e G ra d u a te S c h o o l o f th e O hio S t a t e U n iv e r s ity Hy J a c k C a r l G ro s s k o p f* B. S., M. S c. «*****««The Ohio S t a t e U n iv e r s ity 1972 A pproved by 1 Ad v i s e r / Depar^rtnent o f D a iry T ech n o lo g y

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ACKNOWLEDGMENTS S in c e r e a p p r e c i a t i o n i s e x p re s s e d t o D r. W. J. H a rp e r, D ep artm en t o f Food S c ie n c e and N u t r i t i o n, f o r d i r e c t i n g t h i s e x p e r im e n ta l w o rk, an d f o r h i s a s s i s t a n c e and en co u rag em en t i n t h e p r e p a r a t i o n o f t h i s m a n u s c r ip t. S in c e r e a p p r e c i a t i o n i s a ls o e x p r e s s e d t o D r. T. K r i s t o f f e r s e n, A c tin g C hairm an o f th e D ep artm en t o f Food S c ie n c e and N u t r i t i o n, f o r p r o v id in g th e o p p o r tu n ity f o r g ra d u a te s t u d y, and f o r h i s i n t e r e s t and en co u rag em en t th ro u g h o u t my g r a d u a te p ro g ram. My s in c e r e th a n k s a r e e x te n d e d a l s o t o th e e n t i r e s t a f f o f th e D ep artm en t o f Food S c ie n c e and N u t r itio n f o r t h e i r c o o p e r a tio n d u rin g my g ra d u a te s tu d y, and t o M r. Jam es V. Chambers f o r a s s i s t a n c e in th e c o n tin u o u s c u l t u r e w ork. The f i n a n c i a l a s s i s t a n c e p ro v id e d by th e N a tio n a l S c ie n c e F o u n d a tio n and a d m in is te r e d th ro u g h t h e O hio S t a t e U n iv e r s ity R e searc h F o u n d a tio n i s a ls o g r a t e f u l l y acknow led g ed. I am in d e b te d t o my w i f e, L ynne, f o r h e r en co u rag em en t and u n d e r s ta n d in g th ro u g h o u t my g ra d u a te p ro g ra m, and f o r h e r a s s i s t a n c e i n th e p r e p a r a t i o n o f t h i s m a n u s c rip t. ii

CONTENTS Page ACKNOWLEDGMENTS....... i i T A B L E S... ILLUSTRATIONS... i v,:v INTRODUCTION....... 1 LITERATURE REVIEW... 2 P ro n a se... 2 Im m o b ilized E n z y m e s... 10 SCOPE OF IN V ESTIG A TIO N... U8 EXPERIMENTAL PROCEDURE... 50 RESULTS...... 5 6 P r o te i n P u r i t y... 56 E f f e c t o f S to ra g e on BAEE A c t i v i t y... 59 Bound Enzyme Q u a n t i t a t i o n... 62 M oib ture C o n te n t D e te rm in a tio n... 65 P ro n a se Bound t o G la ss... 65 E f f e c t o f T e m p e r a tu r e... 68 E f f e c t o f p H....... 76 E f f e c t o f C alcium C h l o r i d e... 79 E f f e c t o f S u b s tr a te C o n c e n tr a tio n... 82 S to ra g e S t a b i l i t y....... 108 T herm al S t a b i l i t y... 109 E f f e c t o f Flow R a te on A c t i v i t y o f P o ly m e ric P ro n a s e... I l l E f f e c t o f P ro n a se P r e tr e a tm e n t o f Skim m ilk on B iol o g i c a l O x i d a t i o n... 118 D IS C U S S IO N... 127 SUMMARY... 133 BIBLIOGRAPHY... 136 V I T A... 1>*9 i l l

TABLES Table Page 1. P r o p e r t i e s o f P r o te in a s e s P r e s e n t in P eak s on C M -C ellu lo se C h ro m a to g ra m... 5 2. R e a c tiv e Amino A cid R e s id u e s i n t h e Common I n s o l u b i l i z i n g M e th o d s...*... 18 3. F u n c tio n a l G roups A v a ila b le f o r S ila n e C o u p lin g A g en ts... 17 H. A p p lic a tio n s o f I n s o l u b i l i z e d E n z y m e s... 1+5 5. M o is tu re C o n te n t o f I n s o lu b le P ro n a se... 65 6. Sum m ation o f B in d in g S t u d i e s....... 67 7. Q-^q V alu e s f o r P o ly m e ric P ro n a s e on F o u r M ilk P r o t e i n s... 75 8. C om parison o f K in e tic D a ta f o r S o lu b le and I n s o lu b le P ro n a se... 108 9. E f f e c t o f S o lv e n t on E n zym atic A c t i v i t y... 109 1 0. E f f e c t o f T e m p e ra tu re on th e T herm al S t a b i l i t y o f S o lu b le and P o ly m e ric P r o n a s e... 110 1 1. L in e a r R e g re s s io n A n a ly s is o f R e sid e n c e Time and Time R e q u ire d t o Reach a C o n sta n t E nzym atic R e a c tio n V e lo c ity f o r P o ly m e ric P ro n a se A c tin g on M ilk P r o t e i n s... 115 1 2. B iom ass P e rfo rm a n c e D a t a... 12 U iv

ILLUSTRATIONS Figure Page 1. Some Common C r o s s - lin k in g R e a g e n ts... 16 2. C u r tiu s A zid e M ethod f o r C o u p lin g Enzymes t o C e l l u l o s e... 20 3. C o u p lin g o f P r o te in s t o S e p h a ro se b y Means o f ^ C yanogen Brom ide... 21 U. C o u p lin g o f P r o te i n s t o an E th y le n e -M a le ic A n h y d rid e ( l : l ) Copolym er (EMA)... 22 5. C a rb o d iim id e M ethod f o r B in n in g Enzymes t o a W a te r - I n s o lu b le S u p p o rt... 2k 6. P r e p a r a tio n o f W a te r - I n s o lu b le Enzymes by D iazo C o u p lin g o f t h e Enzyme t o a G la ss S u p p o rt..,. 25 7. I s o th io c y a n a te M ethod f o r C o u p lin g Enzymes t o G l a s s... 27 8. S ta r c h G el E l e c tr o p h o r e s is and P o ly a c ry la m id e G el E l e c tr o p h o r e s i s o f P u r i f i e d P r o te i n s... 57 9. A D i f f e r e n t i a l S p e c t r a l Scan from 330 nm t o 250 nm C om paring New an d O ld BAEE S a m p l e s... 60 10. R e a c tio n R a te f o r V a rio u s P ro n a se A liq u o ts from t h e C o u p lin g R e a c tio n on B A E E... 63 1 1. E f f e c t o f T e m p e ra tu re on th e R e a c tio n V e lo c ity o f S o lu b le P ro n a se w ith F o u r M ilk P r o t e i n s... 70 1 2. E f f e c t o f T e m p e ratu re on th e V e lo c ity o f P o ly m e ric P ro n a se w ith D i f f e r e n t M ilk P r o te in S u b s t r a t e s... 73 13. E f f e c t o f ph on t h e R e a c tio n V e lo c ity o f P o ly m e ric P ro n a se w ith D i f f e r e n t M ilk P r o te in s... 77 1 ^, E f f e c t o f C alcium C h lo r id e on t h e V e lo c ity o f P o ly m e ric P ro n a se w ith D i f f e r e n t M ilk P r o te i n s... 80 v

ILLUSTRATIONS (contd.) Figure Page 1 5. V e lo c ity v s. S u b s tr a te P l o t f o r S o lu b le P ro n a se R e a c tin g w ith C a se in and a - c a s e i n... 83 16. V e lo c ity v s. S u b s tr a te P l o t f o r S o lu b le P ro n a se on a - la c ta lb u m in and B - l a c t o g l o b u l i n... 86 1 7. L in e v e a v e r-b u rk e P lo t f o r t h e R e a c tio n o f S o lu b le P ro n a s e w ith a - c a s e i n... 88 1 8. L in e v e a v e r-b u rk e P lo t f o r S o lu b le P ro n a se Whole C a s e in R e a c tio n........ 90 1 9. L in e v e a v e r-b u rk e P lo t f o r t h e R e a c tio n o f S o lu b le P ro n a se w ith a - la e ta lb u m in... 92 2 0. L in e v e a v e r-b u rk e P lo t f o r S o lu b le P ro n a se { J -la c to g lo b u lin R e a c t i o n... 9 2 1. V e lo c ity v s. S u b s tr a te P lo t f o r t h e P o ly m e ric P ro n a se R e a c tio n on F o u r M ilk P r o te in s a t One M in u te R e s id e n c e T i m e... 97 2 2. L in e w e a v e r-b u rk e P lo t f o r P o ly m e ric P ro n a se C a se in R e a c t i o n * 99 2 3. L in ew eav er-b u rk e P lo t f o r P o ly m e ric P ro n a se a - c a s e i n R e a c tio n... 101 2U. L in ew eav er-b u rk e P lo t f o r t h e R e a c tio n o f P o ly m e ric P ro n a s e w ith B - la c to g lo b u lin... 103 2 5. L in e w e a v e r-b u rk e P lo ts f o r P o ly m e ric P ro n a se on a - l a c t a l b u m i n... 105 2 6. E f f e c t o f Flow R ate on th e Time R e q u ire d t o Come t o a S te a d y R e a c tio n V e lo c ity f o r th e P o ly m e ric P ro n a se 3 - la c t o g lo b u l i n R e a c tio n... 112 2 7. Time R e q u ire d f o r th e A c t i v i t y o f P o ly m e ric P ro n a se t o R each a C o n sta n t V alu e a t D i f f e r e n t Flow R a te s f o r 6 - la c t o g lo b u l i n and a - la c ta lb u m in... 116 vi

ILLUSTRATIONS (contd.) Figure Page 2 8. E f f e c t o f R e s id e n c e Time on t h e Maximum V e lo c ity A c h ie v a b le a t a G iven Flow R a te, v i t h a C o n s ta n t S u b s tr a te C o n c e n tr a tio n, f o r P o ly m e ric P ro n a s e R e a c tin g w ith 8 - l a c t o g l o b u l i n and a - la c ta lb u m in... 119 2 9. L in e w e av er-b u rk e P l o t f o r t h e P o ly m e ric P ro n a se - 8 - l a c t o g l o b u l i n R e a c tio n B ased upon V a lu es O b ta in e d from L in e a r U nw eighted R e g re s s io n A n a ly s is o f D a ta f o r B - la c to - g lo b u lin a t V a rio u s S u b s tr a te C o n c e n tr a tio n s... 121 3 0. H igh V o lta g e E l e c tr o p h o r e s is o f Sam ples O b ta in e d from a B io fe rm e n te r G iven P ro n a se T r e a te d Skim m ilk o v e r a T h re e Day P e r io d... 125 vii

INTRODUCTION C u r r e n t ly, t h e fo o d in d u s t r y p ro d u c e s w a s te s w hich r e p r e s e n t a b o u t UOJf o f t h e t o t a l b i o l o g i c a l oxygen demand on w a ste tr e a tm e n t f a c i l i t i e s. F u r th e r m o r e, p r o t e i n s p r e s e n t i n th e fo o d w a s te t r e a t m ent sy ste m can re d u c e c o n s id e r a b ly t h e e f f i c i e n c y o f a tr e a tm e n t p l a n t. T h is r e d u c tio n in e f f i c i e n c y b y fo o d p r o t e i n s can r e s u l t from i n t e r a c t i o n s w ith t h e m ic r o f lo r a o f t h e b i o l o g i c a l tr e a tm e n t p l a n t, th e r e b y d e c r e a s in g t h e r a t e o f oxygen t r a n s p o r t, w ith th e s u b se q u e n t p r o d u c tio n o f u n d e s ir a b l e foam s. P r e - d e g r a d a tio n o f p r o t e i n macromolecules w ould t h e o r e t i c a l l y i n c r e a s e t h e e f f i c i e n c y o f th e tr e a tm e n t p l a n t by re d u c in g m acro - m o le c u le - m ic r o f lo r a i n t e r a c t i o n s. H ow ever, s o lu b le enzym es a r e e x p e n s iv e, and i t w ould b e b o th u n e co n o m ic a l and i m p r a c tic a l t o u se them in a w a s te tr e a tm e n t p l a n t. A f e a s i b l e m ethod o f p r e t r e a t i n g fo o d w a s te s w ould b e t o a t t a c h enzym es t o a s o l i d s u p p o r t. But b e f o r e t h i s m ethod can b e a p p l i e d, th e optim um e n v iro n m e n ta l c o n d itio n s m ust b e known t o a s s u r e maximum e n z y m a tic a c t i v i t y. T h is i n v e s t i g a t i o n was u n d e r ta k e n t o c h a r a c t e r i z e th e a c t i v i t y o f im m o b iliz e d p ro n a se on s e l e c t e d m ilk p r o t e i n s i n o r d e r t o u s e t h i s te c h n iq u e f o r p o s s i b l e fo o d tr e a tm e n t a p p l i c a t i o n. 1

LITERATURE REVIEW Prona.se P ro n a se i s a c o m m e rc ia lly a v a i l a b l e p r o te in a s e p r e p a r a t i o n v h ic h i s p ro d u c e d in t h e c u l t u r e medium o f S tre p to m y c e s g r is e u s K - l. S in c e i t d e g ra d e s p r o t e i n s by h y d r o ly z in g t h e p e p t i d y l - p e p t i d e b o n d, th e Enzyme Com m ission h a s c l a s s i f i e d p ro n a s e in t h e g ro u p o f enzym es a c t i n g on p e p tid e bonds (1 3 2 ). Ncraoto and N a ra h a sh i ( i l l ) f i r s t r e p o r t e d t h e i s o l a t i o n o f a p r o te a s e from S tre p to m y c e s g r is e u s K -l i n 1959* T h e ir enzyme was conc e n t r a t e d b y ammonium s u l f a t e p r e c i p i t a t i o n, p u r i f i e d by a d s o r p tio n on a c a tio n - e x c h a n g e r e s i n, and f i n a l l y c r y s t a l l i z e d from d i l u t e a c e to n e s o l u t i o n. The enzyme c r y s t a l l i z e d i n n e e d le s w ith t h e f o llo w in g comp o s i t i o n : c a rb o n - 52.0j, h y d ro g en - 6.8?!, n itr o g e n - lu.0 #, s u l f u r - t r a c e, and c a lc iu m - 0.8 ^ ( 2 0 ). The p r o t e i n h ad a m o le c u la r w e ig h t o f 2 0,0 0 0 +_ 800 and an i s o e l e c t r i c p o in t a t ph 5.5. T h e ir enzyme was s t a b l e from ph 5 t o 10 an d p r o te c t e d s tr o n g l y by c a lc iu m i o n s. To i n v e s t i g a t e th e s u b s t r a t e s p e c i f i c i t y o f t h i s enzym e, Nomoto, N a ra h a s h i, and M urakam i (1 1 7 ) u t i l i z e d many s y n t h e t i c s u b s t r a t e s a s d i p e p t i d e s, t r i p e p t i d e s, am ino a c id a m id e s, am ino a c id e s t e r s, o rg a n ic a c id e s t e r s, and t h e i r a n a lo g o u s com pounds. T h e ir s tu d y showed t h a t t h e p r o te a s e h a d an e x tre m e ly b ro a d s p e c i f i c i t y to w a rd s t h e s u b s t r a t e s s tu d ie d and was c a p a b le o f h y d r o ly z in g many p e p t i d e s, am ides and e s t e r s in c lu d in g t h e r e i n th e m a jo r ity o f th e s p e c i f i c s u b s t r a t e s o f p e p s in,

t r y p s i n, c h y m o try p s in, p a p a in, c a th e p s in C, c a r b o x y p e p tid a s e, le u c in e a m in o p e p tid a s e, a m i n o t r i p e p t i d a s e, g l y c y l - l e u c in e d ip e p tid a s e, and im in - 3 o p e p tid a s e. The p r o te a s e was c a p a b le a l s o o f h y d r o ly z in g th e p e p tid e s in v o lv in g D-am ino a c i d r e s i d u e s. E x p e rim e n ts w ith s y n t h e t i c p e p tid e s i n d i c a t e d t h a t th e p r o te a s e p r e f e r e n t i a l l y h y d ro ly z e d th e p e p tid e bonds c o n ta in in g am ino g ro u p s o f L - le u c in e and L -p h e n y la la n in e (1 0 1 ). P e p tid e bonds r e s i s t i n g th e a c t i o n o f p r o te a s e w ere th o s e o f g ly c y l - g l y c i n e and g l y c y 1 - p r o l i n e, and b o n d s in v o lv in g a c y l r e s id u e s o f N -a c y l am ino a c id s and N -a c y l p e p tid e s ( 1 1 7 ). In e x p e rim e n ts w ith p r o t e i n s, th e a v e ra g e num ber o f p e p tid e bonds o f egg alb u m in h y d ro ly z e d by t h i s enzyme e q u a lle d 8755 o f th e t o t a l num ber o f b o n d s i n th e m o le c u le, w h ile 75^ o f th e t o t a l num ber o f b o n d s o f c a s e in w ere h y d ro ly z e d ( l l h ). F u r th e r work b y Nom oto, N a r a h a s h i, and Murakami show ed t h a t t h e i r enzyme was b o th an e n d o p e p tid a s e and an e x o p e p tid a s e w ith u n u s u a lly w ide s id e c h a in s p e c i f i c i t y (1 1 2, 1 1 3, 1 1 5, 1 1 6, 1 1 7 ). H ow ever, th e h o m o g en eity and s p e c i f i c i t y o f p ro n a se rem a in ed co n t r o v e r s i a l. P a ssa g e o f th e enzyme th ro u g h an A m b e rlite IRC-50 r e s i n gave tw o s e p a r a te p e a k s, each e x h i b i t i n g p r o t e o l y t i c a c t i v i t y and e a ch e x h i b i t i n g h e te r o g e n e ity by m oving b o u n d ary e l e c t r o p h o e s i s ( 107). L a te r, H ira m a tsu and O uchi (62) d e m o n s tra te d th e e x i s t e n c e o f t h r e e p e a k s w ith p r o t e o l y t i c a c t i v i t y by s t a r c h g e l zone e l e c t r o p h o r e s i s. Upon re e x a m in in g th e s e r e s u l t s, Nomoto e t. a l. ( 1 1 8 ) r e p o r te d th e f r a c t i o n a t i o n o f p ro n a s e by colum n ch ro m ato g rap h y i n t o f o u r co m p o n en ts, each e x h i b i t i n g p r o t e o l y t i c a c t i v i t y. S u b s e q u e n tly, p ro n a s e h as b een f r a c t i o n a t e d b y colum n ch ro m ato g rap h y w ith C M -c e llu lo s e, DEAE-Sephadex and

A m b e rlite CG-50 r e s i n i n t o 13 p r o t e o l y t i c enzym es (1 0 8, lu 2 ). T hese 13 p r o t e o l y t i c enzym es w ere g ro u p ed i n t o f i v e c l a s s i f i c a t i o n s d e p e n d in g upon t h e i r optim um ph f o r p r o t e o l y s i s and t h e i r s u b s t r a t e s p e c i f i c i t y : f o u r n e u t r a l p r o t e i n a s e s, t h r e e a l k a l i n e p r o t e i n a s e s, t h r e e am inopept i d a s e s, tw o c a r b o x y p e p tid a s e s, and an e l a s t o l y t i c enzym e. When com p arin g r e p o r t s on t h e s p e c i f i c i t i e s o f p ro n a s e en zy m es, t h e v a r i a b i l i t y o f d i f f e r e n t p ro n a s e p r e p a r a t i o n s s h o u ld b e k e p t in m in d. T rop e t. a l. (l^ O, l U l ), u t i l i z i n g B g ra d e p ro n a se * d e m o n s tra te d v e r y l i t t l e c h y m o tr y p s in - lik e a c t i v i t y when a s s a y e d a g a i n s t N - a c e ty l- t y r o s i n e e t h y l e s t e r, and t h i s a c t i v i t y c o u ld n o t b e d e te c te d i n any o f t h e s e p a r a te d f r a c t i o n s. H ow ever, Ryan (1 2 6 ) c l e a r l y d e m o n s tra te d t h e p r e s e n c e o f a o - c h y m o tr y p s in - lik e enzyme i n a d i f f e r e n t p ro n a s e p r e p a r a t i o n. D i f f e r e n t im m u n o d iffu sio n p a t t e r n s w ere a l s o n o te d f o r d i f f e r e n t p ro n a s e p r e p a r a t i o n s (U ). T hese d i f f e r e n c e s in t h e num ber o f enzym es p ro d u c e d and t h e i r q u a n t i t i e s may b e a t t r i b u t e d t o m u ta tio n s o f S tre p to m y c e s g r is e u s o r t o v a r i a t i o n s i n g ro w th c o n d itio n s (1 2 6 ). M ic r o b ia l p r o te in a s e s h ave b een c l a s s i f i e d i n t o t h r e e g ro u p s by t h e i r optimum ph f o r p r o t e o l y s i s, i. e. a c i d, n e u t r a l, and a l k a l i n e p r o te in a s e s ( l O l ). M ost o f t h e n e u t r a l p r o te in a s e s a re known t o be i n h i b i t e d b y e t h y l e n e d i a m i n e t e t r a a c e t i c a c id (EDTA), b u t u n a f f e c te d by d iis o p r o p y l p h o s p h o f l u o r i d a t e, is o p r o p y lm e th y lp h o s p h f lu o r id a te, o r p o t a t o i n h i b i t o r ( 2 0 ), On th e o t h e r h a n d, a l k a l i n e p r o te in a s e s a re n o t a f f e c t e d by th e fo rm e r b u t a r e c o m p le te ly i n h i b i t e d by t h e l a t t e r tw o a g e n t s, As a r e s u l t t h e n e u t r a l p r o te in a s e s a r e c a l l e d m e ta llo - enzym es and t h e a l k a l i n e p r o t e i n a s e s, s e r i n e enzym es. T a b le 1 l i s t s

5 T a b le 1 P r o p e r t i e s o f p r o t e i n a s e s p r e s e n t i n p e a k s on C M -c e llu lo s e chro m ato g ram F r a c t io n P eak Numbers P r o p e r t i e s _ I ^ I l f I I I 3 r v f Optimum ph ( c a s e i n as s u b s t r a t e ) 7.5-8.0 7.5-8.0 9.0-1 0.0 9.0-1 0.0 S t a b l e ph ra n g e 5.0-9.0 5.0-9.0 3.0-9.0 3.0-9.0 H e a t s t a b i l i t y (6 0 C, 10 m in., % o f r e s i d u a l a c t i v i t y E x t i n c t i o n a t 278 nm (1 2 ) 89 95 50 89 1 6.2 * 1 1.5 1 0.2 P e r c e n t i n h i b i t i o n DFP 0 100 100 EDTA 97 9U 0 0 P o t a t o i n h i b i t o r 0 10 U0 90 P e p tid e h y d r o l y s i s ( S o l i d arro w show s th e b o n d s p l i t ) BAEE +++ TAME +++ + Z -G ly -P ro * L e u -G ly - +++ _ Z -G lu*t yr +++ - - xv G e la tin l i q u e f y i n g a c t i v i t y + + +++ D a ta ta k e n from I la ra h a s h i, ShibiQ ^a, an d Y a n a g ita ( 1 0 8 ). " ^ P ro te in a s e s e p a r a t e d fro m a m in o p e p tid a s e on D E A E -c e llu lo se 2 P r o t e i n a s e p r e s e n t in p e a k I I on C M -c e llu lo s e ^ P r o te in a s e s e p a r a t e d fro m c a r b o x y p e p tid a s e on A m b e rlite CG-50 r e s i n ^ P r o te in a s e p r e s e n t i n p e a k IV on C M -c e llu lo s e

some o f t h e p r o p e r t i e s o f p r o te in a s e s s e p a r a te d b y C M -c e llu lo s e, DEAE- S ep h ad ex, and A m b e rlite CG-50 r e s i n. The n e u t r a l p r o te in a s e s p r e s e n t in p e a k s I and I I h a d s i m i l a r p r o p e r t i e s w ith r e s p e c t t o ph optim um to w a rd s c a s e in s u b s t r a t e, s t a b l e ph r a n g e, and b e h a v io r a g a in s t i n h i b i t o r s. I n s t r i k i n g c o n t r a s t a r e th e p r o p e r t i e s o f th e a l k a l i n e p r o t e i n a s e s i n p e a k s I I I and IV, The s t a b l e ph ra n g e s w ere 5 t o 9 f o r th e n e u t r a l p r o te in a s e and 3 t o 9 f o r t h e a l k a l i n e p r o t e i n a s e. One o f t h e a l k a l i n e p r o te in a s e s i n p e a k I I I d e m o n s tra te d t h e a b i l i t y t o h y d ro ly z e N -b e n z p y l-l -a rg in in e e t h y l e s t e r (BAEE) and N - d - to s y l- L - a r g in in e m e th y l e s t e r (TAME) w hich a r e s u b s t r a t e s f o r t r y p s i n ( 1 1 0 ), c a th e p s in B ( 5 l)» an d p a p a in ( 7 8 ). T h is p r o t e i n a s e, u n lik e th e o th e r en zy m es, h ad p o te n t g e l a t i n - l i q u i f y i n g a c t i v i t y and h ad l e s s th e r m a l s t a b i l i t y com pared w ith th e o th e r enzym es i n p r o n a s e. R e c e n tly, T rop and B irk (lu 2 ) h a v e named t h i s enzyme p ro n a se t r y p s i n. F u r th e r w ork h a s i n d i c a te d a s i m i l a r i t y in th e a c t i v i t y o f b o th b o v in e t r y p s i n and p ro n a s e t r y p s i n on v a r io u s s u b s t r a t e s. By b lo c k in g th e e-am in o g ro u p o f th e l y s i n e r e s i d u e in a p e p t i d e, t h e s u b s t r a t e i s r e n d e re d immune t o h y d r o ly s i s by b o th enzym es (lu 2 ). P ro n a s e t r y p s i n a c t i v a t e s p r o - e l a s t a s e ( l l U ), tr y p s in o g e n and ch y m o try p sin o g e n A and h y d ro ly z e s p o ly L - ly s in e i n a s i m i l a r m anner t o t r y p s i n. The a c t i v i t y o f p ro n a s e t r y p s i n i s i n h i b i t e d by lmm d iis o p r o p y lp h o s p h o f lu o r id a te and by n a t u r a l l y o c c u rin g t r y p s i n i n h i b i t o r s. P ro n a se t r y p s i n was i n h i b i t e d b y a l l b o v in e t r y p s i n i n h i b i t o r s t e s t e d. Bowman-Birk so y b ean i n h i b i t o r i n h i b i t e d a b o u t 3056 o f p ro n a s e t r y p s i n 's a c t i v i t y to w a rd c a s e in and 100J6 o f i t s a c t i v i t y to w a rd

b e n z o y l a r g i n in e e t h y l e s t e r (19)* C h ick en o v o i n h i b i t o r i n h i b i t s b o th t h e t r y p s i n l i k e a c t i v i t y o f p ro n a s e to w a rd s b e n z o y 1 - a r g in in e e t h y l e s t e r an d t h e c h y m o try p s in lik e a c t i v i t y to w a rd s a c e t y l t r y o s i n e e t h y l e s t e r, w ith t h e c h y m o try p s in lik e a c t i v i t y b e in g i n h i b i t e d m ore s tr o n g l y th a n t h e t r y p s i n l i k e a c t i v i t y. T rop and B irk ( 1 U2 ) h av e shown t h a t t h e c a s e i n o l y t i c a c t i v i t y o f t h e i s o l a t e d t r y p s i n l i k e com ponent o f p ro n a s e can b e i n h i b i t e d up t o 80 t o 100JS, d e p e n d in g upon t h e i n h i b i t o r, by so y b ean and K u n itz p a n c r e a t i c t r y p s i n i n h i b i t o r, b y lim a b e a n, and by c h ic k e n ovom ucoid. S i m i l a r i t i e s in s i z e, a c t i v i t i e s, and i n i n h i b i t i o n by n a t u r a l l y o c c u rin g t r y p s i n i n h i b i t o r h av e l e d W ahlby (lt+u, IU5 ) t o s u g g e s t a p o s s i b l e s i m i l a r i t y i n c o m p o sitio n an d s t r u c t u r e o f t h e a c t i v e s i t e. A p a r t i a l seq u e n ce a n a l y s is o f th e r e s id u e s aro u n d th e a c t i v e s e r y l ( l 8 ), and h i s t i d y l r e s id u e s ( l 8 ) o f p ro n a s e t r y p s i n y i e l d e d th e f o llo w in g am ino a c i d s : -C y s-g ln -G ly -A sp -S e r - G Iy -G ly -P ro -V a l-t h r-a la -A la -H is - C y s-v a l- I n a d d i t i o n t o t h e p a r t i a l seq u e n c e a n a l y s is o f t h e a c t i v e s i t e, J u r a s e k e t. a l. ( 6 9 ) d e te rm in e d t h a t p ro n a s e t r y p s i n c o n ta in e d s ix d i s u l f i d e b r id g e s w h e re as mammalian t r y p s i n c o n ta in e d o n ly t h r e e d i s u l f i d e b r id g e s. P ro n a s e e l a s t a s e r e c e n t l y was i d e n t i f i e d b y T rop and B irk ( 1U2 ), and was a p p a r e n tly a hom ogeneous e n d o p e p tid a s e w h ich h y d ro ly z e s th e -L e u -T rp - and - A la - H is - p e p tid e b o n d s. I t i s a lm o st e n t i r e l y u n a f f e c te d by n a t u r a l t r y p s i n i n h i b i t o r s, and i s c o m p le te ly i n h i b i t e d by d i i s o - p r o p y lp h o s p h o r o f lu o r id a te, a p r o p e r ty s h a re d by p a n c r e a t i c e l a s t a s e

8 and o t h e r s e r i n e enzym es (1 ^ 2 ). H ow ever, b e n z eth o n iu m c h lo r id e i n h i b i t s ab o u t 85JE o f t h e e l a s t o l y t i c a c t i v i t y o f t h i s enzym e. To d e m o n s tra te t h e p r e s e n c e o f s e v e r a l p r o t e o l y t i c enzym es in p r o n a s e, N a r a h a s h i, and Y a n a g ita (1 0 7 ) o b s e rv e d t h e e f f e c t s o f v a r io u s m e ta l io n s on t h e a c t i v i t y o f p ro n a s e a g a in s t c a s e i n, c a rb o b e n z o x y g ly - c y l - L - le u c in e (CG L), L - le u c y lg ly c in e (L G ), an d L -le u c y l-b -n a p th y la m id e (LNA), T h e ir r e s u l t s showed t h a t t h e p r o te in a s e a c t i v i t y w ith c a s e in a s t h e s u b s t r a t e was somewhat p r o te c t e d by c a lc iu m and s tr o n tiu m i o n s, and c o n s id e r a b ly i n h i b i t e d by c o b a lt and z in c i o n s, w ith th e o th e r d i v a l e n t m e ta l io n s a s m anganese and m agnesium h a v in g no in f lu e n c e (1 0 7 ). S tu d ie s on t h e a c t i o n o f th e p e p tid a s e s i n d i c a t e d t h a t tw o ty p e s o f p e p t i d a s e s w ere p r e s e n t, a m in o p e p tid a s e, w hich h y d ro ly z e d LG and LNA, and c a r b o x y p e p tid a s e, w hich h y d ro ly z e d CGL (1 0 7 ). B oth a c t i v i t i e s in c r e a s e d re m a rk a b ly in th e p r e s e n c e o f c a lc iu m an d c o b a lt i o n s, i n d i c a t i n g b o th ty p e s o f p e p tid a s e s w ere m e ta llo e n z y m e s. B in d in g o f th e m e ta l w ith EDTA h ad th e e f f e c t o f rem oving t h e a c t i v a t i n g m e ta l io n s from th e enzym e, th e r e b y r e n d e r in g t h e enzyme i n a c t i v e. R e s to r a tio n o f a c t i v i t y c o u ld be a c h ie v e d by r e a c t i v a t i o n w ith c o b a lt o r c a lc iu m i o n s. The a m in o p e p tid a se was fo u n d t o be h e a t s t a b l e below 80 C and was n o t i n a c t i v a t e d b y up t o 9M u r e a even a t room te m p e r a tu r e. H ow ever, t h e enzyme was v e ry l a b i l e on d i a l y s i s a g a in s t d i s t i l l e d w a te r and w ith r e a g e n ts w hich bound m e ta l io n s. In th e p re s e n c e o f c a lc iu m io n s th e a m in o p e p tid a s e had a ph o p tim a aro u n d ph 8.3 and a t ph 8.0 and 9-5 in t h e p re s e n c e o f c o b a lt io n s w ith LG as th e s u b s t r a t e.

I n c o n t r a s t, t h e c a r b o x y p e p tid a s e v a s l e s s h e a t s t a b l e th a n t h e a m in o p e p tid a s e, and e x h ib ite d m ore s t a b i l i t y when m e ta l io n s w ere rem oved. The ph optim um f o r c a r b o x y p e p tid a s e v a s ph 7.5 i n th e p re s e n c e o f e i t h e r c a lc iu m o r c o b a lt i o n s. One o f t h e t h r e e a l k a l i n e p r o te in a s e s p o s s e s s e d a h ig h a c t i v i t y to w a rd N - b e n z o y l-l -a rg in in e e t h y l e s t e r (BAEE), N - a - to s y l- L - a r g in in e m e th y l e s t e r (TAME), N - a -b e n z o y l- L -ly s in e m e th y l e s t e r (BIM E), and N - b e n z o y l-d L - a rg in in e - ]D - n itro - a n ilid e (BAjdNA), w hich a r e s u b s t r a t e s o f t r y p s i n (1 0 6 ). The o th e r tw o a l k a l i n e p r o te in a s e s h y d ro ly z e N - a c e ty l- L - ty r o s in e e t h y l e s t e r. The t h r e e a l k a l i n e p r o te in a s e s w ith e s t e r o l y t i c a c t i v i t i e s w ere d e s ig n a te d a s a l k a l i n e p r o te in a s e a, b, and c, r e s p e c t i v e l y ; p r o te in a s e s a and c h y d ro ly z e d ATEE w h ile b h y d ro ly z e d BAEE. S e p a r a tio n o f t h e s e enzym es from p ro n a s e h a s b e en a t t a i n e d by s u c e s s iv e a p p l i c a t i o n o f io n -e x c h a n g e c h ro m a to g ra p h y. F u r th e r p u r i f i c a t i o n o f a l k a l i n e p r o te in a s e b on a S ephadex G-75 co lu m n, w hich h ad b e e n e q u i l i b r a t e d in lmm h y d r o c h lo r ic a c id c o n ta in in g 0.1 M sodium c h l o r i d e, ph 3. 0, w hose tw o s e p a r a t e p e a k s, each w ith a lm o st th e same s p e c i f i c a c t i v i t i e s to w a rd s c a s e in and BAEE, b u t d i f f e r e n t m o le c u la r w e ig h ts. The tw o a l k a l i n e p r o te in a s e s a r e c a l l e d b and b *. A lk a lin e p r o te in a s e b h a d a s i m i l a r i t y t o t r y p s i n aro u n d th e a c t i v e s i t e. The _/T c K v a lu e s a r e 9.0 x 10~ M f o r BAEE, 7.7 x 1 0 M f o r TAME, and l.u x m 1 0 " ^ f o r BAgNA. The m o le c u la r w e ig h t o f t h e p u r i f i e d a l k a l i n e p r o t e i n a s e b v a s e s tim a te d t o b e a b o u t 2 0,0 0 0 b y t h e g e l - f i l t r a t i o n m ethod w ith S ephadex G-75 ( l ). I n c o n c lu s io n p ro n a s e h a s b een found t o b e a h e te ro g e n e o u s m ixt u r e o f enzym es p o s s e s s in g w ide s u b s t r a t e s p e c i f i c i t y. Some w ork h a s

b e e n c o n d u c te d t o p u r i f y and c h a r a c t e r i z e s p e c i f i c p ro n a s e en zy m es, b u t m ore w ork n eed s t o b e done on a c t i v e s i t e a n a ly s is and a m in o -a c id s e q u e n c in g. Im m o b iliz ed Enzymes S y n th e tic o r g a n ic p o ly m e rs p o s s e s s c e r t a i n c h a r a c t e r i s t i c s, su ch a s c h e m ic a l i n e r t n e s s, i n s o l u b i l i t y i n aq u eo u s s o l u t i o n s, and o th e r u n iq u e p r o p e r t i e s, w hich h a v e l e d t o t h e i r w id e -s p re a d u t i l i z a t i o n. On th e o t h e r h a n d, n a t u r a l p o ly m ers,- su ch a s enzy m es, a n t i g e n s, and a n t i b o d i e s, a r e l a b i l e, s o lu b le i n aq ueous s o l u t i o n s, and h av e d i s t i n c t i v e b i o l o g i c a l a c t i v i t i e s. I n th e p a s t, th e i n t e r e s t s o f s c i e n t i s t s from tw o d i s c i p l i n e s, n a t u r a l p o ly m er c h e m is tr y, an d i n p a r t i c u l a r p r o t e i n c h e m is tr y, and s y n t h e t i c p o ly m e r c h e m is try h av e n o t o v e rla p p e d. Howe v e r, r e c e n t l y, i n t e r d i s c i p l i n a r y i n v e s t i g a t i o n s i n t o t h e a r e a o f im m o b iliz e d enzym es h av e b e e n c o n d u c te d b y b o th g ro u p s. E nzym es, a s d e f in e d by W eb ster ( 5 0 ), a re any o f a v e ry la r g e c l a s s o f com plex p r o te in a c e o u s s u b s ta n c e s t h a t a r e p ro d u c e d b y l i v i n g c e l l s and a c t as c a t a l y s t s in p ro m o tin g a t t h e c e l l te m p e ra tu re u s u a lly r e v e r s i b l e r e a c t i o n s w ith o u t th e m s e lv e s u n d e rg o in g m arked d e s t r u c t i o n i n t h e p r o c e s s. I n i n d u s t r y, th e c a t a l y t i c a c t i v i t y and s p e c i f i c i t y o f enzym es a r e w id e ly u t i l i z e d i n su ch d iv e r s e c o n c e rn s as t h e s p e c i f i c b i o l o g i c a l s y n th e s e s o f many p h a r m a c e u tic a ls ( 2 7 ), in c h e e s e m aking (2 U ), and i n d e te r g e n t a d d i t i v e s ( 8 0 ). H ow ever, s o lu b le enzym es i n h e r e n t l y p o s s e s s c e r t a i n d is a d v a n ta g e s : ( a ) enzym es a r e l a b i l e ; (b ) o n ce enzym es a re u t i l i z e d in s o l u t i o n, th e y c a n n o t b e r e c o v e r e d ;

( c ) u s u a l l y enzym es m ust b e f r e e from o t h e r c o n ta m in a tin g enzy m es, and a s a r e s u l t t h e s e p r e p a r a t i o n s may be e x p e n s iv e ; and (d ) i n some c a s e s r e s i d u a l e n z y m a tic a c t i v i t y may c a u se u n d e s ir a b le c o m p lic a tio n s. Many o f th e s e l i m i t a t i o n s can b e overcom e by u t i l i z i n g p o ly m e ric enzym es ( 8 7 ), i. e. n a t u r a l enzym es s u p p o rte d on s o l i d c a r r i e r s o r 11 c h e m ic a lly bonded t o s y n t h e t i c p o ly m e rs. T h ese im m o b iliz e d enzym es: ( a ) a r e r e a d i l y s e p a r a b le from p ro d u c ts and r e a c t a n t s by f i l t r a t i o n, th e r e b y p e r m ittin g r e p e a te d u t i l i z a t i o n o f t h e same enzyme p r e p a r a t i o n ; (b ) may b e u s e d o v e r lo n g p e r io d s o f tim e (1 2 5, 1 5 2 ); ( c ) may b e em ployed w ith o u t t h e d a n g e r o f one d i g e s t i n g a n o th e r ; (d ) p e rm it a g r e a t e r l a t i t u d e o f r e a c t i o n c o n d itio n s ( 3 6 ) ; ( e ) n eed n o t be p u re p r e p a r a t i o n s ; and ( f ) a r e m ore e a s i l y a d a p te d t o a u to m a tio n, c o n v e n ie n t, and e c o n o m ic a l th a n s o lu b le enzyme p r e p a r a t i o n s. Im m o b iliz ed enzym es a l s o p ro v id e s im p le, u s e f u l in v i t r o m odels f o r m em brane-bound enzym es su ch as th o s e p a r t i c i p a t i n g i n p h o to sy n t h e s i s, a c t i v e t r a n s p o r t, r e s p i r a t i o n, and p r o te in b i o s y n t h e s i s (7 2 ). U n t i l r e c e n t l y, t h e s e enzyme sy ste m s h av e b e e n s tu d ie d a lm o st e x c lu s i v e l y i n s o l u t i o n. W ith t h e r e c o g n itio n t h a t enzymes i n t h e l i v i n g c e l l a r e n o rm a lly a tta c h e d t o s u r f a c e s, t h e te rm a l l o t o p y h a s b e e n in tr o d u c e d t o d e s c r ib e t h e d i f f e r e n c e s b etw een th e p r o p e r t i e s o f m em brane-bound enzym es and t h e p r o p e r t i e s o f th e same enzym es in s o l u t i o n (lo U ). M ethods o f Im m o b iliz a tio n S e v e r a l m ethods h av e b een em ployed t o im m o b iliz e enzym es on w a te r i n s o l u b l e c a r r i e r s. T h ese in c lu d e p h y s ic a l a d s o r p tio n o n to

m a tr ic e s l i k e g l a s s ( 9 5 ), c h a r c o a l ( 1 0 9 ), b e n t o n i t e ( 5 7 ), o r t o o r g a n ic Io n -e x c h a n g e rs (1 5 1 ). O th e r i n s o l u b i l i z a t i o n m ethods in c lu d e p o ly c o n d e n s a tio n o f t h e enzyme w ith r e a c t i v e b i f u n c t i o n a l r e a g e n t s ; a d s o r p tio n f o llo w e d b y c o v a le n t c r o s s - l i n k i n g o f t h e p r o t e i n by a p p r o p r ia te b i f u n c t i o n a l r e a g e n t s ; i n c l u s i o n o r e n tra p m e n t o f t h e enzyme in th e p o re s o f a g e l, t h e p o re s o f w h ich a r e l a r g e enough t o a llo w s u b s t r a t e t o p a s s f r e e l y, b u t s m a ll enough t o r e t a i n th e enzym e; and c o v a le n t b in d in g o f t h e p r o t e i n t o a s u i t a b l e, w a t e r - i n s o l u b l e c a r r i e r, v i a f u n c t io n a l g ro u p s w hich a r e n o t e s s e n t i a l f o r b i o l o g i c a l a c t i v i t y. T h ese m ethods have b e en sum m arized i n a num ber o f re v ie w a r t i c l e s (1»3, Uk, 1*7, 7 1, 7 3, 8 7, 8 9, 9 2, 1 2 8, 1 3 1 ). 12 Enzymes Im m o b iliz e d by A d s o r p tio n : A d s o rp tio n a s a p h y s ic a l te c h n iq u e o f i n s o l u b l i z i n g enzym es o f te n le a d s t o d e n a t u r a t i o n. To m in im ize d e n a tu r a tio n a s u i t a b l e a d s o rb e n t s h o u ld p o s s e s s a h ig h a f f i n i t y f o r t h e enzyme and be s t a b l e u n d e r o p e r a t i o n a l c o n d itio n s. B oth c h a rg e d r e s i n s and n e u t r a l s u r f a c e s can b e u t i l i z e d t o a d s o rb - p r o t e i n s. B a rn e t and B u ll (8 ) a c h ie v e d a m ore p erm an en t enzyme a t t a c h m ent b y u s in g Dowex 5 0, a s u lf o n a te d p o ly s ty r e n e c a r r i e r, a s a s u p p o r t. S i m i l a r l y, M itz (9 9 ) h a s a tta c h e d c a t a l a s e, p e p s in, and p r o te a s e t o d ie th y la m in o e t h y l c e l l u l o s e (D E A E -c e llu lo s e ) w ith good r e t e n t i o n o f t h e i r a c t i v i t i e s. S e v e r a l io n -e x c h a n g e r e s i n s su ch a s D E A E -c e llu lo se, DEAE-Sephadex, and C M -c e llu lo se have b e e n u sed as s o l i d s u p p o r ts f o r enzyme im m o b iliz a tio n b y n o n -c o v a le n t b o n d in g (1 3 5, 1 3 8, 1 3 9 ). A d so rp t i o n o f enzym es o n to v a r io u s in o r g a n ic m a t e r ia ls as g l a s s b e a d s, q u a r t z, d i a l y s i s t u b i n g, and M illip o r e f i l t e r s h a s a ls o b e e n d e s c r ib e d (3 8, 122).

When c o m p le te enzyme im m o b iliz a tio n i s d e s i r e d, h o w e v e r, p h y s ic a l 13 a d s o r p tio n te c h n iq u e s a r e o f l i m i t e d r e l i a b i l i t y. Even i f u n d e r a g iv e n s e t o f c o n d itio n s no a c t i v e enzyme i s e l u t e d from an a d s o r b e n t, c h a n g in g t h e io n ic s t r e n g t h, ph o r te m p e r a tu r e, o r a d d i t i o n o f su b s t r a t e, may b r in g a b o u t e i t h e r a p a r t i a l o r a t o t a l d e s o r p tio n o f th e enzyme from t h e a d s o rb e n t ( 7 3 ). Work on t h e a d s o r p tio n o f p r o te in s on v a r io u s s u r f a c e s h a s b een sum m arized i n tw o r e c e n t re v ie w s (U l, 1 3 1 ). Enzymes Im m o b ilized by O c c lu s io n in C r o s s - lin k e d P o ly m e ric M a tr ic e s : Enzymes can b e o c c lu d e d w ith in a c r o s s - l i n k e d g e l m a trix by c a r r y in g o u t t h e p o ly m e r iz a tio n r e a c t i o n le a d in g t o g e l fo rm a tio n in a n aq u eo u s s o l u t i o n c o n ta in in g t h e enzyme ( 7 3 ). S u b se q u e n t m e c h a n ic a l d i s p e r s io n o f t h e g e l c o n ta in in g th e enzyme can b e a c h ie v e d t o o b ta in p a r t i c l e s o f d e f i n i t e s i z e. I n m ost o f th e c a s e s r e p o r t e d, a c ry la m id e was u s e d a s t h e monomer w ith c r o s s - l i n k i n g a f f e c t e d by N,N -m eth y len e b i s (a c ry la m id e ) ( 1 3, 5 3, 6 l ). H ow ever, e x p e rim e n ts w ith c e l l u l o s e ' n i t r a t e and c e l l u l o s e a c e t a t e h av e a ls o b e e n d e s c r ib e d, an d i t was c la im e d t h a t no le a c h in g o f t h e enzyme o c c u rre d d u rin g u s a g e (8 2, 8 7 ). Enzyme im m o b iliz a tio n by o c c lu s io n im p o ses m in im al c o n s t r a i n t s on t h e p o ly m e ric enzyme and d o es n o t in v o lv e c o v a le n t bond fo rm a tio n w ith th e s u p p o r tin g m a tr ix. T h e r e f o r e, i n th e o r y, t h i s p r i n c i p l e can b e a p p lie d t o any enzym e. H ow ever, s e v e r a l i n t r i n s i c l i m i t a t i o n s (7 3 ) o f t h i s m ethod n eed t o b e m e n tio n e d, (a ) B ecau se o f th e b ro a d d i s t r i b u t i o n i n t h e p o re s iz e o f s y n th e t i c g e ls o f t h e p o ly a c ry la m id e t y p e, c o n tin u o u s le a k a g e o f th e o c c lu d e d enzyme i s d i f f i c u l t t o a v o id, (b ) The e n z y m a tic r e a c t i o n o c c u rs o n ly w ith in t h e v i c i n i t y o f t h e g e l

1U m a tr ix. The c a t a l y t i c r e a c t i o n i s th u s l i m i t e d t o s u b s t r a t e s t h a t can d i f f u s e r e a d i l y i n t o t h e g e l. ( c ) The f r e e r a d i c a l s g e n e r a te d i n t h e c o u rs e o f p o ly m e r iz a tio n may a f f e c t t h e a c t i v i t y o f t h e e n tra p p e d enzym e. F o r ex am p le, a l d o l a s e v a s 25$ i n a c t i v a t e d d u r in g th e p r e p a r a t i o n o f t h e p o ly a c ry la m id e i n c l u s i o n c o n ju g a te (1 * 0. H ow ever, th e c o m p a ra tiv e ly h ig h a c t i v i t i e s (9*0 o f t h e i n c l u s i o n c o n ju g a te s o f a lc o h o l d e h y d ro g e n a s e, g lu c o s e o x id a s e, c h y m o try p s in, and e n o la s e r e f l e c t t h e g e n e r a l m ild n e s s o f th e m eth o d. I n c lu s io n a p p e a rs l e s s s u i t a b l e f o r enzym es h a v in g la r g e s u b s t r a t e s as r ib o n u c le a s e (9 * 0, p re su m a b ly t h e r e s u l t o f d i f f u s i o n and s t e r i c h in d r a n c e. Enzymes Im m o b iliz e d b y I n te r m o le c u la r C r o s s - l in k i n g : Many a c t i v e, i n s o l u b i l i z e d enzym es h av e b e e n form ed by r e a c t i o n w ith c r o s s - l i n k i n g r e a g e n t s, B i- o r m u l t i f u n c t i o n a l c r o s s - l i n k i n g r e a g e n ts can b e u t i l i z e d f o r t h e im m o b iliz a tio n o f b i o l o g i c a l l y a c t i v e p r o te in s by tw o m eth o d s (1 3 1 ): ( a ) th r e e - d im e n s io n a l n e t-w o rk fo rm a tio n as a r e s u l t o f i n t e r m o l e c u l a r c r o s s - l i n k i n g o f th e b i o l o g i c a l l y a c t i v e p r o t e i n ; and (b ) im m o b iliz a tio n o f t h e b i o l o g i c a l l y a c t i v e p r o t e i n b y s u i t a b l e b i - o r m u l t i f u n c t i o n a l c r o s s - l i n k i n g r e a g e n ts t o an i n s o l u b l e c a r r i e r. The p r o p e r t i e s o f b i f u n c t i o n a l p r o te in c r o s s - l i n k i n g r e a g e n ts and t h e i r u t i l i z a t i o n f o r t h e i n t r o d u c t i o n o f new i n tr a m o le c u la r c o v a le n t b o n d s, and f o r c o v a le n t l i n k i n g o f tw o s p e c ie s o f p r o te in m o le c u le s a r e d e s c r ib e d by R ic h a rd (12*0 and by Zahn (1 6 3 ). T he b i f u n c t i o n a l r e a g e n ts w hich a r e u sed in t h e in te r m o le c u la r c r o s s - l i n k i n g o f p r o t e i n s may be d iv id e d i n t o tw o g ro u p s (7 3 ): ( a ) h o m o b ifu n c tio n a l r e a g e n ts p o s s e s s in g tw o i d e n t i c a l f u n c t io n a l

g ro u p s, su ch as b i s - d ia z o b e n z id in e 2, 2* d i s u l f o n i c a c id ( F ig u r e l ) ; 15 and (b ) h e t e r o b i f u n c t i o n a l r e a g e n ts p o s s e s s in g tw o d i f f e r e n t f u n c t io n a l g r o u p s, su ch as t o l u e n e - 2 - is o c y a n a te - U - is o th io c y a n a te (F ig u re 1 ). B ecau se h e t e r o b i f u n c t i o n a l r e a g e n ts p o s s e s s d i f f e r e n t c h e m ic a l r e a c t i v i t i e s f o r t h e i r tw o f u n c t i o n a l g r o u p s, th e y a r e p a r t i c u l a r l y u s e f u l in b in d in g p r o t e i n s t o s u i t a b l e s u p p o r ts. H ow ever, t h e s e n s i t i v i t y o f many enzym es t o c h e m ic a l m o d if ic a tio n can l i m i t t h e a p p l i c a b i l i t y o f t h i s m eth o d. Enzymes Im m o b ilized b y C o v a le n t B in d in g : The b in d in g o f enzym es t o w a t e r - i n s o l u b l e c a r r i e r s by c o v a le n t b o n d s s h o u ld be c a r r i e d o u t v i a f u n c t i o n a l g ro u p s on th e p r o t e i n w hich a r e n o n e s s e n t i a l f o r b i o l o g i c a l a c t i v i t y. O b v io u s ly, t h e b in d in g r e a c t i o n s h o u ld b e p e rfo rm e d u n d e r c o n d itio n s t h a t do n o t c a u se d e n a t u r a t i o n. Any change i n th e a c t i v i t y o f an enzyme c o v a le n tly bound t o a p o ly m er may be a t t r i b u t e d t o tw o s o u r c e s. F i r s t, ch an g es r e s u l t i n g from c o v a le n t i n t e r a c t i o n s w ith t h e p o ly m er and s e c o n d, ch an g es a r i s i n g fro m n o n - c o v a le n t i n t e r - ' a c t i o n s. I n d e te rm in in g th e n a tu r e o f t h e c o v a le n t bonds by w h ich a * g iv e n p r o t e i n s h o u ld b e i n s o l u b i l i z e d an d s e l e c t i n g t h e a p p r o p r ia te w a t e r - i n s o l u b l e c a r r i e r u t i l i z a t i o n can b e made o f th e in f o r m a tio n a v a i l a b l e on th e e f f e c t s o f c h e m ic a l m o d if ic a tio n o f p r o te in s on t h e i r b i o l o g i c a l a c t i v i t y ( 3 2, 1 3 3, 13T) and on th e am ino a c id r e s i d u e s i n t h e a c t i v e s i t e o f enzym es ( 2 6 ). An i n s o l u b i l i z i n g r e a g e n t w hich i s p o t e n t i a l l y r e a c t i v e w ith an e s s e n t i a l am ino a c id r e s id u e in an en zy m e's a c t i v e s i t e may n o t, h o w e v e r, alw ay s le a d t o i n a c t i v i t y as i f when t h e r e s id u e i s s t e r i c a l l y

? " 2 < H CHp I n CHw-C/* 2 V H G lu ta ra ld e h y d e 6 n : F NOo 1, 5 - D if lu o r o - 2, 4 - d in itr o b e n z e n e *03 S S<% sn ca - M c n s D ip h e n y l-4, 4 '- d i i s o t h i o c y a n a t e - 2, 2 - d i s u l f o n i c a c id _0 3 S SO3 cr'ng^-^npcr B le d ia z o b e n z id in e - 2, 2 - d is u l f o n i c a c id CH- ( S c NO CNS T o lu e n e -2 - is o c y a n a t e 4 - is o t h i o c y a n a t e T r i c h l o r o - 6 - t r i a z i n e ( c y a n u r ic c h lo r id e ) F ig u re 1 : Some common c r o s s - l i n k i n g r e a g e n ts

i n a c c e s s i b l e. A ls o, t h e a c t i v e s i t e may b e p r o te c t e d w ith a s p e c i f i c r e a g e n t o r c o m p e titiv e i n h i b i t o r (1 2 0, 1 3 l) d u rin g i n s o l u b i l i z a t i o n t o s t a b i l i z e t h e enzyme i n i t s a c t i v e c o n fo rm a tio n (1 7 )- The f u n c t i o n a l g ro u p s o f p r o t e i n s s u i t a b l e f o r c o v a le n t b in d in g u n d e r m ild c o n d itio n s in c lu d e t h e te r m in a l c a rb o x y l and am ino g ro u p s o f en zy m es, and t h e s u b s t i t u e n t s o f c e r t a i n am ino a c id r e s i d u e s (9*0 i a r g i n in e ( g u a n id y l g r o u p ), l y s i n e (a and e am ino g r o u p s ), h i s t i d i n e (im id a z o y l g r o u p ), c y s te in e ( s u l f h y d r y l g r o u p ), s e r i n e (h y d ro x y l g r o u p ), t y r o s i n e (p h e n o l g r o u p ), a s p a r t i c a c i d (c a rb o x y l g r o u p ), and g lu ta m ic a c id (c a rb o x y l g ro u p ). T a b le 2 shows t h e r e a c t i v e am ino a c id r e s i d u e s i n th e common i n s o l u b i l i z i n g m eth o d s. N a tu r a l and s y n t h e t i c o r g a n ic p o ly m ers a r e u sed as c a r r i e r s f o r c o v a le n t b in d in g o f enzym es t o a r t i f i c i a l m a t r i c e s. T hese can be d iv id e d i n t o e l e c t r i c a l l y n e u t r a l c a r r i e r s su ch as c e l l u l o s e, S ep h ad ex, and S e p h a ro s e, and p o l y e l e c t r o l y t e c a r r i e r s su ch as C M -c e llu lo s e, and e th y le n e - m a le ic a c i d co p o ly m ers (EMA), and am ino e t h y l c e l l u l o s e ( U l). 'R e c e n tly, W e e ta ll (1^*9) u sed in o r g a n ic c a r r i e r s su ch as g l a s s w ith f u n c t i o n a l g ro u p s a tta c h e d t o im m o b iliz e enzym es (T a b le 3 ). T a b le 3 F u n c tio n a l g ro u p s a v a i l a b l e f o r s i l a n e c o u p lin g a g e n ts - ch2 =ch2 -CHg-CN - ch2 ~nh2 -CHg-CH-CHgO -CH2-NH-CH2-CH3 -CHgOH - ch2 -CH SH 0 CH g-cl -CH NH-C- 2 -NH, i B a ta ta k e n from W e e ta ll (1 5 1 )

18 Table 2 R e a c tiv e Amino A cid R e sid u e s in t h e Common I n s o l u b i l i z i n g M ethods I n s o l u b i l i z i n g r e a g e n t «R e a c tiv e am ino a c id r e s id u e s C h lo r o - s y m - tr ia z in y l d e r i v a t i v e Lys D ia z o - d e r iv a tiv e L y s, H is, T y r, A rg, Cys I s o - t h io c y a n a t e d e r i v a t iv e L y s, Arg N -E th y l-5 -p h e n y 1 i s oxaz o liu m -3 - s u lp h o n a te L y s, A sp, G lu D iim id e L y s, T y r, C ys, A sp, G lu A cid a z id e t y s, T y r, Cys, S e r M a le ic a c id o r m a le ic a n h y d rid e co p o ly m er N -C arb o x y -a-am in o a c id a n h y d rid e Lys Lys G lu ta ra ld e h y d e L y s, H is, T y r, Cys C y c lic im in o c a rb o n a te I ^ s D a ta ta k e n from T a b ach n ik and S o b o tk a (1 3 6 ) * In i n s t a n c e s v h en Lys i s r e a c t i v e a - N ^ g r o u p (s ) may r e a c t a l s o ; s i m i l a r l y, te r m in a l COOH g r o u p (s ) may co n d en se v hen Asp and ( o r ) Glu a r e r e a c t i v e. ** Formed by t h e r e a c t i o n o f cyanogen b ro m id e v i t h p o ly s a c c h a r id e s.

19 The g l a s s was a c t i v a t e d by c o u p lin g w ith *y-ami n o p ro p y l t r i e t h o x y s i l a n e, and th e a m in o a k ly ls ila n e - g la s s d e r i v a t i v e was c o n v e r te d t o i s o t h i o - c y a n a te d e r i v a t i v e o r was r e a c t e d w ith g - n itr o b e n z o ic a c id and th e n i t r o g ro u p re d u c e d and d i a z o t i z e d ( U l). The am ino g ro u p s o f enzym es h a v e b e e n u t i l i z e d t o o b ta in c o v a le n t lin k a g e t o s e v e r a l c a r b o x y lic p o ly m ers v i a th e c o rre s p o n d in g a z id e s (1 0 0, 150) (F ig u re 2 ), by a c t i v a t i o n o f th e p o ly m er c a rb o x y ls by c a rb o d iim id e (lu b, 1 U5 ) o r by W oodw ard^ R eagent K, N - e th y l- S - p h e n y l- is o x a z o liu r a - 3 1- s u l f o n a t e (1 2 1 ). More r e c e n t l y s u c c e s s f u l enzyme im m o b iliz a tio n h a s b e e n o b ta in e d by c e l l u l o s e a c t i v a t e d by symt r i c h l o r o t r i a z i n e ( c y a n u r ic c h l o r id e ) ( 7 b ), and by S ep h ad ex, o r S e p h a ro s e, a c t i v a t e d b y cyanogen b ro m id e (1 2 2 ) ( F ig u r e 3 ). Sty a c y l a t i o n o f th e am ino g ro u p s o f a p r o t e i n w hich a r e n o t e s s e n t i a l f o r i t s b i o l o g i c a l a c t i v i t y, i t h a s b een p o s s i b l e t o i n t r o d u ce new f u n c t io n a l g ro u p s w hich can b e u t i l i z e d i n b in d in g th e m o d ifie d p r o t e i n t o t h e a p p r o p r ia te c a r r i e r. F o r e x a m p le, new t y r o s y l r e s i d u e s w ere in tr o d u c e d i n t o t r y p s i n by i n i t i a t i n g th e p o ly m e r iz a tio n o f N - c a rb o x y - ty r o s in e a n h y d rid e w ith t h e enzyme ( 3 7 ). The f u l l y a c t i v e p o l y t y r o s y l t r y p s i n o b ta in e d was em ployed s u c c e s s f u l l y i n th e p r e p a r a t i o n o f a w a t e r - i n s o l u b l e p o l y t y r o s y l t r y p s i n d e r i v a t i v e by c o u p lin g i t w ith an i n s o l u b l e p o ly d ia z o n iu m c a r r i e r ( 7 ). New s u lf h y d r y l g ro u p s can be in tro d u c e d i n t o p r o te in s b y a c y la t i o n w ith N - a c e ty l h o m o c y ste in e t h i o l a c t o n e ( l l ). Such s u lf h y d r y l e n r ic h e d p r o te in s c a n b e p r e c i p i t a t e d from s o l u t i o n by c r o s s - l i n k i n g w ith a b i f u n c t i o n a l o rg a n o ra e rc u ria l compound ( 9 0 ).

l-chgoh -f CI-CHg-COOH + NaOH--------* CHgO-CHgCOOH + C I^O H * I C H ^ O - C H g C O O C H g + H 2 N - N H 2 ------------» U ch^ o- ch^ - c o - nh- nh2 + no2 i-c H g-0-c H 2 C0N3 -J- ghn-enzyme [ CH -O -CH gco N H -Enzym e F ig u re 2: C u r tiu s A zid e m ethod f o r c o u p lin g enzym es to c e l l u l o s e (1 0 0 ).

CH-1 OH \ \ CH-OH CH- + BrCN * y5h- 0-C 5N OH XC H - O v / I C=NH 4- HpN CH-0 -Enzyme \>H-Ox >N-Enzym e / L r H - H g O C H - 0 -C -^ -E n2yrne -O H F ig u r e 3 : C o u p lin g o f p r o t e i n s t o S e p h a ro s e by m eans cy an o g en b ro m id e ( 1 2 2 ).

22 F ig u re U sh o v s t h a t a p o ly m e ric a c y l a t i n g r e a g e n t, e th y le n e - m a le ic a n h y d rid e ( l : l ) copoly m er (EMA), h a s b een s u c c e s s f u l l y u s e d i n t h e p r e p a r a t i o n o f p o ly a n io n ic w a t e r - i n s o l u b l e d e r i v a t i v e s o f enzy m es, enzyme i n h i b i t o r s, and a n tig e n s ( 8 3, 1 4 6 ). EM A-papain and EMA- c h y m o try p sin c o n ju g a te s h av e b e en c o n v e rte d t o p o ly a lc o h o l o r p o ly - c a t i o n i c d e r i v a t i v e s b y c o u p lin g th e p o ly c a r b o x y lic enzyme d e r i v a t i v e w ith N,N -d im e th y la m in o -p ro p y la m in e o r p ro p a n o la m in e, r e s p e c t i v e l y, i n t h e p r e s e n c e o f c a rb o d iim id e ( 7 3 ). The p o ly a n io n ic c h a r a c t e r o f EMA- t r y p s i n and EM A -chym otrypsin c o n ju g a te s c o u ld a l s o b e p a r t l y n e u t r a l i z e d by in tr o d u c in g v a r y in g am ounts o f N,N -d im e th y la n iin o -e th y la m in e i n t o th e c o u p lin g m ix tu re (3 1*, 3 5 ). -CHg-CH -C H -C H 5 - COCT H nzyme-nh2 IH o - c I COO" -C H -C H ^ C H-C- H -C H F ig u r e 4 : C o u p lin g o f p r o t e i n s t o a n e th y le n e - m a le ic a n h y d rid e (1 :1 ) copoly m er (EMA) ( 8 3 ).

23 A lk y la tio n and a r y l a t i o n r e a c t i o n s can b e u t i l i z e d i n b in d in g enzym es t o s u i t a b l e c a r r i e r s. H ow ever, w o rk e rs (9&» 127) h av e shown t h a t a l k y l a t i n g r e a g e n ts su ch as io d o a c e tic a c id and 2, h - d i n i t r o - flu o ro b e n z e n e r e a c t, ev en u n d e r m ild c o n d i t i o n s, n o t o n ly w ith th e a -a m in o, c -a m in o, and t h i o l g ro u p s o f p r o t e i n s, b u t a ls o w ith th e s u l f u r o f m e th io n in e, t h e p h e n o lic h y d ro x y l o f t y r o s i n e, and th e im id a z o le o f h i s t i d i n e. R e c e n tly, G o ld s te in (U5 ) h a s c o v a l e n t l y bound enzym es v i a t h e i r c a rb o x y l g r o u p s, t o a m in o e th y l c e l l u l o s e o r o th e r r e s i n s c o n ta in in g p rim a ry a l i p h a t i c am ines u t i l i z i n g c a rb o d iim id e ( F ig u re 5 ). A n o th er c o u p lin g r e a c t i o n w hich h a s b e e n u s e d t o p r e p a re w a te r - i n s o l u b l e a n t i g e n s, a n t i b o d i e s, and enzym es i s th e r e a c t i o n b etw een a p r o t e i n and a p o ly d ia z o n iu m s a l t (F ig u re 6 ). V a rio u s f u n c t io n a l g ro u p s o f t h e p r o t e i n a p p e a r t o b e in v o lv e d i n th e c o u p lin g r e a c t i o n s in c e i t h a s b e e n shown t h a t low m o le c u la r w e ig h t d ia zo n iu m s a l t s r e a c t r a t h e r n o n - s p e c i f i c a l l y w ith p r o t e i n s. They com bine n o t o n ly w ith th e p h e n o l and a m id a z o le g ro u p s a n d, somewhat m ore s lo w ly, w ith am ino g ro u p s (3 3, 1 3 6 ), b u t a ls o w ith o th e r g ro u p s on t h e p r o t e i n m o le c u le. Howe v e r, azo c o u p lin g does p o s s e s s c e r t a i n i n t r i n s i c a d v a n ta g e s ( l 3 l ) : ( a ) i t i s e x tre m e ly r a p id ev en a t low te m p e r a tu r e s ; (b ) i t c an b e p e rfo rm e d in n e u t r a l aq u eo u s s o l u t i o n s ; and ( c ) th e s t a b l e a ro m a tic am ine p r e c u r s o r may b e q u a n t i t a t i v e l y d i a z o tiz e d r e a d i l y and sim p ly b e f o r e u s e. C am pbell e t. a l. ( 2 2 ), who c o u p le d b o v in e serum abum in w ith d ia z o tiz e d - a m in o b e n z y lc e llu lo s e w ere th e f i r s t t o r e p o r t an im m o b iliz e d enzyme c o u p le d by t h i s p r o c e d u re. S in c e th e n p o ly d ia z o n iu m

9 N Support C-OH + C 6 QI i 0 NH S u pport-c -O -6 -j- HUN-Enzyme O P 0 NH Support-6-N-E nzym e + C=0 + H* H P ISIH F ig u re 5; C a rb o d iim id e m ethod f o r b in d in g enzym es to a w a t e r - i n s o l u b l e s u p p o rt (4 5 ),

25 G lass-o -Si-O - + R 0 -^ i-r -N H 2 ------ > G lass-0 -S i-0 -$ i-r -N H 2 + 20 N -^ V c -C I 0 Glass-0-Si-0-j i-r-isi 6 N02 --------- G la s s-0 -S i-0 -S i-r -fy -C -@ NH2 --------- H G lass-o -Si-O -qi-r -N -C -^-N gc f+enzym e H 08S 0 - ^ 0 W ^ N - - N. En2yme n F ig u r e 6 ; P r e p a r a tio n o f w a t e r - i n s o l u b l e enzym es by d ia z o c o u p lin g o f th e enzym e to a g l a s s s u p p o rt ( 1 4 9 ).

26 s a l t s o f t h e f o llo w in g c a r r i e r s h av e "been u s e d f o r t h i s p u rp o s e : ( a ) m -am in o b en zylo x y m eth y l e s t e r o f c e l l u l o s e ( 2 5 ), p o ly - -a m in o - s ty r e n e ( 1 0 8 ), (b ) a copoly m er o f -a m in o p h e n y la la m in e and le u c in e ( 1 7 ), and ( c ) S-MDA (1*9), a r e s i n p r e p a r e d b y c o n d e n s a tio n o f d ia ld e h y d e s t a r c h ( a co m m e rc ia lly a v a i l a b l e p e r i o d a t e - o x i d a t i o n p ro d u c t o f s t a r c h ) w ith ji,]3'd ia jn in o d ip h e n y lm e th a n e, and s u b se q u e n t r e d u c tio n o f t h e s c h i f f b a s e o f t h e p o ly m e ric p r o d u c t. I s o c y a n a te s r e a c t p r im a r i l y w ith th e am ino g ro u p s o f p r o t e i n u n d e r m ild c o n d itio n s t o y i e l d t h e c o rre s p o n d in g u r e id o d e r i v a t i v e s ( l ). B ra n d e n b e rg e r ( 2 l ) u t i l i z e d t h i s p r i n c i p l e t o p r e p a r e a c r o s s - lin k e d p o l y - l i - i s o c y a n a te - s t y r e n e. P r e lim in a r y s t u d i e s on i t s u t i l i z a t i o n i n p r o t e i n b in d in g h av e b e e n d e s c r ib e d b y M anecke and c o -w o rk e rs (9 1, 9 3 ). I s o th io c y a n a te s a l s o r e a c t w ith am ino g ro u p s t o y i e l d th e c o rre s p o n d in g t h i o u r e i d o d e r i v a t i v e s (F ig u re 7 ). K ent and S la d e (7 7 ) and Axen and P o r a th ( 5 ) h av e r e p o r te d u s in g p o ly m e ric c a r r i e r s c o n ta in in g i s o t h i o c y a n a t e g ro u p s t o b in d p e p tid e s and p r o t e i n s. O rg a n o m e rc u ria l compounds su ch a s a n a lo g u e s o f -m e rc u rib e n z o ic a c id h a v e b e e n u s e d f o r b in d in g p r o t e i n s ( 6 0 ). K ent and S la d e (7 6 ) u s e d p o ly s ty r y lm e r c u r ie a c e t a t e as a c a r r i e r f o r t h e c o v a le n t b in d in g o f a n t i b o d i e s. H ow ever, c o v a le n t lin k a g e v i a an o rg a n o m e rc u ria l bond h a s tw o d is a d v a n ta g e s (1 3 1 )- F i r s t, t h e m e r c u r y - s u lf u r bond u n d e rg o e s ex ch an g e i n t h e p r e s e n c e o f s u lf h y d r y l compounds so t h a t t h e p r e s e n c e o f t h e l a t t e r may c a u se s o l u b i l i z a t i o n. M o reo v er, b e c a u s e m ost enzym es c o n ta in e i t h e r l i t t l e o r no f r e e s u lf h y d r y l g r o u p s, p r i o r

2T. i? Glass- Si- 0-Si-R-NHo + CI-C-CI i i 2 I I Glass S i-0 -S i-,-r-n»c=s + 9HN-Enzyme» I I 2 Glass- S i- 0 - Si R-N-C- N- Enzyme I I F ig u re 7: I s o th lo c y a n a te m ethod f o r c o u p lin g enzym es to g l a s s (1 3 1 ).

e n ric h m e n t o f th e p r o t e i n w ith s u lf h y d r y l g ro u p s (1 1 ) w i l l o f te n h e n e c e s s a r y b e f o r e c a r r y in g o u t b in d in g t o t h e o r g a n o m e rc u ria l c a r r i e r s. 28 C a r r i e r s U n t i l r e c e n t l y v i r t u a l l y a l l p o ly m e ric c a r r i e r s w ere d e r i v a t i v e s o f c e l l u l o s e, s y n t h e t i c p o ly am in o a c i d s, o r p o ly s ty r e n e. L im ita tio n s ( l 5 l ) in t h e u t i l i z a t i o n o f o r g a n ic p o ly m ers as s u p p o rt sy ste m s i n c lu d e : * 4 ( a ) S u s c e p t i b i l i t y t o ph an d s o lv e n t c o n d i t i o n s many p o ly m e rs change c o n f ig u r a tio n u n d e r d i f f e r i n g o p e r a t i o n a l c o n d i t i o n s, th u s c r e a t i n g ch an g es i n fle w r a t e s i f colum ns a re u s e d. (b ) S u s c e p t i b i l i t y t o e n z y m a tic a t t a c k, e i t h e r from m ic ro o rg a n ism s o r by enzym es b e in g im m o b iliz e d. ( c ) O p e r a tio n a l s t a b i l i t y - many enzym es im m o b iliz e d on o r g a n ic p o ly m e rs h av e p o o r s t a b i l i t y u n d e r o p e r a t i o n a l c o n d itio n s. (d ) P a r t i c l e s i z e and c o n d itio n Many p o ly m e r m a tr ic e s a r e o f e x tre m e ly s m a ll p a r t i c l e s i z e an d g e la tin o u s i n n a t u r e. To overcom e th e s e l i m i t a t i o n s C o rn in g G la s s Works h a s d e v e lo p e d m ethods o f c o v a le n tly a t t a c h i n g enzym es t o in o r g a n ic m a t e r ia ls r a n g in g from g l a s s t o s t a i n l e s s s t e e l. A d v an tag es (1 5 1 ) o f enzym es a tta c h e d t o o rg a n ic p o ly m e rs in c lu d e : ( a ) S t r u c t u r a l s t a b i l i t y th e in o rg a n ic m a t e r i a l s a r e n o t s u s c e p t i b l e t o ph and s o lv e n t c o n d itio n s. T h e r e f o r e, th e y w i l l n o t ch ange s iz e o r c o n f ig u r a tio n d u rin g u s a g e. (b ) The in o r g a n ic m a tr ic e s a r e n o t s u s c e p t i b l e t o m ic r o b ia l a t t a c k.

29 ( c ) I n o r g a n ic m a t e r i a l s can e a s i l y b e sh ap ed p e r m ittin g a w ide v a r i e t y o f c o n f i g u r a t i o n s. (d ) Enzymes c o u p le d t o in o r g a n ic m a t e r i a l s a p p e a r t o h av e g r e a t e r o p e r a t i o n a l s t a b i l i t y. In c o n s id e r in g a g iv e n c a r r i e r, a f u n c t i o n a l g ro u p w h ich i s h ig h ly r e a c t i v e w ith low m o le c u la r w e ig h t compounds may r e a c t s lu g g i s h l y when in c o r p o r a te d i n t o a p o ly m e r, e s p e c i a l l y when r e a c t i n g w ith f u n c t io n a l g ro u p s o f a h ig h m o le c u la r w e ig h t s u b s t r a t e. In p r i n c i p l e f u n c t io n a l g ro u p s c l o s e ly a tta c h e d t o t h e back b o n e o f a p o ly m e ric c a r r i e r, and h e n c e l e s s a c c e s s i b l e, s h o u ld e x h i b i t lo w er r e a c t i v i t y th a n s i m i l a r g ro u p s s i t u a t e d on t h e en d s o f lo n g, f l e x i b l e, h y d r o p h ilic s id e c h a in s. C e r ta in p h y s ic a l c h a r a c t e r i s t i c s o f th e c a r r i e r su c h a s s o lu b i l i t y, m e c h a n ic a l s t a b i l i t y, s w e llin g c h a r a c t e r i s t i c s, and s u r f a c e a r e a t o g e t h e r w ith i t s e l e c t r i c c h a rg e and h y d r o p h ilic o r h y d ro p h o b ic n a t u r e, p la y an im p o rta n t r o l e i n d e te r m in in g th e m axim al am ount o f p r o t e i n w hich c a n b e c o v a le n tly bound and t h e s t a b i l i t y and b i o l o g i c a l a c t i v i t y o f th e i n s o l u b l e p r o d u c t. M inim al s o l u b i l i t y, h ig h m e c h a n ic a l s t a b i l i t y, and a d e q u a te p a r t i c l e s iz e a r e d e s i r a b l e c h a r a c t e r i s t s w h ich a r e e s s e n t i a l f o r t h e p r e p a r a tio n o f b i o l o g i c a l l y a c t i v e, bound p r o t e i n s t h a t can b e r e a d i l y an d c o m p le te ly rem oved from t h e r e a c t i o n m ix tu re b y f i l t r a t i o n o r c e n t r i f u g a t i o n. The c h e m ic a l n a tu r e o f t h e c a r r i e r m a tr ix may a l s o re m a rk e d ly a f f e c t i t s a f f i n i t y f o r a g iv e n p r o te in a n d, a s a r e s u l t, i t a b i l i t y t o b in d c o v a le n tly t h e same p r o t e i n th ro u g h i t s r e a c t i v e g ro u p s (lu 6 ). S in c e an enzyme m o le c u le c o n ta in s one o r a s m a ll num ber o f a c t i v e s i t e s,

c o m p le te i n a c t i v a t i o n may o c c u r i f b in d in g t o a c a r r i e r ta k e s p la c e v i a f u n c t i o n a l g ro u p s o f t h e s e s i t e s. 30 S t a b i l i t y o f W a te r - I n s o lu b le Enzymes S to ra g e and O p e r a tio n a l S t a b i l i t y : S to ra g e a t h C f o r s e v e r a l m onths r e s u l t e d in l i t t l e l o s s o f a c t i v i t y f o r aqueous s u s p e n s io n s o f e th y le n e - m a le ic a c id enzyme d e r i v a t i v e s o f t r y p s i n ( 8 3 ), c h y m o try p sin ( 1 5 6 ), p a p a in ( 1 5 6 ), s u b t i l l i s i n Novo ( 1 5 6 ), s u b t i l l i s i n C a rls b e rg (1*5), f o r C M -c e llu lo s e d e r i v a t i v e s o f c h y m o try p sin ( 7 5 ), b ro m e la in ( 1 5 7 ), and f i c i n ( 6 5 ), and f o r S e p h a ro se d e r i v a t i v e s o f c h y m o try p sin ( 5 ). L yo- p h i l i z a t i o n o f th e s e p o ly m e ric enzym es d id n o t g r e a t l y re d u c e t h e i r a c t i v i t y a f t e r p ro lo n g e d s to r a g e a t h C and a t roam te m p e r a tu re ( ^ 7, 8 3, 1 3 1, 1 5 6 ). When -sm in o b e n z y l c e l l u l o s e s, S-MDA r e s i n s, and le u c in e -j> - a m in o p h e n y la la n in e co p o ly m ers w ere u sed a s p o ly m e ric c a r r i e r s f o r p a p a in ( 1 3 1 ), s u b t i l l i s i n Novo (U 5 ), s u b t i l l i s i n C a r ls b e rg (U9 ), and p o l y t r y r o s y l t r y p s i n ( 7 ), s to r a g e i n t h e c o ld f o r s e v e r a l m onths h a d l i t t l e a f f e c t on e n z y m a tic a c t i v i t y. H ow ever, th e s e p o ly m e ric enzym es w ere c o m p le te ly i n a c t i v a t e d on a i r d r y in g o r by l y o p h i l i z a t i o n, p ro b a b ly d u e t o th e h y d rc p h o b ic n a tu r e o f t h e c a r r i e r ( 1 3 1 ). M anecke (9 1 ) fo u n d t h a t w ith a copoly m er o f m e th a c r y lic a c id and m e th a c r y lic a c id f l u o r o d i n i t r o a n i l i d e, in s o lu b le c o n ju g a te s o f p e p s in, i n v e r t a s e, and a lc o h o l d e h y d ro g e n a se l o s t m ost o f t h e i r a c t i v i t y w ith in s e v e r a l w eeks v hen s to r e d a t lt C, Num erous w o rk e rs (1 3 1, 3 9, 5*0, h a v e a s c e r ta in e d t h a t enzym es o c c lu d e d i n p o ly a c ry la m id e o r s t a r c h g e l s, as w e ll a s e n z y m e -c o llo d io n

m em branes o f p a p a in and a l k a l i n e p h o s p h a ta s e, r e t a i n e d t h e i r a c t i v i t y 31 f o r s e v e r a l m onths a t r e f r i g e r a t e d s t o r a g e. L y o p h iliz e d p o ly a c ry la m id e an d s t a r c h g e l p r e p a r a tio n s c o n ta in in g th e above enzym es c o u ld b e r e h y d r a te d e a s i l y w ith t h e c o n c o m ita n t r e c o v e ry o f m ost o f t h e e n z y m a tic a c t i v i t y ( b l ). H ow ever, t h e e n z y m e -c o llo d io n membranes s h ra n k i r r e v e r s i b l y on l y o p h i l i z a t i o n and l o s t t h e i r a c t i v i t y p re su m a b ly due t o d e c r e a s e d p e r m e a b ility t o s u b s t r a t e ( 3 9 ). ^ Of t h e 50 in s ta n c e s in w hich d i r e c t co m p ariso n s o f t h e s t a b i l i t i e s o f t h e r e s p e c t i v e s o lu b le and in s o lu b le d e r i v a t i v e s w ere m ade, M elro se (9*0 c o n c lu d e d t h a t th e l a t t e r was m ore s t a b l e i n 30 c a s e s, t h e fo rm er i n 8 c a s e s, and t h a t t h e r e was l i t t l e d i f f e r e n c e in s t a b i l i t i e s in 12 c a s e s. P resu m ab ly t h e bound enzym es a r e m ore r e s i s t a n t t o d e n a tu r a tio n (9**) and i n th e c a s e o f i n s o l u b i l i z e d p r o t e a s e s, t h e i r s t a b i l i t i e s a r e e n h an ced f u r t h e r b y re d u c e d a u t o l y s i s. W e e ta ll (1 5 1 ) i n v e s t i g a t e d t h e o p e r a t i o n a l s t a b i l i t y o f a l k a l i n e p h o s p h a ta s e and L -am ino a c id o x id a s e bound t o g l a s s. A f te r 25 day s a t room te m p e r a tu re no s i g n i f i c a n t l o s s i n a c t i v i t y was n o te d f o r p o ly m e ric a l k a l i n e p h o s p h a ta s e. H ow ever, i n s o l u b i l i z e d L -am ino a c id o x id a s e d id e x h i b i t a s l i g h t l o s s in a c t i v i t y a f t e r 28 day s a t room te m p e r a tu r e. A n alogous r e s u l t s w ere o b s e rv e d w ith c o n tin u o u s, s t i r r e d ta n k r e a c t o r s. S i m i l a r l y, b o th c ru d e and c r y s t a l l i n e p e p s in bound t o p o ro u s g l a s s p a r t i c l e s r e t a i n e d 100# o f t h e i r a c t i v i t y a f t e r s to r a g e f o r 30 d ay s a t 6 C i n m o is t c a k e s. U nder o p e r a t i o n a l c o n d itio n s i n a colum n, no lo s s i n a c t i v i t y was n o te d a f t e r Uo d a y s. R oyer (1 2 5 ) h a s im m o b iliz e d p ro n a s e on a ry la m in e g l a s s and h as

32 o b s e rv e d l i t t l e change i n a c t i v i t y a f t e r 3 m onths vhen t h e p o ly m e ric enzyme was k e p t r e f r i g e r a t e d and i n a m o is t c o n d itio n. F i n a l l y, W e e ta ll ( lb 3 ) com pared w a t e r - i n s o l u b l e enzymes c o u p le d t o o rg a n ic p o ly m ers w ith w a t e r - i n s o l u b l e enzym es c o u p le d t o in o r g a n ic c a r r i e r s i n c lu d in g c o l l o d a l s i l i c a, g l a s s, and a lu m in a. He c o n c lu d e d t h a t enzym es c o v a l e n t l y c o u p le d t o in o r g a n ic c a r r i e r s h ad g r e a t e r s to r a g e s t a b i l i t y th a n enzym es c o v a le n tly a tta c h e d t o o rg a n ic p o ly m ers when s to r e d f o r s e v e r a l w eeks a t U and 2 3 C., d ry o r in d i s t i l l e d w a te r. He a l s o o b s e rv e d t h a t enzym es c o v a le n tly c o u p le d t o in o r g a n ic c a r r i e r s by s u lfo n a m id e lin k a g e w ere n o t as s t a b l e d u rin g s to r a g e a s enzym es c o u p le d by azo lin k a g e (1 U3 ). T h erm al S t a b i l i t y : Im proved th e rm a l s t a b i l i t i e s as com pared t o t h e c o rre s p o n d in g n a t i v e enzym es hav e b e en r e p o r te d f o r t h e fo llo w in g enzym es a t t a c h e d t o o r g a n ic c a r r i e r s : C M -c e llu lo se f i c i n ( 6 5 ), g lu c o s e o x id a s e im m o b iliz e d on c e llo p h a n e s h e e ts ( 2 l ), and D E A E -c e llu lo se - l a c t i c d e h y d ro g e n a se ( l 6 l ). P o ly m e ric enzym es e x h i b i t i n g lo w e r th e r m a l s t a b i l i t i e s th a n t h e c o rre s p o n d in g n a tiv e enzym es in c lu d e : p a p a in bound t o le u c in e - -a m in o p h e n y la la n in e co p o ly m er (131) and a l k a l i n e p h o s p h a ta s e ( 3 9 ), and g lu c o s e -6 - p h o s p h a te d e h y d ro g e n a se (bo) im m o b iliz e d o n to c o llo d io n m em branes. A s i m i l a r lo w e rin g o f th e rm a l s t a b i l i t y h a s b e e n r e p o r t e d f o r s e v e r a l o f t h e p o ly a n io n ic, p o l y c a t i o n i c, and p o ly a l c o h o l i c d e r i v a t i v e s o f p a p a in ( b 5 ). W e e ta ll ( lb 9 ) s tu d i e d th e r a t e o f th e r m a l i n a c t i v a t i o n o f t r y p s i n and p a p a in bound t o g l a s s. T h erm al i n a c t i v a t i o n o f b o th t r y p s i n and

p a p a in i n t h e i r s o lu b l e form s o c c u r re d w ith in 30 m in u te s a t a p p r o x i 33 m a te ly 60 C (1 9 ) an d in c r e a s e d a t h ig h e r te m p e r a tu r e s. W ith th e t r y p s i n - g l a s s d e r i v a t i v e s p r e p a re d by a z o - and s u lfo n a m id e c o u p lin g, t h e th e r m a l i n a c t i v a t i o n c u rv e s w ere s i m i l a r. I r r e v e r s i b l e d e n a tu r a tio n b eg an a t 53 C and 50 C, r e s p e c t i v e l y, v h en h e ld a t th o s e te m p e r a tu re s f o r 30 m in u te s. H ow ever, t h e p a p a in g l a s s d e r i v a t i v e was e x tre m e ly s t a b l e. When c o n t i n u a l l y a s s a y e d a t 88 C, no d e t e c t a b l e d e n a tu r a tio n o c c u rre d f o r a t l e a s t 80 m in u te s (ll+ 9 ). D ependence o f S t a b i l i t y on ph: Im proved s t a b i l i t i e s to w a rd a l k a l i n e ph*s up t o 1 0.7 have b e e n r e p o r te d f o r t h e E M A -d e riv a tiv e s o f t r y p s i n ( 8 3 ), p a p a in ( 1 5 6), an d c h y m o try p sin ( 1 5 6 ). C o n v e rs e ly, p o ly - c a t i o n i c d e r i v a t i v e s e x h ib ite d im p ro v ed s t a b i l i t i e s in t h e a c id ph ra n g e (1+5). I t h a s b e e n s u g g e s te d by L evin ( 8 3 ) an d by G o ld s te in ( U8) t h a t t h e s e phenom ena c o u ld be r e l a t e d t o l o c a l ph e f f e c t s in d u c e d by t h e p o l y e l e c t r o l y t e c a r r i e r. G o ld s te in (1+9) and S ilm an (1 3 1 ) o b se rv e d l i t t l e change in th e ph d ep en d en ce o f th e s t a b i l i t y o f p a p a in bound t o n e u t r a l c a r r i e r s as _-am inobenzyl c e l l u l o s e o r S-MDA r e s i n s v h en com pared t o t h e n a t i v e enzym e. I n c o n t r a s t j> -am inobenzyl c e l l u l o s e and S-MDA d e r i v a t i v e s o f p o l y t y r o s y l t r y p s i n (1+9) e x h i b i t e d m axim al s t a b i l i t y a t n e u t r a l and a l k a l i n e ph v a l u e s, i n c o n t r a d i s t i n c t i o n t o n a t i v e t r y p s i n o r p o ly t y r o s y l t r y p s i n w hich a r e m ost s t a b l e a t low ph v a lu e s (1+3, 9 2 ). W e e ta ll ( 1 5 1 ), L in e ( 8 8 ), and R oyer (1 2 5 ) h a v e shown t h a t enzym es im m o b iliz e d t o p o ro u s g l a s s p a r t i c l e s g e n e r a l l y e x h i b i t a b ro a d e n in g i n t h e i r maximum ph optim um.

3k I n c o n c lu s io n i t c an b e i n d i c a t e d t h a t th e m o d ifie d s t a b i l i t y p a t t e r n o f c o v a le n tly bound enzym es i s d e te rm in e d n o t o n ly by th e p h y s ic a l and c h e m ic a l c h a r a c t e r i s t i c s o f t h e c a r r i e r, b u t a l s o by th e n a tu r e o f t h e c h e m ic a l m o d if ic a tio n o f t h e enzyme m o ie ty b ro u g h t a b o u t b y t h e c o v a le n t b in d in g (l+ l). F a c to r s A f f e c tin g t h e K in e tic B e h a v io r The o b s e rv e d k i n e t i c b e h a v io r o f an im m o b iliz e d enzyme sy ste m i s t h e sum m ation o f t h e s u p e r p o s itio n o f t h e c a r r i e r c h a r a c t e r i s t i c s upon t h e s p e c i f i c enzyme k i n e t i c p a r a m e te r s. The e f f e c t s o f im m o b iliz a tio n on t h e a p p a r e n t k i n e t i c b e h a v io r o f t h e enzyme m ig h t b e r e s o lv e d a s fo llo w s C ^ l ) : ( a ) E f f e c ts o f d i f f u s i o n l i m i t a t i o n s th e k i n e t i c p a ra m e te rs o f enzym ic r e a c t i o n s m ig h t b e d e te rm in e d by t h e r a t e o f d i f f u s i o n o f s u b s t r a t e a c r o s s t h e u n s t i r r e d l a y e r s u rro u n d in g th e i n s o l u b l e m a tr ix, (b ) S t e r i c e f f e c t s when an im m o b iliz e d enzyme a c t s on a h ig h m o le c u la r w e ig h t s u b s t r a t e, s t e r i c r e s t r i c t i o n s im posed by t h e m a trix m ig h t m a rk e d ly a f f e c t t h e c o u rse o f t h e c a t a l y t i c r e a c t i o n. Cc) M ic ro e n v iro n m e n ta l e f f e c t s t h e s e a r e m ost p ro n o u n ced in t h e c a s e o f p o l y e l e c t r o l y t e c a r r i e r s, w here th e c h a rg e d m a tr ix im poses a m odif i e d m ic ro e n v iro n m e n t on t h e im m o b iliz e d enzym e. E f f e c t o f d i f f u s i o n : The v e l o c i t i e s o f r e a c t i o n s o f im m o b ilized enzyme p a r t i c l e s in aq ueous s o lu t i o n s a r e m a rk ed ly in c r e a s e d by r a p i d s t i r r i n g o f t h e i r s u s p e n s io n s ( 6 5 ), w hich a f f e c t s t h e th ic k n e s s o f th e u n s t i r r e d s o lv e n t l a y e r (59)* Hornby e t. a l. (6 6 ) d e r iv e d an e q u a tio n t o a c c o u n t f o r t h e e f f e c t o f d i f f u s i o n and e l e c t r i c a l p a ra m e te rs in

35 m o d ify in g t h e u s u a l M ic h a e lls -M e n te n c o n s ta n t o f th e enzym e: Km *Km + D ") ^RT-ZxF g rad * E q * ^ w h e re, K = t r u e M ic h a e lis -M e n te n c o n s ta n t m K* ~ a p p a re n t M ic h a e lis -M e n te n c o n s ta n t m x» e f f e c t i v e th ic k n e s s o f t h e d i f f u s i o n l a y e r v = maximum r e a c t i o n v e l o c i t y o f t h e f r e e enzyme S D s d i f f u s i o n c o n s ta n t o f th e s u b s t r a t e R - g a s c o n s ta n t T = a b s o lu te te m p e r a tu re o f t h e sy stem Z = e le c tr o c h e m ic a l v a le n c e o f t h e s u b s t r a t e g ra d = g r a d ie n t o f e l e c t r i c a l p o t e n t i a l F - F a ra d a y c o n s ta n t In t h e c o u rs e o f th e e n z y m a tic r e a c t i o n a s u b s t r a t e c o n c e n tr a tio n g r a d i e n t b u i l d s up a c r o s s t h e u n s t i r r e d b o u n d ary l a y e r ( 3 9 ). S a tu r a t i o n o f t h e p o ly m e ric enzyme o c c u rs a t h ig h e r s u b s t r a t e c o n c e n tr a tio n s th a n th o s e r e q u ir e d f o r t h e c o rre s p o n d in g n a t i v e enzyme i n s o l u t i o n : t h i s le a d s t o an i n c r e a s e i n th e v a lu e o f th e a p p a re n t M ic h a e lis c o n s t a n t, K^, w hich i s i n ag reem en t w ith t h e above e q u a tio n. From t h i s e q u a tio n i t a l s o can b e shown t h a t K i s i n v e r s e l y r e l a t e d t o th e m d i f f u s i o n te rm D, w h ich h a s b een a d e q u a te ly v e r i f i e d by e x p e rim e n ts o f o th e r w o rk e rs (0 5, 1 2 3, 1 6 2 ). When an enzyme i s d i s t r i b u t e d th ro u g h o u t a s w o lle n p o ly m e ric c a r r i e r, s u b s t r a t e c o n c e n tr a tio n g r a d ie n ts can a l s o b e e s ta b lis h e d

36 w i t h i n t h e r e g io n o f t h e im m o b iliz e d enzyme p h a s e. I n t h i s c a s e Goldman e t. a l. {1*2) h a v e shewn t h a t maximum a c t i v i t y w i l l be a t t a i n e d o n ly when t h e l o c a l s u b s t r a t e c o n c e n tr a tio n g r e a t l y ex c e e d s th e o f t h e n a tiv e enzym e. The e f f e c t o f an u n s t i r r e d l a y e r s u rro u n d in g a r i g i d p a r t i c l e on t h e M ic h a e lis c o n s ta n t o f an a t t a c h e d enzyme i s i l l u s t r a t e d in th e f o llo w in g ex am p les (1 3 1 ): (a ) H ornby e t. a l. (6 6 ) r e p o r te d a IT m o f 5.6 x 10 f o r c h y m o try p sin a t t a c h e d t o C M -c e llu lo se u s in g a c e t y l - -U L - ty r o s in e e t h y l e s t e r a s s u b s t r a t e. A v a lu e o f = 2.7 x 10 M was g iv e n by th e same a u th o r s f o r n a t i v e c h y m o try p s in. (b ) G o ld s te in e t. a l. (1 5 6 ) r e p o r t e d, f o r S-M DA-papain c o n ju g a te s a c t i n g on b e n z o y lg ly c in e e t h y l e s t e r, a o f 3.1+ x 1 0 ~ ^ com pared t o a o f 1.8 x lo ^ S i f o r n a t i v e p a p a in. G o ld s te in (1 5 6 ) a l s o r e p o r te d a Km = _2 1,7 x 10 M f o r S -M D A -su b tilo p e p tid a se A c o n ju g a te s a c t i n g on a c e t y l - L - ty r o s in e e t h y l e s t e r, w hich com pares t o a r e c o rd e d _2 10 M f o r n a t i v e s u b tilo p e p t i d a s e A. o f 0.5*+ x S t e r i c E f f e c t s : I m m o b iliz a tio n o f an enzyme o n to an o r g a n ic o r in o r g a n ic c a r r i e r may le a d t o s t e r i c r e s t r i c t i o n s on i t s r e a c t i v i t y w ith h ig h m o le c u la r w e ig h t s u b s t r a t e s. M ost o f th e w ork on t h e k i n e t i c b e h a v io r o f p o ly m e ric enzymes to w a rd la r g e s u b s t r a t e s h a s b e e n lim i t e d t o p r o t e a s e s. G e n e r a lly, im m o b iliz e d p r o te a s e s had lo w er s p e c i f i c a c t i v i t i e s to w a rd p r o t e i n s th a n t h e c o rre s p o n d in g n a tiv e enzym es (1 3 1, ll+ 6 ). F o r ex a m p le, p o ly m e ric p a p a in (1 3 1 ) and d e r i v a t i v e s o f p o ly t y r o s y l t r y p s i n (7 ) h y d ro ly z e d c a s e in a t i n i t i a l r a t e s w h ich w ere 30 t o 59X o f th o s e e x p e c te d on th e b a s i s o f t h e i r a c t i v i t y to w a rd

b e n z o y l- L - a r g in in e e t h y l e s t e r. O th e r p o ly m e ric enzym es e x h i b i t i n g low p r o t e o l y t i c a c t i v i t y in c lu d e t r y p s i n ( 1 0 0 ), b ro m e la in ( 1 5 7 ), f i c i n ( 6 5 ), p a p a in ( 1 5 6 ), and s u b t i l o p e p t i d a s e A (U 9 ). I n th e m a jo r ity o f th e s e c a s e s, s t e r i c h in d ra n c e in d u c e d by t h e c a r r i e r c o u ld b e a t t r i b u t e d as t h e c a u se f o r t h e lo w e rin g o f p r o t e o l y t i c a c t i v i t y (1+7)* O f te n, low p r o t e o l y t i c a c t i v i t y was acco m p an ied by a d e c r e a s e in t h e t o t a l num ber o f p e p tid e b o n d s s u s c e p t i b l e t o h y d r o ly s is (1 5 6 ). T h is e f f e c t v a s p a r t i c u l a r l y p ro n o u n c ed in t h e c a s e o f p o l y e l e c t r o l y t e enzyme d e r iv a t i v e s w here e l e c t r o s t a t i c i n t e r a c t i o n s b etw een t h e c h a rg e d c a r r i e r and th e c h a rg e d, h ig h m o le c u la r w e ig h t p r o t e i n s u b s t r a t e w ere su p e rim p o se d on t h e s t e r i c r e s t r i c t i o n s ( ^ 1, ^ 7, 1 1 9 ). R e s u lts by L e v in e t. a l. ( 8 3 ), s u g g e s t t h a t t h e num ber o f p e p tid e bonds s p l i t by p o ly m e ric t r y p s i n c o u ld b e c o n t r o l l e d b y v a r y in g t h e c h a rg e d e n s ity on t h e p o ly e l e c t r o l y t e enzyme d e r i v a t i v e. F u rth e rm o re, S ilm an and K a tc h a ls k i (1 3 1 ) i n d i c a t e d t h a t t h e s i t e s o f a t t a c k as w e ll a s t h e r a t e s o f c le a v a g e o f a h ig h m o le c u la r w e ig h t s u b s t r a t e m ig h t b e a f f e c t e d by t h e c h e m ic a l n a tu r e o f t h e p o ly m e ric c a r r i e r. O n g 's w ork (1 1 9 ) w ith t r y p s i n s u b s t a n t i a t e s th e fo rm e r. He e s t a b l i s h e d t h a t w h ereas t r y p s i n h y d ro ly z e d t h e 15 l y s y l p e p tid e bonds in p e p s in o g e n, th e IM A -try p sin d e r i v a t i v e n e v e r c le a v e d m ore th a n 10 b o n d s. T hese f in d in g s w ere c o n firm e d by p e p tid e m apping. The d a t a i n d i c a t e s t h a t t h e c h e m ic a l n a tu r e o f th e c a r r i e r, a t le a B t i n th e c a s e o f p o l y e l e c t r o l y t e enzyme d e r i v a t i v e s, may im pose a d d i t i o n a l r e s t r i c t i o n s on t h e s p e c i f i c i t y o f t h e bound enzyme ( 7 1 ). T h ese r e s t r i c t i o n s p ro b a b ly r e s u l t from c h a rg e i n t e r a c t i o n s b etw een

38 c a r r i e r an d d i f f e r e n t r e g i o n s, o r d i f f e r e n t se q u e n c e s on t h e h ig h m o le c u la r w e ig h t s u b s t r a t e m o le c u le ( 7 1 ). M ic ro e n v iro n m e n ta l E f f e c t s : Enzymes im m o b iliz e d on s o l i d s u p p o rts a r e s i t u a t e d i n a m ic ro e n v iro n m e n t v e ry d i f f e r e n t from th e aq u eo u s s o lu t i o n s i n w hich th e b e h a v io r o f p u r i f i e d enzym es u s u a lly i s exam in ed. C hanges i n t h e l o c a l d i e l e c t r i c c o n s t a n t, e l e c t r o s t a t i c f i e l d, io n ic s t r e n g t h, o r s u b s t r a t e c o n c e n tr a tio n o f th e m ic ro e n v iro n m ent c o u ld a f f e c t t h e mode o f a c tio n o f t h e enzym e. The m ost th o ro u g h ly i n v e s t i g a t e d e f f e c t s o f m ic ro e n v iro n m e n t on th e k i n e t i c b e h a v io r o f im m o b iliz e d enzym es a r e th o s e o f an e l e c t r o s t a t i c f i e l d p ro d u c e d by h ig h ly c h a rg e d c a r r i e r s (1*1). G o ld s te in e t. a l. (1+8) an d G o ld s te in and K a tc h a ls k i (1*7) r e p o r te d t h a t p o ly a n io n ic d e r i v a t i v e s o f s e v e r a l p r o t e o l y t i c enzym es a c t i n g on t h e i r s p e c i f i c, low m o le c u la r w e ig h t s u b s t r a t e s had t h e i r p H - a c t i v i ty p r o f i l e s d is p la c e d to w a rd s m ore a l k a l i n e p H -v a lu e s by 1 t o 2.5 ph u n i t s a t low io n ic s t r e n g t h ( r / 2 0,0 1 ) as com pared t o t h e n a tiv e enzym es. I n c o n t r a s t, p o l y c a t i o n i c d e r i v a t i v e s o f th e same enzym es e x h ib ite d d i s p la c e m e n t o f p H - a c t i v i ty p r o f i l e s to w a rd m ore a c i d i c p H -v a lu e s (1+7). T h ese a n o m a lie s w ere a b o lis h e d a t h ig h i o n i c s t r e n g t h ( r / 2 ^ l ). G o ld s te in e t. a l. (1+8) a l s o h a s r e p o r te d t h a t t h e a p p a re n t M ic h a e lis c o n s ta n t o f a p o ly a n io n ic d e r i v a t i v e o f t r y p s i n (E M A -try p sin ) w ith p o s i t i v e l y c h a rg e d s u b s t r a t e b e n z o y l- L - a r g in in e am ide was lo v e r a t low io n ic s t r e n g t h, th a n t h a t o f th e n a tiv e enzym e. T h is e f f e c t was a b o lis h e d a t h ig h io n ic s t r e n g t h. S im ila r r e s u l t s h av e b een r e p o r te d f o r th e p o ly a n io n ic d e r i v a t i v e s o f p a p a in (1+6), f i c i n ( 6 5 ), and b ro m e la in.

T hese phenom ena h a v e "been e x p la in e d as r e s u l t i n g from an u n e q u a l d i s t r i b u t i o n o f io n i c s p e c ie s b etw een t h e c h a rg e d p o ly e le c tr o y te -e n z y m e 39 p a r t i c l e and th e s u rro u n d in g s o l u t i o n (U 8). The l o c a l h y d ro g e n -io n c o n c e n tr a tio n i n t h e r e g io n o f th e c h a rg e d enzyme d e r i v a t i v e c o u ld b e d e s c r ib e d by e q u a tio n 2 ( 7 1 ): w h e re, i o zeip/kt Eq. (2 ) h* v - e a^+ an d a + = h y d ro g en io n a c t i v i t i e s i n t h e p o l y e l e c t r o l y t e - enzyme d e r i v a t i v e p h a se and t h e e x t e r n a l s o l u t i o n r e s p e c t i v e l y V* = e l e c t r o s t a t i c p o t e n t i a l in t h e dom ain o f th e c h a rg e d im m o b iliz e d enzyme p a r t i c l e e = p o s i t i v e e l e c t r o n c h a rg e z = p o s i t i v e o r n e g a tiv e i n t e g e r o f v a lu e u n i t y i n t h e c a s e o f h y d ro g en i o n s. K T - B o ltzm an n c o n s ta n t = a b s o lu te te m p e r a tu re I t can b e c o n c lu d e d from t h i s e q u a tio n t h a t t h e l o c a l ph w i l l b e lo w e r i n t h e r e g io n o f t h e p o ly a n io n ic enzyme p a r t i c l e th a n in th e e x t e r n a l s o l u t i o n. F o r p o l y c a t i o n i c enzym es t h e r e v e r s e i s t r u e. T h u s, t h e p H - a c t i v i ty p r o f i l e o f a p o ly m e ric enzyme w i l l b e d is p la c e d to w a rd s m ore a l k a l i n e ph v a lu e s f o r a n e g a t i v e l y c h a rg e d c a r r i e r and to w a rd s m ore a c id ph v a lu e s f o r a p o s i t i v e l y c h a rg e d c a r r i e r. The d ep en d en ce o f enzym ic a c t i v i t y on ph i s r e l a t e d t o th e d i s s o c i a t i o n o f i o n i z i n g g ro u p s p a r t i c i p a t i n g i n t h e enzym ic c a t a l y s i s m echanism s (1 0, 2 8 ). The d is p la c e d p H - a c tiv ity p r o f i l e s o f a

l+o p o l y e l e c t r o l y t e enzyme d e r i v a t i v e can t h e r e f o r e h e a l t e r n a t i v e l y r e p r e s e n te d in te rm s o f ch an g es in t h e v a lu e o f t h e a p p a re n t a c id ic d i s s o c i a t i o n c o n s ta n ts (pka ( a p p ) ) o f th e a c t i v e s i t e i o n iz in g g ro u p e f f e c t e d hy th e p o l y e l e c t r o l y t e m ic ro e n v iro n m e n t o f t h e enzyme d e r i v a t i v e a s e x p re s s e d i n e q u a tio n 3 (7 1 ) * ApKa = PKa - P *a» * U3 E q* (3 ) w h e re, pka and pk^ = a p p a re n t d i s s o c i a t i o n c o n s ta n ts f o r t h e n a tiv e enzyme and p o l y e l e c t r o l y t e enzyme d e r i v a t iv e r e s p e c t i v e l y, c a l c u l a t e d from t h e a p p r o p r ia te p H - a c t i v i ty p r o f i l e. T h is m o d el, sum m arized i n e q u a tio n s 2 and 3, e x p la in s s a t i s f a c t o r i l y m ost o f t h e known d a t a on t h e ph -dependence o f a c t i v i t y o f p o ly m e ric enzym es. The ch an g es in th e v a lu e s o f t h e a p p a re n t M ie h a e lis c o n s ta n ts o f p o l y e l e c t r o l y t e enzyme d e r i v a t i v e s a c tin g on c h a rg e d low m o le c u la r w e ig h t s u b s t r a t e s can h e r e l a t e d t o t h e u n e q u a l d i s t r i b u t i o n o f su b s t r a t e betw een t h e c h a rg e d enzyme p a r t i c l e and t h e e x t e r n a l s o lu t i o n by t h e fo llo w in g e q u a tio n ( 7 l ) : j g j i _ w h e re, [ s ] ^ and [ S ] = t h e s u b s t r a t e c o n c e n tr a tio n s i n t h e r e g io n o f t h e p o l y e l e c t r o l y t e enzyme p a r t i c l e and t h e e x t e r n a l s o lu t i o n r e s p e c t i v e l y. When p o l y e l e c t r o l y t e enzyme d e r i v a t i v e and s u b s t r a t e p o s s e s s o p p o s ite

c h a rg e s ([s]1 > [S f) t th e enzym e d e r i v a t i v e w i l l a t t a i n V max a t a lo w e r s u b s t r a t e c o n c e n tr a tio n com pared t o t h e n a tiv e enzym e, and th e K f o r t h e im m o b iliz e d enzym e w i l l be lo w e r (71)* The o p p o s ite w i l l in b e t r u e when t h e s u b s t r a t e an d p o l y e l e c t r o l y t e enzyme p a r t i c l e p o s s e s s Ul th e same c h a rg e ( [ S ] 1 < [ s ] ), and th e K o f t h e p o ly m e ric enzyme w i l l b e h ig h e r. T h u s, t h e v a lu e o f t h e e x t e r n a l s u b s t r a t e c o n c e n tr a tio n [ S ], a t w hich half-m axim um v e l o c i t y i s a t t a i n e d, le a d s t o an a p p a re n t M ic h a e lis c o n s ta n t K, r e l a t e d t o th e M ic h a e lis c o n s ta n t o f th e n a tiv e m enzyme b y t h e e x p r e s s io n ( 7 1 ):, - m * / kt (, m m A r e c e n t re v ie w a r t i c l e by K a tc h a ls k i e t. a l. (7 3 ) d is c u s s e s in m ore d e t a i l t h e e f f e c t o f t h e m ic ro e n v iro n m e n t on t h e mode o f a c t i o n o f im m o b iliz e d enzym es. Enzyme Colum ns Im m o b iliz e d enzyme colum ns have b e e n em ployed t o r e g u l a t e th e e x te n t o f c o n v e r s io n o f s u b s t r a t e t o p r o d u c t, f o r th e c o n tin u o u s p r e p a r a t i o n o f p r o d u c t, and i n au to m ated a n a l y t i c a l p r o c e d u r e s. B a r - E li and K a tc h a ls k i (7 ) and l a t t e r L i l l y (8 5 ) h av e i n v e s t i g a te d t h e k i n e t i c b e h a v io r o f p o ly m e ric enzym es i n co lu m n s. In 1963 B a r - E li and K a tc h a ls k i u s e d t h e i n t e g r a t e d form o f th e M ic h a e lis - M en ter e q u a tio n ( e q u a tio n 6 ), d e r iv e d f o r enzym es in s o l u t i o n, t o o b ta in e q u a tio n 7. (s0 - S t) + k I n (S Q/S t ) = h 3E0t Eq. (6)

w here, Sc - i n i t i a l s u b s t r a t e c o n c e n tr a tio n S-t = s u b s t r a t e c o n c e n tr a tio n a t t i m e, t» M ic h a e lis * c o n s ta n t E0 = enzym e c o n c e n tr a tio n k 3 *= r a t e c o n s ta n t fo** d i s s o c i a t i o n o f [ES] com plex w h e re, (S 0 - Sh ) + i n (S D/S t ) = k 3ED (h /V ) E q. (T) h/v = r e s id e n c e tim e o f s u b s t r a t e i n th e enzyme colum n Sj! = s u b s t r a t e c o n c e n tr a tio n o f e lu e n t The above e q u a tio n c o r r e l a t e s t h e e x te n t o f s u b s t r a t e c o n v e rs io n w ith t h e enzyme c o n c e n t r a t i o n, th e h e ig h t o f t h e co lu m n, and th e l i n e a r r a t e o f flo w o f s u b s t r a t e th ro u g h t h e colum n i n c e n tim e te r s p e r u n i t tim e ( 7 5 ). The e f f i c i e n c y o f o p e r a tio n o f b io c h e m ic a l r e a c t o r s u s in g im m o b iliz e d enzym es i n s u s p e n s io n i n a c o n tin u o u s - f e e d s t i r r e d ta n k h a s b e e n i n v e s t i g a t e d an d com pared t o a p ack ed b e d (Ul, 8U, 86), L i l l y and S h a rp (8U) showed t h a t in a ta n k c o n ta in in g a s u s p e n s io n o f p o ly m e ric c h y m o try p sin a g i t a t e d by a t u r b i n e i m p e l l e r, b o th th e a p p a re n t an d V Y v a r i e d w ith th e am ount o f a g i t a t i o n, i n d i c a t i n g t h a t t h e r a t e o f th e r e a c t i o n was p a r t l y d i f f u s i o n c o n t r o l l e d. H ow ever, in a p a c k e d b e d t h i s l i m i t a t i o n c o u ld b e overcom e by i n c r e a s i n g th e l i n e a r v e l o c i t y o f t h e s u b s t r a t e s o lu t i o n th ro u g h t h e b ed ( U l, 8U ).

1*3 A p p lic a tio n A ff i n i t y C h ro m ato g rap h y : T h is te c h n iq u e i s u t i l i z e d t o p u r i f y one o f t h e com ponents o f a sy ste m c o n s is t i n g o f tw o o r m ore s p e c ie s v h o se r e v e r s i b l e i n t e r a c t i o n s r e f l e c t a f f i n i t y w ith a h ig h d e g re e o f " b io lo g ic a l s p e c i f i c i t y ( 2 3 ). The b a s ic p r i n c i p l e i s t o im m o b iliz e one o f t h e com ponents o f th e i n t e r a c t i n g sy stem t o an i n s o l u b l e s u p p o rt w hich can th e n s e l e c t i v e l y i n t e r a c t w ith th e o th e r com ponents i n th e m edium. E l u tio n can b e a c h ie v e d by any p ro c e d u re w hich r e s u l t s i n d i s s o c i a t i o n o f th e co m p lex. A f f i n i t y ch ro m ato g rap h y h as b een em ployed t o p u r i f y enzym es such as f la v o k in a s e ( 3 ), r ib o n u c le a s e ( 1 5 9 ), t r y p s i n ( 3 0 ), and mushroom t y r o s i n a s e ( 8 l ). F r i t z, W erle and t h e i r co w o rk ers ( 3 1*) hav e r e v e r s e d t h e g e n e r a l p ro c e d u re b y u s in g bonded p r o te a s e s t o i s o l a t e p h a rm a c o lo g i c a l l y im p o rta n t i n h i b i t o r s o f t h e s e enzym es from b i o l o g i c a l f l u i d s. T h is m ethod a l s o h a s b e e n u s e d in t h e a r e a o f im m unology w here i t i s f r e q u e n t l y im p o rta n t t o s e l e c t one p a r t i c u l a r ty p e o f a n tib o d y m o le c u le o u t o f a h ig h ly com plex m ix tu r e. T h is can be a c c o m p lish e d q u i t e sim p ly b y u s in g m a tr ic e s w ith t h e a n tig e n c o rre s p o n d in g t o th e d e s ir e d a n t i body c o v a le n tly a t t a c h e d. C o n v e rs e ly, m a trix -b o u n d a n tib o d ie s c an be em ployed t o i s o l a t e t h e i r s p e c i f i c a n tig e n s. Enzyme E l e c tr o d e : G u ilb a u lt and M o n talv o (1 0 4 ) h av e em bedded u r e a s e in a p o ly a c ry la m id e membrane a b o u t 0.1 m i l l i m e t e r t h i c k on an e l e c t r o d e s e n s i t i v e t o ammonium io n s. In th e p re s e n c e o f u r e a s e, u r e a and w a te r r e a c t t o form ammionium io n s and b ic a r b o n a te. The c o n c e n tr a tio n o f ammonium io n s t h a t b u i l d s up a t th e s u r f a c e o f th e

uu e l e c t r o d e y i e l d s a d i r e c t m easu re o f t h e u r e a p r e s e n t in th e sam p le. Enzyme e l e c t r o d e s o f t h i s ty p e have o p e ra te d c o n t i n u a l l y a t room te m p e r a tu re f o r t h r e e w eeks w ith o u t l o s s o f a c t i v i t y {lok). Food A p p l i c a t i o n s : F e r r i e r e t. a l, ( 3 l ) u s e d im m o b iliz e d p e p s in i n a colum n t o a c t upon skim m ilk a c i d i f i e d t o ph 5-6. t h e m ilk t o 30 C, a t y p i c a l skim m ilk c u rd f o r fo rm ed. Upon w arm ing H ow ever, p a s s a g e o f skim m ilk o r whey th ro u g h t h e colum n s lo w ly i n a c t i v a t e d th e in s o l u b l e enzym e, S h ip p e a t. a l. (1 3 0 ) p a s s e d m ilk o v e r t r y p s i n im m o b iliz e d on p o ro u s g l a s s t o r e t a r d t h e dev elo p m en t o f o x id iz e d f l a v o r. T h e ir r e s u l t s i n d i c a t e d t h a t m ilk t r e a t e d w ith p o ly m e ric t r y p s i n i s m ore s t a b l e t o o x id a tio n th a n u n t r e a t e d m ilk. T a b le k i n d i c a t e s o th e r p o s s i b l e a p p l i c a t i o n s o f im m o b iliz e d enzyme te c h n o lo g y.

Table k A p p lic a tio n s o f I n s o lu b iliz e d Enzymes I n s o lu b iliz e d Enzyme A p p lic a tio n 1. G lucose o x id ase 2. G lucose o x id ase and p e ro x id a se 3. C at a la s e P e ro x id a se 5. H exokinase, phosp h o g lu - c o iso m erase, p h o sp h o fru c to - k in a s e, a ld o la s e 6. A ld o lase and g ly c e r a ld e - hydephosphate dehydrogenase T* P y ru v ate k in a se and l a c t a t e dehydrogenase 8. NAD p y ro p h o sp h o ry lase co n tin u o u s 'r e a g e n t l e s s ' a ssa y o f g lu c o se c o n tin u o u s, tv o - s te p d e g ra d a tio n : D -glucose + 0 2 = D -glucono-6 - la c to n e + H2O2 H2O2 + H d oner = 2H2O + o x id iz e d doner in v iv o enzyme replacem en t in a c a ta la sa e m ic m ice p re p a ra tio n o f enzyme-bound p ap er s t r i p s f o r th e d e te c tio n o f H2O2 ( to 1 0-6M) c o n tin u o u s, f o u r - s te p s y n th e s is and d e g ra d a tio n : D -glucose t o D -glucose 6-p h o sp h a te t o D -fru c to se 6- p h osphate t o D -fru c to se 1,6 -d ip h o s p h a te t o d ih y d ro x y aceto n e phosp h ate and D -g ly cerald eh y d e 3-p h o sp h ate c o n tin u o u s, tv o - s te p d e g ra d a tio n : D -fru c to se 1,6 -d ip h o sp h a te to D -g ly cerald eh y d e 3 -p h o sp h ate t o 3 -p h o sp h o -D -g ly ceric a c id c o n tin u o u s, tw o -ste p s y n th e s is : p h o sp h o en o lp y ru v ate t o p y ru v a te t o L - la c ta te co n tin u o u s s y n th e s is o f NAD 9. C h o lin e s te ra s e s c o n tin u o u s assay o f a n ti- c h o lin e s te r a s e compounds \n

Table U (Continued) Applications of Insolubilized Enzymes I n s o lu b iliz e d Enzyme A p p lic a tio n 10. Amylases 11. G a la c to s id e se 12. I n v e rta s e 13. C arb o x y p ep tid ase co n tin u o u s p ro d u c tio n o f h ig h g lu co se sy ru p s c o n tin u o u s h y d ro ly s is o f 0 -D -g a la c to s id e s co n tin u o u s h y d ro ly s is o f su c ro se co n tin u o u s r e s o lu tio n o f c h lo ro a c e ty l-d L -a la n in e l k, Rennin c l o t t i n g m ilk 15. T ry p sin s e le c tiv e d e g ra d a tio n (a n d /o r a c tiv a tio n ) o f ( i ) myosin ( i i ) heavy meromyosin ( i i i ) fib rin o g e n ( iv ) proth ro m b in and r e l a t e d c l o t t i n g f a c to r s (v ) chym otrypsinogen ( v i) pepsin o g en and carb o x y m eth y lated pep sin o g en ( v i i ) p r e k a llik r e in l 6. T ry p sin and rib o n u c le a s e s t a b i l i z a t i o n o f each in o rd e r t o stu d y th e r e v e r s ib le re d u c tio n o f t h e i r d is u lp h id e bonds IT, Chym otrypsin 18. P apain c l o t t i n g o f m ilk ( i ) s e le c tiv e d e g ra d a tio n s o f a n tib o d ie s ( i i ) c h ill- p r o o f in g o f b e e r 4 o\

Table I (Continued) Applications of Insolubilized Enzymes I n s o lu b iliz e d Enzyme A p p lic a tio n 19. Thrombin s e le c tiv e d e g ra d a tio n o f fib rin o g e n 2 0. R enin a c t iv a tio n o f a n g io te n sin o g e n 21. S tre p to k in a s e s e le c tiv e a c tiv a tio n o f plasm inogen 22. K a llik r e in a c t iv a tio n o f p r e k a llik r e in 23. A sp arag in ase ( i ) co n tin u o u s r e s o lu tio n o f D,L -a sp arag in e ( i i ) tre a tm e n t o f lymphosarcoma D ata ta k e n from M elrose (9M -

SCOPE OF INVESTIGATION A r e p o r t on t h e d is p o s a l o f d a ir y w a s te s (5 8 ) h a s shown t h a t c e r t a i n fo o d c o n s t i t u e n t s su ch a s m ilk p r o t e i n s te n d t o c r e a t e p ro b le m s i n b i o l o g i c a l tr e a tm e n t p r o c e s s e s. D i f f i c u l t i e s h av e b e e n e n c o u n te re d i n t h e b i o d e g r a d a b i l i t y o f c e r t a i n fo o d c o n s t i t u e n t s, p a r t i c u l a r l y B - l a c t o g l o b u l i n, and i n t h e i r a b i l i t y t o i n t e r a c t w ith o th e r m acrom o le c u le s a n d /o r t h e m ic r o f lo r a o f t h e b i o l o g i c a l tr e a tm e n t s y s te m, r e s u l t i n g i n im peded d e g r a d a tio n, fo a m in g, o r i n t e r f e r e n c e w ith oxygen t r a n s p o r t, th e r e b y re d u c in g th e e f f i c i e n c y o f t h e w a ste d i s p o s a l p l a n t. A tte m p ts t o im prove th e w a s te d is p o s a l p l a n t 's e f f i c i e n c y by p r e t r e a t i n g t h e w a s te m a t e r ia l w ith s o lu b le p r o te a s e p r e p a r a t i o n s have m et w ith some s u c c e s s. H ow ever, b e c a u s e o f t h e i r e x p e n s e, s o lu b le p r o te a s e s h av e b e e n em ployed f o r t h i s p u rp o se o n ly as a l a s t r e s o r t. P r e d e g r a d a tio n o f t h e s e fo o d c o n s t i t u e n t s by enzym es a tta c h e d t o i n s o l u b l e s u p p o rts may o f f e r a s o l u t i o n t o t h e s e p ro b lem s by e lim in a t i n g d i f f i c u l t i e s a s s o c i a t e d w ith m a c ro m o le c u le -m ic ro flo ra i n t e r a c t i o n s. H ow ever, l i t t l e w ork h a s b een c o n d u c te d t o d e te rm in e th e e f f e c t s o f v a r io u s m ilk c o n s t i t u e n t s on t h e a c t i v i t y o f im m o b iliz e d enzym es. T h e r e f o r e, th e m a jo r p u rp o se o f t h i s s tu d y was t o i n v e s t i g a t e th e a c t i o n o f an im m o b iliz e d p r o t e a s e, p r o n a s e, on c e r t a i n p u r i f i e d m ilk p r o t e i n s. The s p e c i f i c o b j e c t i v e s w ere: ( a ) To im m o b iliz e p ro n a s e on a p o ro u s g l a s s s u p p o rt and d e te rm in e i t s a c t i v i t y, ua

U9 <b) To d e te rm in e t h e optim um c o n d itio n s f o r t h e maximum e n z y m a tic d e g r a d a tio n o f s e l e c t e d, p u r i f i e d m ilk p r o t e i n s. ( c ) To d e te rm in e t h e a p p l i c a b i l i t y o f u t i l i z i n g p ro n a s e as a p r e tr e a tm e n t p r o c e s s f o r b i o l o g i c a l w a s te s.

EXPERIMENTAL PROCEDURE im m o b iliz a tio n o f P ro n a se t o G la s s D i a z o t i z a t i o n : One gram o f a ry la m in e g l a s s pow der (C o rn in g G la s s W orks, C o rn in g, New Y o rk ), 10 ml o f 2 N HC1, and 20 m l d i s t i l l e d w a te r w ere m ixed in an ic e - c o o le d E rle n m e y e r f l a s k. F i v e - t e n t h s ml o f 0.5 N NaNOg was ad d ed and s t i r r i n g was c o n tin u e d f o r 15 m in u te s. The g l a s s d e r i v a t i v e was f i l t e r e d on a f r i t t e d d i s c, w ashed w ith 200 ml o f 3% s u lfa m ic a c i d s o lu t i o n t o d e s tr o y any e x c e s s n i t r o u s a c i d, and f i n a l l y w ashed w ith 100 ml d i s t i l l e d w a te r. C o u p lin g : The g la s s d e r i v a t i v e was added t o 50 m l o f a r e f r i g e r a t e d s o lu t i o n o f p ro n a s e (Sigm a C hem ical Company, S t. L o u is, M is s o u ri) c o n ta in in g 50 rag o f enzyme in.05 N T r i s b u f f e r, ph 7. 0. The m ix tu re w as s t i r r e d f o r 60 m in u te s. A liq u o ts o f t h e enzyme s o l u t i o n w ere rem oved p e r i o d i c a l l y f o r a n a l y s i s. F o llo w in g c o u p lin g, th e g l a s s - enzyme d e r i v a t i v e s o lu t i o n was f i l t e r e d on a f r i t t e d d i s c, w ashed w ith 100 ml o f T r is b u f f e r, ph 7. 0, and 100 m l o f d i s t i l l e d w a t e r, and s to r e d u n d e r w a te r a t l* C. A ssay P ro c e d u re The s p e c tro p h o to m e tr ic a s s a y p ro c e d u re d e s c r ib e d i n M ethods o f E nzym atic A n a ly s is (12) was u t i l i z e d t o d e te rm in e th e am ount o f enzyme im m o b iliz e d t o th e g l a s s s u p p o rt. 50

51 A c t i v i t y o f S o lu b le P ro n a se on S e le c te d M ilk P r o te i n s A p re d e te rm in e d am ount o f t h e d e s ig n a te d p r o t e i n was d is s o lv e d in.0 5 N T r i s b u f f e r, ph 7. 0. F o u r m l o f t h i s p r o t e i n s o l u t i o n was p la c e d i n a t e s t tu b e and one m l o f p ro n a s e ( l mg/1 m l ), p r e v io u s ly d is s o lv e d i n T r i s b u f f e r, ph 7. 0, was a d d ed. The b la n k c o n ta in e d f o u r m l o f t h e d e s ig n a te d p r o t e i n and one ml o f d i s t i l l e d w a te r. A ll t e s t tu b e s w ere p la c e d on a D u b n o ff m e ta b o lic s h a k e r b a th a t room te m p e ra t u r e, and th e r e a c t i o n was a llo w e d t o c o n tin u e f o r 30 m in u te s. T h en, f i v e m l o f 25)5 (w /v ) t r i c h l o r o a c e t i c a c id was ad d ed t o a l l t u b e s, m ixed f o r f i v e seco n d s on a V o rte x -G e n ie m ix e r, and a llo w e d t o s ta n d q u ie s c e n tly f o r 20 m in u te s a t room te m p e r a tu r e. The c o n te n ts w ere f i l t e r e d th ro u g h tw o s h e e ts o f Whatman No. h2 f i l t e r p a p e r, and t h e f i l t r a t e was m easu red s p e c tr o p h o to m e tr ic a lly a t 280 nm w ith a H ita c h i P e rk in -E lm e r 139 UV-Vis s p e c tro p h o to m e te r. A ll e x p e rim e n ts w ere ru n in d u p l i c a t e. A c t i v i t y o f P o ly m e ric P ro n a se on S e le c te d M ilk P r o te in s A j a c k e te d g l a s s co lu m n, P h a rm a c ia ty p e K 9 / 1 5, w ith a d ia m e te r o f 0.9 cm, a le n g th o f 15 cm, and a b ed volum e o f 9.5 ml was u s e d i n i n v e s t i g a t i o n s w ith im m o b iliz e d p r o n a s e. The v o id volum e o f t h e enzyme colum n was d e te rm in e d u s in g a 0. IjC b lu e d e x tr a n s o l u t i o n. I n d e te rm in in g t h e e n z y m a tic a c t i v i t y o f p o ly m e ric p ro n a s e on s e l e c t e d m ilk p r o t e i n s, w a te r was a llo w e d t o flo w th ro u g h t h e colum n c o n ta in in g t h r e e gram s o f th e e n z y m e -g la ss p r e p a r a t i o n u n t i l t h e w a te r l e v e l h a d re a c h e d th e to p o f th e enzyme colum n. T hen, th e

p r o t e i n s o l u t i o n, p r e v io u s ly m ade-up i n.05 N T r i s " b u ffe r, ph 7. 0, 52 was added and "between 15 and 20 ml o f e lu a n t w ere d is c a r d e d. Flow r a t e th ro u g h t h e colum n was k e p t c o n s ta n t b y u s in g a B u c h le r p e r i s t a - t i c, m u l t i - c h a n n e l, p o l y s t a l t i c i n f u s io n pump a t t h e i n l e t t o t h e colum n and c o n t r o l l e d b y a screw clam p a t t h e o u t l e t o f t h e colum n. F o u r m l sam p les w ere c o l l e c t e d in d u p l i c a t e i n g ra d u a te d t e s t tu b e s an d p la c e d in i c e u n t i l a l l sam p le s h a d b e e n c o l l e c t e d. The b la n k s o l u t i o n c o n s is te d o f f o u r ml o f t h e p r o t e i n s o l u t i o n n o t t r e a t e d by t h e enzym e. T h e n, f o u r m l o f 25$ (w /v ) t r i c h l o r o a c e t i c a c id was added t o e a c h sam p le, m ixed f o r f i v e seco n d s on a V o rte x -G e n ie, and a llo w e d t o s ta n d q u i e s c e n t ly a t room te m p e r a tu re f o r 20 m in u te s. The c o n te n ts w ere f i l t e r e d th ro u g h tw o s h e e ts o f Whatman No. k2 f i l t e r p a p e r, and t h e f i l t r a t e was m easu red s p e c tr o p h o ta m e tr ic a lly a t 280 nm a g a i n s t th e u n t r e a t e d p r o t e i n b la n k u s in g a Coleman 12U d o u b le beam s p e c tr o p h o to m e te r. D e te rm in a tio n o f P r o te i n P u r i t y The p u r i f i e d m ilk p r o t e i n s u s e d i n t h i s s tu d y ( f i - l a c t o g l o b u lin, a - l a c t a l b u m in, a - c a s e i n, and w hole c a s e in ) w ere p u rc h a s e d from N u t r i t i o n a l B io c h e m ic a ls, C le v e la n d, O hio. S ta r c h g e l e l e c t r o p h o r e s i s ( 1 0 3 ), and v e r t i c a l s la b p o ly a c ry la m id e g e l e l e c t r o p h o r e s i s ( 1 0 2 ), w ere u t i l i z e d t o d e te rm in e th e p u r i t y o f t h e p u rc h a s e d p r o t e i n sam p le s. M o is tu re C o n te n t o f I n s o lu b le P ro n a se F o u r m o is t p o ly m e ric p ro n a s e sam p les w ere p la c e d on s e p a r a t e w atch g l a s s e s, p r e v io u s ly w eig h ed on a M e ttle r H10T a n a l y t i c a l b a la n c e. The w a tc h g l a s s e s w ith th e sam p les w ere w e ig h e d, an d t h e

sam p les v e r e d r ie d f o r UO m in u te s a t 110 C i n a M odel l 6 T h e lc o oven* The sam p les w ere t r a n s f e r r e d immediately t o a d e s s l c a t o r, c o o le d f o r 10 m in u te s and w eig h ed. The m o is tu re c o n te n t was c a l c u l a t e d as f o llo w s : % moisture = Ms plejret - wt. sample dry x 10Q w t. sam p le w et 53 T herm al S t a b i l i t y T h re e d i f f e r e n t te m p e r a tu re s (3 0 C, 50 C, and T0 C) v e r e u t i l i z e d f o r 6 h o u rs e a c h t o stu d y th e e f f e c t o f h e a t on t h e b i o l o g i c a l a c t i v i t y o f p o ly m e ric p ro n a s e. Two p o ly m e ric p ro n a s e colum ns v e re u s e d, e a c h c o n ta in in g t h r e e gram s o f t h e g l a s s enzyme d e r i v a t i v e, one f o r s t u d i e s a t 30 and 50 C, and t h e o th e r f o r e x p e rim e n ts a t 70 C. Bound enzyme in t h e colum n was s u b je c te d t o t h e d e s ig n a te d te m p e r a tu re f o r one h o u r and th e n r e a c t e d w ith 25 ml o f c a s e i n, ph 7.1, a t a flo w r a t e o f 1 m l/2.5 m in u te s. Two 5 m l sam p les w ere c o l l e c t e d a f t e r 10 ml o f p r o t e i n e lu a n t h ad b e e n d is c a r d e d. The two s a m p le s, t o g e t h e r w ith t h e p r o te in sam ple t o be u s e d a s a b la n k v e re k e p t i n i c e u n t i l a l l sam p les w ere c o l l e c t e d. T h en, a l l sam p les w ere t r e a t e d w ith I* m l o f 25% t r i c h l o r o a c e t i c a c id ( w /v ), a llo w e d t o s ta n d q u i e s c e n t ly f o r 20 m in u te s a t room te m p e r a tu r e, f i l t e r e d th ro u g h tw o s h e e ts o f Whatman No, 1*2 f i l t e r p a p e r and r e a d a t 280 nm, S o lu b le p ro n a se (2 m g/m l) was k e p t i n a w a te r b a th f o r th e d e s ig n a te d tim e and th e n rem oved and one ml a llo w e d t o r e a c t w ith fo u r m l o f t h e p r o t e i n s u b s t r a t e u n d e r th e same c o n d itio n s as s t a t e d f o r t h e im m o b iliz e d enzyme d e r i v a t i v e. The re m a in d e r o f t h e enzyme

5U s o l u t i o n was r e t u r n e d t o t h e w a te r h a th f o r a n o th e r h o u r. F o llo w in g t h e r e a c t i o n, t h e same p ro c e d u re as d e s c r ib e d above was u s e d t o o b ta in t h e f i l t r a t e w hich was re a d a t 280 nm on a Colem an 12U d o u b le beam s p e c tr o p h o to m e te r, u s in g a c a s e in sam ple n o t t r e a t e d w ith s o lu b le p ro n a s e as t h e b la n k. B io lo g ic a l O x id a tio n o f Skim m ilk T r e a te d w ith P ro n a se T h ree l i t e r s o f sk im m ilk, p u rc h a s e d from t h e U n iv e r s ity d a ir y * was a d ju s te d t o ph 7.1 w ith 0.1 H NaOH and t r e a t e d w ith t h r e e gram s o f p ro n a s e f o r ^5 m in u te s a t 30 C. A g ita tio n o f t h e m ix tu re was a c h ie v e d by u s in g a Y ankee v a r i a b l e sp e e d r o t a t o r. The d e g r a d a tio n o b ta in e d, w ith s o lu b le p ro n a s e w ould b e e q u iv a le n t t o t h a t o b ta in e d a t a flo w r a t e o f 0.0 8 3 m l/m in f o r p o ly m e ric p r o n a s e. The p ro n a s e t r e a t e d sk im m ilk was th e n a u to c la v e d f o r 7 m in u te s a t 121 C, c o o le d q u ic k ly i n an ic e b a th t o ^ 5 F, and in tr o d u c e d i n t o th e s u b s t r a t e r e s e r v o i r. The b io f e r m e n ta tio n sy stem was m a in ta in e d a t 2 3 C, ph 6.8 5 + 0.0 3 and 0. 7 ** ppm d is s o lv e d oxygen w ith an a e r a t i o n r a t e o f 2 l i t e r s / m i n u t e a t 5 p s i. When t h e b io f e r m e n ta tio n sy stem h ad a c h ie v e d a s te a d y s t a t e, sam p les w ere c o l l e c t e d a t d i f f e r e n t tim e i n t e r v a l s from v a r io u s lo c a t i o n s and f r o z e n f o r s u b s e q u e n t i n v e s t i g a t i o n s. H igh V o lta g e E le c tr o p h o r e s is The f r o z e n sam p les o b ta in e d from t h e b i o l o g i c a l o x id a tio n s tu d y w ere thaw ed a t room te m p e r a tu r e. One h u n d re d m i c r o l i t e r sam p les w ere s p o tte d 5 in c h e s from t h e b o tto m o f a p a p e r s h e e t m e a su rin g 18 in c h e s by 22 in c h e s and a i r d r i e d. T h e n, th e p a p e r was p la c e d in an

55 e l e c t r o p h o r e t i c cham ber and a p y r id i n e : a c e t i c a c id : w a te r m ix tu re ph 3.U, ( 5 :1 5 :8 5 ) was a d d e d. E l e c tr o p h o r e s i s was c o n d u c te d a t 2500 v o l t s, 150 m a, f o r 1.5 h o u r s. F o llo w in g e l e c t r o p h o r e s i s, t h e p a p e r was a i r - d r i e d and th e n s p ra y e d w ith n in h y d r in t o v i s u a l i z e t h e am ino a c id s and p e p t i d e s.

RESULTS P r e lim in a r y I n v e s t i g a t i o n s P r o te i n P u r i t y C h a r a c t e r i z a t i o n o f an e n z y m e -s u b s tra te r e a c t i o n u n d e r v a r io u s p h y s ic a l c o n d itio n s r e q u i r e s a know ledge o f t h e p u r i t y o f th e *; com ponents in v o lv e d in t h e r e a c t i o n. T h e r e f o r e, v e r t i c a l s l a t p o ly a c ry la m id e g e l e l e c t r o p h o r e s i s and s t a r c h g e l e l e c t r o p h o r e s i s v e r e u t i l i z e d t o d e te rm in e t h e p u r i t y o f c o m m e rc ia lly p u rc h a se d p r o te in s u sed in s u b s e q u e n t i n v e s t i g a t i o n s. I d e n t i f i c a t i o n o f com ponents was made a c c o rd in g t o s ta n d a r d s d e s c r ib e d by Thompson (1 3 7 ) as shown In F ig u re 8. B - la c to g lo b u lin e x h ib ite d one m a jo r zone and t h r e e m in o r whey p r o t e i n zones when s u b je c te d t o e l e c t r o p h o r e s i s on s t a r c h g e l a t ph 8,6 in t h e p re s e n c e o f b o th 7 M u r e a and 2 -m e rc a p to e th a n o l (F ig u re 8a ). W ith a - c a s e i n tw o m a jo r zo n es w ere e v id e n t, a l a r g e r r e g io n c o n ta in in g a - c a s e i n and a s m a lle r a r e a c o n ta in in g a - c a s e i n. A m in o r r e g io n s c o n ta in in g x - c a s e in a l s o was p r e s e n t. Whole c a s e in gave a p a t t e r n i d e n t i c a l t o a - c a s e i n e x c e p t f o r a s m a lle r a - c a s e i n r e g io n. P ro n a se s was n o t s t a i n e d s u f f i c i e n t l y b y th e dye t o e x h i b i t any z o n e s. E l e c t r o p h o r e s is o f a - la c ta lb u m in as shown i n F ig u re 8b show ed m in o r c o n ta m in a t i o n w ith { J -la c to g lo b u lin and b lo o d serum a lb u m in. 6- l a c t o g l o b u l i n c o u ld b e s e p a r a te d i n t o i t s g e n e tic v a r i a n t s, B - la c to g lo b u lin ^ and g - l a c t o g l o b u l i n B, w ith no t r a c e s o f c a s e in

57 F ig u re 8 : S ta rc h g e l e l e c t r o p h o r e s i s and p o ly a c ry la m id e g e l e l e c t r o p h o r e s i s o f p u r i f i e d p r o t e i n s. (a ) S ta r c h g e l e l e c t r o p h o r e s i s, ph 8.6 in t h e p r e s e n c e o f b o th 7 M u r e a and 2- m e rc a p to e th a n o l. ( l ) B - l a c t o g l o b u l i n, ( 2 ) a - c a s e i n, ( 3 ) w h o le c a s e i n, and {!*) p ro n a s e. (b ) S ta r c h g e l e l e c t r o p h o r e s i s, ph 8.6 in t h e p re s e n c e o f b o th 7 M u r e a and 2- m e rc a p to e th a n o l. ( l ) a - la c ta lb u m in. (c ) V e r t i c a l s la b p o ly a c ry la m id e g e l e l e c t r o p h o r e s i s, ph 8.6. ( l ) w h o le c a s e i n, (2 ) a - c a s e i n, (3 ) & - la c to g lo b u lin, and ( h ) p ro n a s e.

Sample slo ts P ronase K -casein B -casein a-la B -casein BLG a -c a s e in s o - c a s e in s BLG B BLG. «1 2 3 h 1 1 2 la ) (t) (c)

59 com ponents b y u t i l i z i n g v e r t i c a l s la b p o ly a c ry la m id e g e l e l e c t r o p h o r e s is a t ph 8.6 i n t h e a b se n c e o f u r e a and 2 -m e rc a p to e th a n o l ( F ig u r e 8c ). S in c e n e i t h e r u r e a n o r 2 -m e rc a p to e th a n o l v e r e em ployed in t h i s m ethod* a - c a s e i n and w hole c a s e in e x h ib ite d d i f f u s e p a t t e r n s f o r a - and 8- c a s e i n s. s s l o t s. A p p a re n tly K -c a s e in p o ly m e riz e d in t h e sam ple P ro n a se showed tw o f a i n t b a n d s, i n d i c a t i n g t h a t t h e dye d id n o t s t a i n t h e p r o t e i n s u f f i c i e n t l y t o i d e n t i f y t h e com pounds. T h ese i n v e s t i g a t i o n s showed t h a t t h e whey p r o t e i n s, B - la c to - g lo b u lin and a - l a c t a l b u m i n, c o n ta in e d l i t t l e c o n ta m in a tin g m a te r ia l an d t h a t t h e c a s e in s w ere a lm o s t i d e n t i c a l i n c o m p o sitio n e x c e p t f o r a l a r g e r a - c a s e i n r e g io n in t h e p u r i f i e d a - c a s e i n. s E f f e c t o f S to ra g e on BAEE A c t i v i t y B e n z o y l- a r g in in e e t h y l e s t e r was u s e d t o d e te rm in e t h e amount o f enzyme bound t o t h e g l a s s s u p p o r t. F o r co m p a riso n b etw een l o t s o f enzym e, i t w ould b e d e s i r a b l e t o u s e one l o t o f BAEE. H ow ever, a p p a re n t d is c r e p a n c ie s i n r e s u l t s w ith f r e s h and aged BAEE, le d t o an i n v e s t i g a tio n o f t h e s to r a g e s t a b i l i t y o f t h e s u b s t r a t e. To s tu d y t h e e f f e c t o f p ro lo n g e d s to r a g e on s u b s t r a t e s t a b i l i t y u n d e r r e f r i g e r a t e d cond i t i o n s, a f r e s h BAEE p r e p a r a t i o n (B) was com pared t o an 8 m onth o ld BAEE sam ple (A ). F ig u re 9 p r e s e n t s a s p e c t r a l sc a n from 330 nm t o 250 nm co m p arin g sam p les A and B. S c h v e rt and T akenaka (1 2 ) found t h a t t h e a b s o r p tio n o f N - b e n z o y l- L - a r g in in e - e th y l e s t e r i s much w eak er a t 25U nm th a n t h a t o f N - b e n z o y l-b -a r g in in e. T h e r e f o r e, when two sam p les a r e a n a ly z e d i n a d o u b le beam s p e c tro p h o to m e te r w ith one o f

60 F ig u re 9. A d i f f e r e n t i a l s p e c t r a l sc a n from 330 nm t o 250 nm co m p arin g new and o ld BAEE s a m p le s. R e fe re n c e c e l l i s new BAEE; sam ple c e l l i s o ld BAEE.

Relative Difference in Absorbance i\> at o Wavelength in nm ro o oj o

62 t h e sam p les a s t h e r e f e r e n c e, no change In a b s o rb a n c e i s t o b e e x p e c te d i f n e i t h e r sam ple h a s b een d e g ra d e d. I f any d e g r a d a tio n p r o d u c ts a r e p r e s e n t, th e y w i l l a p p e a r a s a p eak a t nm. R e s u lts from t h i s p h a s e o f t h e s tu d y i n d i c a t e d t h a t t h e o ld BAEE sam ple had u n d e rg o n e d e g r a d a tio n o v e r t h e e ig h t-m o n th p e r io d e i t h e r th ro u g h c h e m ic a l breakdow n upon s to r a g e o r by m ic r o b ia l a t t a c k o r by a c o m b in a tio n o f t h e tw o p r o c e s s e s. S S in c e a l a r g e q u a n t i t y o f th e e n z y m e -g la ss d e r i v a t i v e was p r e p a r e d i n i t i a l l y u t i l i z i n g f r e s h BAEE, t h e s e f in d in g s d id n o t in f lu e n c e p r e v io u s r e s u l t s. H ow ever, b e c a u s e o f th e s e r e s u l t s, o n ly r e c e n t l y p u rc h a s e d BAEE was u sed t o a n a ly z e f o r e n z y m a tic a c t i v i t y o f s u b s e q u e n t b o u n d enzyme d e r i v a t i v e s. Bound Enzyme Q u a n tita tio n To d e te rm in e th e am ount o f enzyme bound t o t h e a ry la m in e g l a s s i n t h e c o u p lin g r e a c t i o n, a tim e c o u rse was o b ta in e d f o r t h e s o lu b le pronase-b A E E r e a c t i o n. F ig u r e 10 d e p i c t s th e tim e c o u rs e o f t h e r e a c t i o n o f BAEE w ith a l i q u o t s o f t h e p ro n a s e s o lu t i o n rem oved a t 15 m in u te i n t e r v a l s from t h e c o u p lin g r e a c t i o n. P ro n a se rem oved a t 0 tim e from t h e c o u p lin g r e a c t i o n g av e 100^ h y d r o ly s is a f t e r tw o m in u te s r e a c t i o n w ith BAEE. On th e o th e r h a n d, p ro n a se rem oved a t 60 m in u te s o r T5 m in u te s a f t e r th e b in d in g r e a c t i o n h a d begun showed 52JC and 53J& h y d r o l y s i s, r e s p e c t i v e l y, a f t e r tw o m in u te s o f c o n ta c t w ith BAEE. S in c e l i t t l e change was n o te d i n t h e p e r c e n t BAEE h y d ro ly z e d i n t h e a l i q u o t s o f p ro n a se ta k e n a t 60 and 75 m in u te s, i t was c o n c lu d e d t h a t th e c o u p lin g r e a c t i o n was n e a r ly c o m p leted a f t e r

63 F ig u re 10: R e a c tio n r a t e f o r v a r io u s p ro n a s e a l i q u o t s from t h e c o u p lin g r e a c t i o n on BAEE. (T e m p e ra tu re 26 C ; 0.0 0 1 M BAEE; ph 7.1 )

100 6 U 15 min 00 30 min % BAEE hydrolyzed 6 0 40 75 min r 45 min 20 0 60 90 Reaction Time in Seconds 120

65 60 m in u te s, and t h i s tim e was u s e d i n s u b s e q u e n t e n z y m e -g la ss p r e p a r a t i o n s. M o is tu re C o n te n t D e te rm in a tio n I n o r d e r t o d e te rm in e th e e x te n t o f h y d r a tio n o f in n a o b iliz e d p ro n a s e a tta c h e d t o p o ro u s g l a s s, th e m o is tu r e c o n te n t o f f o u r w et sa m p les was d e te rm in e d. R e s u lts a r e g iv e n I n t h e fo llo w in g T a b le : T a b le 5 M o is tu re C o n te n t o f I n s o lu b le P ro n a se Sam ple No. Sam ple w et ( g r,) Sam ple d ry ( g r.) M o is tu re % M o is tu re 1 0.1 2 6 0 0.03U 7 0.0 9 1 3 7 2.5 2 0.1 7 6 6 0.01*67 0.1 3 1 9 7 3.8 3 O.llU? 0.0 3 1 0 0.0 9 1 0 7 ^.6 1* O.llli? 0.0 3 0 0 0.081*5 7 3.8 L i t t l e v a r i a t i o n i n m o is tu r e c o n te n t was n o tic e d b etw een s a m p le s, and t h e a v e ra g e m o is tu re c o n te n t f o r th e f o u r sam p les was 7 3.7?. T h is i n d i c a t e s t h a t t h e m ic ro e n v iro n m e n t aro u n d t h e im m o b iliz e d enzyme m o le c u le h a s a h ig h d e g re e o f h y d r o p h i l i c i t y. P ro n a se Bound t o G lass P ro n a se was bound t o a ry la m in e g la s s b y d ia z o c o u p lin g th ro u g h a t y r o s i n e r e s id u e on t h e enzyme m o le c u le. I n o r d e r t o d e te rm in e th e am ount o f p ro n a s e a tta c h e d t o t h e p o ro u s g l a s s s u r f a c e o f v a r io u s p r e p a r a t i o n s d u rin g t h e b in d in g p r o c e d u re, a s p e c tr o p h o to m e tr ic a s s a y m ethod u t i l i z i n g b e n z o y l - L - a r g i n i n e - e t h y l - e s t e r (BAEE) was em ployed.

66 A sum m ation o f t h e b in d in g r e s u l t s a r e fo u n d i n T a b le 6. I n t h e i n i t i a l i n v e s t i g a t i o n ( T r i a l s 1 t h r u 3 ) o f p o ly m e ric p r o n a s e, o n ly one gram o f a ry la m in e g la s s v a s u t i l i z e d in t h e d ia z o - t i z a t i o n and c o u p lin g r e a c t i o n s. I n s u b s e q u e n t colum n s t u d i e s ( T r i a l s k t h r u 1 0 ) l a r g e r q u a n t i t i e s o f t h e e n z y m e -g la ss d e r i v a t i v e w ere r e q u i r e d. T h e r e f o r e, t h r e e gram s o f a ry la m in e g l a s s w ere r e a c t e d w ith 200 m l o f p ro n a s e ( l mg/1 m l). As a r e s u l t, th e p e r c e n t e.nzyme b o u n d /g g l a s s d e c r e a s e d from an a v e ra g e o f 21.1>f in T r i a l s 1 t h r u 3 t o an a v e ra g e o f 1 3.8 # i n T r i a l s U t h r u 10. To i n s u r e t h a t no enzyme was b e in g e l u t e d from t h e b o u n d p ro n a s e i n t h e g l a s s co lu m n, 150 ml o f d i s t i l l e d w a te r was p a s s e d th ro u g h th e enzyme colum n a t a flo w r a t e o f 2 m l/m in u te. The e lu a n t v a s co n cen t r a t e d t o 5 m l b y u t i l i z i n g a r o t a r y e v a p o r a to r. One m l o f t h i s e lu a n t was r e a c t e d w ith one m l o f a 25# a c id ( v / v ) ; 2 m l w ere r e a c t e d w ith 2 m l s o l u t i o n o f t r i c h l o r o a c e t i c o f BAEE; and 2 ml w ere r e a c te d w ith 2 m l o f c a s e i n, ph 7. 0, p r e c i p i t a t e d w ith *+ ml o f a 25# s o l u t i o n o f t r i c h l o r o a c e t i c a c id ( w /v ), and f i l t e r e d on Whatman No. h2 f i l t e r p a p e r. No m ilk y c o lo r d e v e lo p e d w ith t h e t r i c h l o r a c e t i c a c id s o lu t i o n a f t e r 20 m in u te s, an d no a c t i v i t y was m easu red e i t h e r a t 2 5 *+ nm f o r BAEE o r a t 280 nm f o r t h e t r i c h l o r a c e t i c a c id s o l u t i o n. T h e re f o r e, i t was c o n c lu d e d t h a t t h e r e was no l o s s o f bound enzyme from th e colum n.

Table 6 Summation o f B inding S tu d ie s T r i a l No. I n i t i a l A b s o r b a n c e ^ F in a l A bsorbance (b ) % enzyme b o u n d /.v g g la s s mg enzyme b o u n d /.* g g la s s 1 0.7 7 0.61* 1 6.8 8.1* 2 0.8 2 0.67 1 8.2 9.1 3 0.8 1 0.5 8 28.3 ll*.l It 0.1*0 0.2 0 16.6 1 1.1 5 0.1*1 0.2 0 17*1 11.1* 6 0. 1*0 0.2 5 1 2.5 8.3 T 0.1*3 0.2 8 11.6 7.7 8 0. 1*0 0.25. 12.5 8.3 9 0. 1*1 0.2 6 1 2.1 8.1 10 0.3 1 0.1 8 11*.0 9.3 (A) P ro n ase in s o lu tio n a t b e g in n in g o f b in d in g, ab so rb an ce a t 25U nm w ith BAEE as s u b s tr a te. (B) P ronase in s o lu tio n a t te rm in a tio n o f b in d in g, ab so rb an ce a t 25*+ nm w ith BAEE as s u b s t r a te. (C )rf, 3 ^ I n i t i a l A b s.-f in a l Abs. % bound enzyme c a lc u la te d as " T / '.v------------- x 100* J I n i t i a l Abs. ^ I n sam ples 1* th r u 10 6 6.6 mg s o lu b le enzyme/mg g la s s was used compared t o 50 mg s o lu b le enzyme/mg g la s s f o r sam ples 1 th r u 3. o\ -3

68 C h a r a c t e r i s t i c s o f S o lu b le and P o ly m e ric P ro n a se F o r t h e e v e n tu a l a p p l i c a t i o n o f im m o b iliz e d p r o n a s e t o d a i r y fo o d p l a n t w a s te t r e a t m e n t, in f o r m a tio n on th e c h a r a c t e r i s t i c s o f th e im m o b iliz e d enzyme and a co m p a riso n t o t h e c h a r a c t e r i s t i c s o f th e s o lu b l e form o f t h e enzyme a r e e s s e n t i a l. S i g n i f i c a n t in f o r m a tio n f o r e v e n tu a l a p p l i c a t i o n in c lu d e d e te r m in a tio n o f t h e e f f e c t o f t h e f o llo w in g f a c t o r s : ( a ) ty p e o f s u b s t r a t e and r e l a t i v e a c t i v i t y a g a i n s t v a r io u s m ilk p r o t e i n s, (b ) te m p e r a tu r e, ( c ) ph, ( d ) i o n i c s t r e n g t h, and (e ) flo w r a t e. T h e r e f o r e, s t u d i e s w ere c o n d u c te d w ith s o lu b l e and im m o b iliz e d p ro n a s e a c tin g on f o u r m ilk p r o t e i n s ( P - l a c t o g l o b u l i n, a - l a c t a l b u m in, a c a s e in and w hole c a s e in ) u n d e r v a r io u s e n v iro n m e n ta l c o n d itio n s t o d e te rm in e w hat e f f e c t s im m o b iliz a tio n h a s on p ro n a s e a c t i v i t y and t o com pare th e s e e f f e c t s t o s o lu b le p r o n a s e. E f f e c t o f T e m p e ra tu re L ik e o th e r p r o t e i n s, enzym es r e t a i n t h e i r b i o l o g i c a l a c t i v i t y o n ly w ith in a l i m i t e d te m p e r a tu re r a n g e. E x p o su re o f p r o t e i n m olec u le s t o h ig h te m p e r a tu r e s c a u s e them t o d e n a tu re o r u n f o ld t h e i r c h a r a c t e r i s t i c f o ld e d s t r u c t u r e y i e l d i n g p o ly p e p tid e c h a in s t h a t a r e random ly and i r r e g u l a r l y lo o p e d o r c o i l e d (U l), S in c e t h e th e r m a l s t a b i l i t y w i l l v a ry w ith t h e ty p e o f enzym e, i n v e s t i g a t i o n s w ere made t o d e te rm in e t h e e f f e c t s o f te m p e r a tu r e on t h e th e r m a l s t a b i l i t y o f b o th s o lu b le and i n s o l u b l e p ro n a s e p r e p a r a t i o n s. S o lu b le p ro n a s e r e a c t e d f o r 30 m in u te s w ith t h e d e s ig n a te d p r o t e i n (0.2 6 6 grams p r o te in /1 0 0 m l o f 0.0 5 N T r is b u f f e r, ph 7. 1 ). The b la n k c o n s is te d o f U m l o f t h e d e s ig n a te d p r o t e i n + 1 ml o f T r is b u f f e r. A flo w r a t e

o f 0.5 7 m l/m in u te v a s o b ta in e d f o r t h e p o ly m e ric p ro n a s e - d e s ig n a te d p r o t e i n r e a c t i o n (0.2 5 0 grains p r o te in /1 0 0 ml o f 0.0 5 N T r i s b u f f e r, ph 7. 1 ). The c o n t r o l c o n s is te d o f ^ m l o f t h e d e s ig n a te d p r o t e i n v i t h no enzym e t r e a t m e n t. L i t t l e change i n r e a c t i o n v e l o c i t y v a s o b s e rv e d from 3 0 t o 60 C f o r s o lu b l e p ro n a s e ( F ig u re 1 1 ), v i t h e i t h e r w hole c a s e in o r a - c a s e in a s t h e s u b s t r a t e. H ow ever, la r g e c h a n g e s i n t h e p ro n a s e r e a c t i o n v e l o c i t y w ere o b s e rv e d f o r th e whey p r o t e i n s, 6- l a c t o g l o b u l i n and a - l a c t a l b u m in, o v e r t h e same te m p e r a tu re r a n g e. At te m p e r a tu re s a ro u n d 30 C, su ch a s w ould b e e n c o u n te re d i n w a s te t r e a t m e n t, c a s e in w ould b e d e g ra d e d a t 1.2 3 tim e s f a s t e r th a n p - la c t o g lo b u l i n and 2.5 tim e s f a s t e r th a n a - la c ta lb u m in. H ow ever, b o th o f t h e s e p r o t e i n s w ould b e d e g ra d e d a t a f a s t e r r a t e th a n c a s e in a t te m p e r a tu r e s above 50 C. At 6j C t h e r a t e o f d e g r a d a tio n o f c a s e i n, B - la c to g lo b u lin and a - la c ta lb u m in w ere e s s e n t i a l l y t h e sam e. S in c e enzym es in c r e a s e in r e a c t i o n v e l o c i t y w ith in c r e a s in g t e m p e r a tu r e, one m ethod o f e x p r e s s in g t h i s i n c r e a s e i s w hich i s d e f in e d a s a t J P. A 10, q v a lu e s f o r s o lu b le p ro n a s e v e l o c i t y a t 10 C 10 re m a in e d c o n s ta n t a t 1.0 0 and 1.0 2 f o r a - c a s e i n and w h o le c a s e in from 30 t o 60 C. C o n v e rs e ly, a - la c ta lb u m in an d 6- l a c t o g l o b u l i n e x h ib ite d Q10 v a lu e s o f 1.9 3 and I. I 46 from 3 0 t o 50 C. The Q1q v a lu e s w ith c a s e in as th e s u b s t r a t e in d i c a t e t h a t te m p e r a tu re s t o 50 C a p p e a r t o h a v e l i t t l e e f f e c t upon p ro n a se v e l o c i t y. T h is p ro b a b ly can be a t t r i b u t e d t o t h e random c o i l s t r u c t u r e o f th e c a s e in s w hich i n i t i a l l y e x p o s e s m ost o f t h e s u s c e p t i b l e b o n d s t o enzym ic a t t a c k th e r e b y

70 F ig u re 11: E f f e c t o f te m p e ra tu re on t h e r e a c t i o n v e l o c i t y o f s o lu b le p ro n a s e w ith f o u r m ilk p r o t e i n s, ( T r i s b u f f e r, 0.0 5 N; ph 7.1 ; 0.2 6 6 grains p r o te in /1 0 0 ml b u f f e r ; 30 m in. r e a c t i o n tim e ; c o n t r o l li ml d e s ig n a te d p r o t e i n + 1 ml b u f f e r )

71 ea A Abt /min X 10*3 4 t t a-casein 20-2 0 30 4 0 50 60 70 Temperature C

p r e v e n tin g g r e a t in c r e a s e s i n v e l o c i t y v i t h i n c r e a s i n g te m p e r a tu r e s. 72 H ow ever, t h e v a lu e s f o r t h e whey p r o t e i n s seem t o i n d i c a t e t h a t t h e s e p r o t e i n s may b e u n d e rg o in g c o n f o rm a tio n a l ch an g es and i n t h e c a s e o f 3 - l a c t o g l o b u l i n, monomer f o rm a tio n r e s u l t i n g in t h e a p p e a ra n c e o f m ore bonds s u s c e p t i b l e t o e n z y m a tic h y d r o ly s is and h e n c e, a g r e a t e r r a t e o f h y d r o ly s is by th e s o lu b le enzym e. When t h e te m p e r a tu re was r a i s e d t o 7 0 C, s i g n i f i c a n t i n c r e a s e s in p ro n a s e r e a c t i o n v e l o c i t i e s frc m n e a r ly c o n s ta n t v a lu e s w ere r e c o rd e d v i t h e ach m ilk p r o t e i n - enzym e r e a c t i o n. T h is p ro b a b ly i n d i c a t e s e x te n s iv e d e n a tu r a tio n and d e g r a d a tio n o f b o th t h e enzyme and m ilk p r o t e i n m o le c u le s r a t h e r th a n i n c r e a s e d a c t i v i t y o f t h e s o lu b le p ro n a s e s in c e b o th th e enzyme and m ilk p r o t e i n m o le c u le s w ere s u b je c te d t o 70 C f o r 30 m in u te s. I n s o lu b le p ro n a s e { F ig u re 12) gave s i m i l a r th e r m a l p r o f i l e s f o r w hole c a s e i n, a - c a s e i n, and a - la c ta lb u m in e s p e c i a l l y from 30 t o 50 C. 6 - l a c t o g l o b u l i n, h o w e v e r, had a m arked i n c r e a s e i n th e r a t e o f d e g ra d a t i o n above Uo C w ith t h e g r e a t e s t in c r e a s e in v e l o c i t y o c c u r r in g b etw een U0 and 50 C. S in c e B - la c to g lo b u lin e x i s t s a s a d im e r a t n e u t r a l ph and a t m o d e ra te c o n c e n tr a tio n s w ith in c r e a s in g d i s s o c i a t i o n o f th e d im er o c c u r r in g b e tw e e n 30 and 50 C, i t i s p r o b a b le t h a t t h e m arked change i n im m o b iliz e d p ro n a se v e l o c i t y on B - la c to g lo b u lin can b e a t t r i b u t e d t o d i s s o c i a t i o n o f th e d im er t o t h e monomer fo rm, th e r e b y e x p o s in g m ore s u s c e p t i b l e p e p tid e b o n d s t o enzym ic a t t a c k. Above 55 C B - la c to g lo b u lin b e g in s t o u n f o ld and lo s e i t s g lo b u la r s t r u c t u r e e x p o s in g m ore s u s c e p t i b l e bonds t o h y d r o ly s is and th e r e b y e x p la in in g th e l i n e a r i n c r e a s e i n r e a c t i o n v e l o c i t y from 5 0 t o 70 C. The o t h e r whey p r o t e i n, a - l a c t a l b u m in, show ed n o su ch d r a m a tic in c r e a s e i n th e

73 F ig u re 12: E f f e c t o f te m p e r a tu re on t h e v e l o c i t y o f p o ly m e ric p ro n a s e v i t h d i f f e r e n t m ilk p r o t e i n s u b s t r a t e s. ( T r is b u f f e r, 0.0 5 N; ph 7.1 ; 0.2 5 0 0 gram s p r o te in /1 0 0 ml b u f f e r ; flo w r a t e 0. 5 7 m l/m in, ; c o n t r o l ml d e s ig n a te d p r o te in w ith no enzyme t r e a t m e n t ).

7* 64 56 48 -loctoglobulin x 40 A Abs /min 32 24 casein y a-lactalbum in Temperature C 60 70

75 r a t e o f p r o t e o l y s i s v h en a c te d upon by im m o b iliz e d p r o n a s e. On th e c o n t r a r y, t h e v e l o c i t y o f t h e r e a c t i o n in c r e a s e d in a l i n e a r f a s h io n fro m U0 t o 70 C i n d i c a t i n g t h a t n o t as e x te n s iv e c o n fo rm a tio n a l c h a n g es a r e o c c u r r in g w ith a - la c ta lb u m in a s w ith B - la c to g lo b u lin. The m arked d i f f e r e n c e in t h e r a t e o f d e g r a d a tio n o f a - la c ta lb u m in by t h e s o lu b le an d p o ly m e ric p ro n a s e sy stem s may r e s u l t fro m t h e lo n g e r c o n ta c t tim e b etw een s o lu b le enzyme and s u b s t r a t e and t h e m ore e x te n s iv e h e a t t r e a t m e n t. T h e r e f o r e, a g r e a t e r h y d r o ly s i s o f a - la c ta lb u m in w ould b e e x p e c te d w ith t h e s o lu b le p r o n a s e. F i n a l l y, t h e th e r m a l p r o f i l e f o r p o ly m e ric p ro n a s e on t h e s e f o u r m ilk p r o t e i n s i n d i c a t e s t h a t im m o b iliz e d p ro n a s e i s n o t d e n a tu r e d, ev en a f t e r e x p o s u re t o 70 C T a b le 7 g iv e s t h e Q1Q v a lu e s f o r t h e a c t i o n o f p o ly m e ric p ro n a s e on v a r io u s m ilk p r o t e i n s. T h ese r e s u l t s t o g e t h e r v i t h th o s e shown in F ig u re 12 i n d i c a t e t h a t te m p e r a tu r e s from Uo t o 70 C do n o t m ark ed ly in f lu e n c e t h e r e a c t i o n r a t e o f im m o b iliz e d p ro n a s e on w hole c a s e i n, a - c a s e i n, and a - la c ta lb u m in. T a b le 7 ($10 V a lu es f o r P o ly m e ric P ro n a se on F o u r M ilk P r o te i n s T e m p e ra tu re P r o t e i n R an g e, C V alu e a - la c ta lb u m in 140-50 1.1 3 50-60 1.1 2 6 0-7 0 1.1 1 B - la c t o g lo b u lin 50-60 1.06 6 0-7 0 1. 0U Whole c a s e in 30-140 1.19 140-50 1. 1U 50-60 1. 1I4 a - c a s e i n 30-140 1.21 140-50 1.1 7

76 E f f e c t o f ph The p H - a c t i v i ty r e l a t i o n s h i p o f any g iv e n enzyme dep en d s u p o n : ( a ) t h e p k o f t h e io n i z i n g g ro u p s o f t h e a c t i v e s i t e on t h e enzyme t h a t p a r t i c i p a t e i n b in d in g t h e s u b s t r a t e ; (b ) t h e pk o f th e f u n c t io n a l g ro u p s o f t h e s u b s t r a t e m o le c u le t h a t p a r t i c i p a t e i n b in d in g t o t h e enzym e; (c ) t h e pk o f th e f u n c t i o n a l g ro u p s o f t h e enzyme m o le c u le r e s p o n s ib le f o r t h e c a t a l y t i c a c t ; and (d ) t h e pk o f o th e r g ro u p s o f t h e enzyme m o le c u le w hose s t a t e o f i o n i z a t i o n may d e te rm in e t h e s p e c i f i c, c a t a l y t i c a l l y a c t i v e c o n fo rm a tio n o f th e m o le c u le. T h e r e f o r e, e x p e rim e n ts w ere c o n d u c te d t o d e te rm in e th e p H - a c t i v i ty r e l a t i o n s h i p o f im m o b iliz e d p ro n a s e w ith v a r io u s m ilk p r o te in s b etw een ph 6.0 an d 10. P o ly m e ric p ro n a s e was r e a c te d w ith s e le c t e d m ilk p r o t e i n s (0,2 3 3 gram s p r o te in /1 0 0 ml o f 0.0 5 N T r is b u f f e r ) a t 30 C +_ 1 C w ith a flo w r a t e o f 0.7 ^ ra l/m in. F our ml o f t h e d e s ig n a te d p r o t e i n w ith no enzyme tr e a tm e n t was u s e d as th e c o n t r o l. R e s u lts o f th e s e e x p e rim e n ts on d u p l i c a t e p r o t e i n sam p les a r e shown i n F ig u re 13. P o ly m e ric p ro n a s e h ad an optim um ph ra n g e from 8 t o 10 f o r a l l m ilk p r o t e i n s exam in ed. On 0 - la c t o g lo b u l i n t h e im m o b iliz e d enzyme showed maximum a c t i v i t y b etw een ph 8 and 10 w ith a ph optimum a t 8. A c tin g on a - l a c t a l b u m in, p o ly m e ric p ro n a s e d e m o n s tra te d a ph optim um a t 9. The h ig h e s t a c t i v i t y o f im m o b iliz e d p ro n a s e was a t ph 10 f o r i t s a c t i o n on a - c a s e i n w ith a l a r g e d i f f e r e n c e in t h e v e l o c i t y o f th e r e a c t i o n o c c u r r in g b etw een ph 9 and 10. On w hole c a s e in a r a t h e r b ro a d ph o p tim a b etw een 9 and 10 was o b se rv e d f o r th e p o ly m e ric enzym e. The m a g n itu d e o f t h e e f f e c t o f ph on t h e a c t i v i t y a g a i n s t th e d i f f e r e n t

77 F ig u re 13: E f f e c t o f ph on t h e r e a c t i o n v e l o c i t y o f p o ly m e ric p ro n a s e v i t h d i f f e r e n t m ilk p r o t e i n s. (T e m p e ra tu re 30oc +_ 1 C ; 0.0 5 N T r i s b u f f e r ; 0.2 3 3 gram s p r o te in /1 0 0 ml b u f f e r ; flo w r a t e 0.7U m l/m in.; c o n t r o l H m l o f d e s ig n a te d p r o t e i n w ith no enzyme t r e a t m e n t).

78 o- lact albumin casein 5 7 io ph

79 s u b s t r a t e s v a r i e d. U sin g ph 6.0 a s a r e f e r e n c e t h e a c t i v i t y a t th e ph optim um was 7. 6, 7.1 * 3.1 and 2.1 tim e s g r e a t e r a t t h e ph optim um th a n a t ph 6.0 f o r B - l a c t o g l o b u l i n, w hole c a s e i n, a - la c ta lb u m in and a - c a s e i n r e s p e c t i v e l y. T h e se d a ta show t h a t an a l k a l i n e ph ra n g e o f 9.0 t o 1 0.0 g iv e s a good im m o b iliz e d p ro n a s e r e a c t i o n v e l o c i t y f o r a l l m ilk p r o t e i n s ex am in ed. E f f e c t o f C alciu m C h lo rid e F ig u re ill d e p i c t s t h e r e s u l t s o f t h e e f f e c t s o f c a lc iu m c h lo r id e on t h e v e l o c i t y o f im m o b iliz e d p ro n a se m ilk p r o t e i n r e a c t i o n s. Im m o b iliz e d p ro n a s e d e m o n s tra te d t h e g r e a t e s t b i o l o g i c a l a c t i v i t y w ith a - la c ta lb u m in and re m a in e d s t a b l e t o a c a lc iu m c h lo r id e c o n c e n tr a tio n o f 0.5 0 M. At c o n c e n tr a tio n s above t h i s am ount, th e r e was a g ra d u a l d e c r e a s e i n enzyme a c t i v i t y. W ith B - la c to g lo b u lin as t h e s u b s t r a t e, p o ly m e ric p ro n a s e e x h i b i t e d a r e a c t i o n p r o f i l e s i m i l a r t o a - la c ta lb u m in b u t d is p la c e d f a r t h e r t o t h e l e f t. Maximum s t a b i l i t y was a c h ie v e d t o a c a lc iu m c h lo r id e c o n c e n tr a tio n o f 0.1 0 M w ith a d e c r e a s e i n enzyme a c t i v i t y o c c u r r in g as t h e c a lc iu m c h l o r id e c o n c e n tr a tio n in c r e a s e d t o 0.5 0 M. At h ig h e r m o la r c o n c e n tr a tio n s o f c a lc iu m c h lo r id e t h e r e was l i t t l e change i n enzyme v e l o c i t y. W ith a - c a s e i n a s t h e s u b s t r a t e th e im m o b iliz e d p ro n a se r e a c t i o n v e l o c i t y d e c re a s e d r a p i d l y t o a c a lc iu m c h lo r id e c o n c e n tr a tio n o f 0.3 0 M. As t h e c a lc iu m c h l o r id e c o n c e n tr a tio n v a s in c r e a s e d t o 0.9 0 M, h o w e v er, a l i n e a r i n c r e a s e in th e r e a c t i o n v e l o c i t y was o b se rv e d w ith t h e r e a c t i o n v e l o c i t y f o r t h e im m o b iliz e d p ro n a se a t a maximum a t a 0.9 0 M c a lc iu m c h lo r id e c o n c e n tr a tio n. The p o ly m e ric en zy m e's

80 F ig u re lu ; E f f e c t o f c a lc iu m c h lo r id e on t h e v e l o c i t y o f p o ly m e ric p ro n a s e w ith d i f f e r e n t m ilk p r o t e i n s. (T e m p eratu re 30 C +_ 1 C ; ph 7. 1 ; 0.0 5 N T r is b u f f e r ; flo w r a t e 0.8 5 1 m l/m in.; 0.2 1 0 gram s p r o te in /1 0 0 ml b u f f e r ; c o n t r o l ^ ml o f d e s ig n a te d p r o t e i n w ith no enzyme t r e a t m e n t ).

81 30 a- lactalbumin AAbt/min X 10* a-casein casein CaCL Molarity

82 v e l o c i t y v i t h w h o le c a s e in sh o v ed a s i m i l a r r e a c t i o n p r o f i l e t o a - c a s e i n t o a 0.5 0 M c a lc iu m c h l o r id e c o n c e n tr a tio n. H ig h e r c o n c e n tr a t i o n s p ro d u c e d l i t t l e ch an g e i n enzyme c a t a l y s i s. I n sum m ary, t h e s e r e s u l t s show t h a t p o ly m e ric p ro n a s e i s m ore r e a c t i v e a t low c a lc iu m c h l o r id e c o n c e n tr a tio n s v i t h a l l o f th e p r o t e i n s exam ined e x c e p t a - c a s e i n. E f f e c t o f S u b s t r a t e C o n c e n tr a tio n V c C h a r a c t e r i z a t i o n o f p o ly m e ric p ro n a s e b y v a r y in g t h e s u b s t r a t e c o n c e n tr a tio n o f e a c h m ilk p r o t e i n y i e l d s in f o r m a tio n on t h e tim e c o u rs e o f t h e r e a c t i o n, th e maximum r e a c t i o n v e l o c i t y, and t h e su b s t r a t e c o n c e n tr a tio n r e q u ir e d t o y i e l d h a l f t h e maximum v e l o c i t y ( a p p a re n t K ^ ). F u r th e r m a n ip u la tio n o f t h e k i n e t i c d a ta c o u ld i n d i c a t e w h e th e r one o r m ore enzyme r e a c t i o n s a r e o c c u r r in g. T h e r e f o r e, e x p e rim e n ts w ere c o n d u c te d t o d e te rm in e t h e e f f e c t s o f s u b s t r a t e conc e n t r a t i o n on t h e r e a c t i o n v e l o c i t y o f s o lu b le and in s o l u b l e p r o n a s e. A l l a n a ly s e s w e re c o n d u c te d a t ph 7.1 and room te m p e r a tu r e. R e s u l t s, e x p r e s s e d as A A b so rb a n c e /m in u te r e s id e n c e tim e, a r e p r e s e n te d i n F ig u r e s 15 t h r u 20 f o r s t u d i e s v i t h s o lu b le p ro n a s e and in F ig u re s 21 t h r u 25 f o r i n s o l u b l e p r o n a s e. As shown i n F ig u re 1 5, s o lu b le p ro n a se e x h ib ite d s i m i l a r r e a c t i o n p r o f i l e s f o r b o th w hole c a s e in and a - c a s e i n v i t h an a p p a re n t M ic h a e lis c o n s ta n t o f 0.1*2 gram s and v i t h r e s p e c t i v e a p p a re n t maximum v e l o c i t i e s o f 0,1 5 0 and 0.1 5 6 a b s o rb a n c e u n i t s / m i n u t e. The s i m i l a r r e a c t i o n p r o f i l e s w ould b e e x p e c te d s in c e p r e v io u s e l e c t r o p h o r e t i c i n v e s t i g a t i o n s showed l i t t l e q u a l i t a t i v e d i f f e r e n c e b etw een th e tw o p r o t e i n s. D i f f e r e n t r e a c t i o n p r o f i l e s w ere fo u n d f o r t h e a c tio n o f s o lu b le p ro n a s e

83 F ig u re 15- V e lo c ity v s s u b s t r a t e p l o t f o r s o lu b le p ro n a s e r e a c t i n g w ith c a s e in and a - c a s e i n. (ph 7.1 ; 0,0 5 N T r i s b u f f e r ; 30 C + 1 C ; 30 m in u te r e a c t i o n tim e ).

150 a-: casein casein 100 A Abs/min X I0 3 50 Substrate ^/tooml 20

on t h e whey p r o t e i n s, a - la c ta lb u m in an d B - l a c t o g l o b u l i n, and th e s e a r e p r e s e n te d in F ig u re l 6. S o lu b le p ro n a s e gave an a p p a r e n t maximum v e l o c i t y o f 0.1 0 2 a b s o rb a n c e u n its / m i n u t e f o r B - l a c t o g l o b u l i n, w ith a c o rre s p o n d in g a p p a re n t K,^ o f 0.2 2 g ram s. H ow ever, a t s u b s t r a t e conc e n t r a t i o n s above 0.7*+ g ra m s, s u b s t r a t e i n h i b i t i o n was n o te d. On th e o th e r h a n d, s o lu b le p ro n a s e n e v e r a p p ro a c h e d i t s maximum v e l o c i t y w ith a - la c ta lb u m in as s u b s t r a t e, ev en th o u g h a 2.7 5 # p r o t e i n s o lu t i o n v as u s e d. The a p p a re n t v a lu e s o b ta in e d from F ig u r e s 15 and 16 s u g g e s t t h a t t h e s o lu b le p ro n a s e h a s a low a f f i n i t y f o r t h e s e s u b s t r a t e s. When t h e d a ta p r e s e n te d in F ig u r e s 15 and 16 w ere g ra p h e d i n a L in e v e a v e r-b u rk e p l o t, s o lu b le p ro n a s e w ith t h e whey p r o t e i n s a s s u b s t r a t e s gav e a p p a r e n tly l i n e a r r e s u l t s, v i t h c u r v i l i n e a r p l o t s r e s u l t i n g when th e c a s e in s w ere u s e d a s s u b s t r a t e s as d e p ic te d in F ig u re s 17 t h r u 20. The c u r v i l i n e a r p l o t s f o r t h e a c t i o n o f s o lu b le p ro n a s e on t h e c a s e in s s u g g e s t a m u l t i p l i c i t y o f tw o o r m ore e n z y m a tic r e a c t i o n s o c c u r r in g c o n c o m ita n tly, b u t a t d i f f e r e n t r a t e s. From th e r e s u l t s i n F ig u re s 19 and 20 th e a p p a r e n t vmax f o r s o lu b le p ro n a se a c tin g on B - la c to g lo b u lin and a - la c ta lb u m in w ere c a l c u l a t e d t o be 0.1 7 3 9 an d 0.1 2 9 0 a b s o rb a n c e u n its / m i n u t e w ith a p p a re n t M ic h a e lis c o n s ta n ts o f 0.5 1 3 gram s f o r th e fo rm e r r e a c t i o n and 1.1 3 6 gram s f o r t h e l a t t e r r e a c t i o n. H ow ever, c u r v i l i n e a r p l o t s r e s u l t e d f o r s o lu b le p ro n a se a c t i n g on e a c h m ilk p r o t e i n when th e s e d a t a w ere p l o t t e d on an E a d ie -H o fs te e p l o t, w h ich i s u s e d t o m ag n ify any n o n - l i n e a r i t y e x i s t i n g in t h e L in e v e a v e r-b u rk e p l o t. T h ese d a t a i n d i c a t e t h a t e i t h e r s e v e r a l p r o t e o l y t i c enzym es w ere a c t i v e s im u lta n e o u s ly b u t a t d i f f e r e n t r a t e s

86 F ig u re 16: V e lo c ity v s. s u b s t r a t e p l o t f o r s o lu b le p ro n a se on a - la c ta lb u m in and 6 - la c t o g lo b u l i n (ph 7.1 ; 0.0 5 N T r is b u f f e r ; 30 C +_ IOC; 30 m in u te r e a c t i o n tim e ).

100 0- lactoglobulin 8 0 a-lactalbumin o io 60 X e e 4 0 \ <9 < < 20 Substrate gy^ooml

88 F ig u re IT : L in e w e a v e r-b u rk e p l o t f o r th e r e a c t i o n o f s o lu b le p ro n a s e w ith a - c a s e i n.

89 80i 60 4 0 0 1 2 3 4 5 6 7

90 F ig u re 18: c a s e in r e a c t i o n. L in e w e a v er-b u rk e p l o t f o r s o lu b le p ro n a s e v h o le

91 40 30 20

92 F ig u re 29: L in ew eav er-b u rk e p l o t f o r th e r e a c t i o n o f s o lu b le p ro n a s e w ith a - la c ta lb u m in.

60 80-40- 2 a TFT

F ig u re 20: L in e v e a v e r-b u rk e p l o t f o r s o lu b le p ro n a s e B - la c to g lo b u lin r e a c t i o n.

34 24

t o c a u se p r o t e i n d e g r a d a tio n a n d /o r t h a t one enzyme was h y d r o ly z in g a t d i f f e r e n t r a t e s m ore th a n one p e p tid e bond w hich l i n k s d i f f e r e n t am ino a c id r e s id u e s t o g e t h e r. R e s u lts fro m s tu d i e s o f im m o b iliz e d p ro n ab e a c t i n g on m ilk p r o te in s a r e p r e s e n te d in F ig u re 2 1. W ith 0 - l a c t o g l o b u l i n and c t-c a s e in a s s u b s t r a t e s, im m o b ilized p ro n a s e re a c h e d a maximum v e l o c i t y o f 0,80 and 0.2 2 a b s o rb a n c e u n its /m in u te r e s p e c t i v e l y. P o ly m e ric p ro n a s e was a p p ro a c h in g i t s maximum v e l o c i t y w ith w hole c a s e in b u t was s t i l l e x h i b i t i n g f i r s t o r d e r k i n e t i c s w ith a - la c ta lb u m in. F ig u re s 22 t h r u 25 r e p r e s e n t t h e L in ew eav er-b u rk e p l o t s f o r im m o b iliz e d p ro n a se w ith e ach m ilk p r o t e i n u t i l i z i n g d a ta r e p l o t t e d from F ig u re 21. R e s u lts w ith w hole c a s e i n as t h e s u b s t r a t e i n d i c a t e a c u r v i l i n e a r p l o t, s u g g e s tin g t h e o c c u rre n c e o f m ore th a n one e n z y m a tic r e a c t i o n. Howe v e r, a l i n e a r p l o t was o b ta in e d f o r t h e s u b s t r a t e a - c a s e i n w ith c a lc u la te d v a l u e s f o r a p p a re n t vraax and a p p a r e n t o f 0,500 a b s o rb a n c e u n its /m in u te an d 0.1 2 5 g ram s. T h e se v a lu e s com pare f a v o r a b ly w ith a c a l c u l a t e d, a p p a r e n t o f 0.1 0 0 gram and an a p p a re n t maximum v e l o c i t y 0.2 2 a b s o rb a n c e u n its /m in u te from F ig u re 2 1. R e s u lts w ith t h e whey p r o t e i n s u b s t r a t e s a r e shown in F ig u re s 2U and 2 5. Im m o b iliz ed p ro n a s e e x h ib ite d a l i n e a r p l o t w ith a - la c ta lb u m in w ith a c a l c u l a t e d a p p a re n t maximum v e l o c i t y o f 5.0 0 a b s o rb a n c e u n its /m in u te and an a p p a re n t o f 5.0 0 g ram s. A lth o u g h r e s u l t s c a l c u l a t e d from F ig u re 21 showed an a p p a re n t va a x of 0.8 0 a b s o rb a n c e u n its /m in u te and an a p p a re n t M ic h a e lis c o n s ta n t o f 0.26 gram s f o r im m o b iliz e d p ro n a se w ith t h e s u b s t r a t e B - l a c t o g l o b u l i n, th e L in ew eav e r-b u rk e p l o t i n d i c a t e d a c u r v i l i n e a r r e l a t i o n s h i p. When th e k i n e t i c d a ta f o r th e im m o b iliz e d p ro n a s e -m ilk

97 F ig u re 21: V e lo c ity v s. s u b s t r a t e p l o t f o r t h e p o ly m e ric p ro n a s e r e a c t i o n on f o u r m ilk p r o t e i n s a t one m in u te r e s id e n c e tim e. (ph 7. 1 ; te m p e r a tu r e 50 C +_ 1 C ; 0.0 5 N T r i s b u f f e r ; flo w r a t e 2 m l/ m in.; c o n t r o l U ml o f d e s ig n a te d p r o t e i n w ith no enzyme t r e a t m e n t ).

98 750 1acto globulin 6 0 0 casein 450 a-casein 150 0-0 0-5 Substrate g /fo o m l 1 0

99 r e a c tio n. F ig u re 22: Lineweaver-Burke p lo t f o r polym eric p ro n ase c a s e in

S 100

F ig u re 23: a -c a s e in r e a c tio n. Linew eaver-burke p l o t fo r polym eric pronase

CO CNJ in T in

F ig u re 2U: Linew eaver-burke p lo t f o r th e r e a c tio n of polym eric pronase w ith B -la c to g lo b u lin.

10*1 I S

105 F ig u re 25: a -la c ta lb u m in. Linew eaver-burke p lo ts f o r polym eric pronase on

10

p r o te in r e a c tio n s were p lo tte d on an E ad ie-h o fstee p lo t* th e r e s u l t s showed t h a t more th a n one enzym atic r e a c tio n was o c c u rrin g w ith each m ilk p r o te in. A lthough i t i s reco g n ized t h a t th e a b so lu te v a lu e s o b ta in e d by a n a ly s is o f th e k i n e t i c d a ta a re o f l i t t l e v a lu e, th e v alu es o b ta in e d do p ro v id e an in s ig h t in to d iff e r e n c e s in r e a c tio n r a te s w ith th e d i f f e r e n t m ilk p r o te in system s u sed and betw een th e s o lu b le and po ly m eric p ro n ase. The ap p aren t V and K_ t h a t co u ld be approxim ated max fo r th e v a rio u s system s from F ig u res 15, l 6, 19, 21, and 25 a re 107 summarized in T able 8. In com paring th e a c t i v i t y o f so lu b le and in s o lu b le pronase on th e v a rio u s m ilk p r o te in s, th e so lu b le p ro n a s e 's maximum v e l o c i t y, e x c ep tin g th e r e a c tio n w ith a - c a s e in, is alm ost te n tim es le s s th an th e maximum v e lo c ity fo r polym eric p ro n ase. T his in d ic a te s t h a t polym eric pronase was p re s e n t in a g r e a te r q u a n tity in th e enzyme column th a n t h a t used in th e so lu b le pronase s tu d ie s. C om paratively good agreement in ap p a re n t K ^'s were o b tain e d f o r s o lu b le and polym eric p ro n ase re a c tin g w ith c a s e in (0.U2 g. and 0.30 g.) and 8- la c to g lo b u lin (0.2 2 g, and 0.26 g. ). However, s o lu b le pronase gave much h ig h e r I ^ 's f o r a -c a s e in and a -la c ta lb u m in (0. t 2 g, and 1.11 g.) th an d id polym eric pronase "(0.125 g. and 0.5 g. ). F in a lly, th e s e r e s u l t s in d ic a te t h a t th e m ajor m ilk p r o te in s are degraded a t somewhat d i f f e r e n t r a t e s by each enzyme sy stem, w ith a -la c ta lb u m in b e in g th e most d i f f i c u l t p r o te in to degrade.

Table 8 108 Comparison o f K in e tic D ata f o r S o lu b le and In s o lu b le Pronase S u b s tra te A pparent V max (AAbs/min) S olu b le Polym eric P ronase Pronase S oluble Pronase A pparent Km (grams) Polym eric Pronase»* a -la c ta lb u m in.125 > 0.9 1.11 0.5 # B -la c to g lo b u lin.102 0.80.22 0.26 * a -c a s e in.156 0.22. 1*2 0.125 * c a se in.150 0.55.h2 0.3 «Based on V/S p l o t s, b u t n o n -lin e a r on Linew eaver-burke p lo ts. Based on l i n e a r Linew eaver-burke p l o t. S to rag e S t a b i l i t y S ince th e so lv e n t system may a f f e c t th e a c t i v i t y o f th e immobiliz e d enzyme, experim ents were perform ed w ith p o la r s o lv e n ts, nonp o la r s o lv e n ts and d rie d polym eric pronase t o determ ine th e e f f e c t o f so lv e n t on th e a c t i v i t y o f polym eric p ro n ase. A ll so lv e n t system s were s to r e d a t U C f o r th e two week p e rio d ex cep tin g cyclohexane and benzene which were s to re d a t 25 C. Polym eric pronase a c t i v i t y was determ ined u t i l i z i n g 10 ml of c a se in (0.500 gram s/25 ml) in 0.05 N T r is b u f f e r, ph 7.1 a t 30 C. R e su lts from t h i s in v e s tig a tio n are p re se n te d in Table 9. A fte r two weeks o f sto ra g e a l l enzyme p re p a ra tio n s s to r e d under p o la r o r n o n -p o la r so lv e n ts e x h ib ite d g r e a te r th an 90J( o f t h e i r o r ig in a l a c t i v i t y. In c o n tr a s t th e a i r - d r i e d bound

enzyme p re p a ra tio n showed only 20% o f i t s o r ig in a l a c t i v i t y, in d ic a tin g 109 s u b s t a n t i a l in a c tiv a tio n o f th e enzyme. The polym eric enzyme s to re d under w ater showed l i t t l e lo s s in enzymic a c t i v i t y even a f t e r 7 weeks s to ra g e under r e f r i g e r a te d c o n d itio n s. I t can "be concluded from th e s e ex p erim en tal r e s u l t s t h a t benzene, T r is b u f f e r, ph 7.0, and w ater a l l a re s u ita b le s o lv e n ts f o r polym eric p ro n ase. T able 9 E ffe c t o f S olv en t on Enzymatic A c tiv ity S olvent 0 day ab s. ll* day a b s. % o r ig in a l a c t i v i t y W ater 10. 6k 10.60 9 9.6 T r is b u f f e r ph 7.0 11.20 11.12 9 9.3 Benzene 10. 6U 10.56 99.2 M ethanol 11.20 1 1.OU 98.5 Cyclohexane 9.92 9.60 96.7 Frozen 10.21* 9.3 6 91.1+ A ir d rie d 1 0.2k 2.08 20.3 Therm al S t a b i l i t y As shown p re v io u sly in t h i s s tu d y, th e optimum a c t i v i t y o f p ro n ase a g a in st m ilk p r o te in s was in excess o f 50 C. S ince e le v a te d te m p e ra tu re s over p rolonged p e rio d s o f tim e may in a c tiv a te enzyme m olecules by cau sin g c o n fo rm atio n al changes in th e a c tiv e or b in d in g s i t e s, experim ents were conducted a t th re e d i f f e r e n t te m p e ra tu re s to d eterm in e th e th erm a l s t a b i l i t y o f bo th bound and s o lu b le p ro n a se.

C asein ( l g ram /7 6.2 ml "buffer) a t ph 7.1, 0.05 N T ris b u f f e r, 30 C + 1 C was used as th e s u b s tr a te. R e s u lts a re p re s e n te d in T able 10. 110 T able 10 E f f e c t o f Tem perature on th e Thermal S t a b i l i t y of S o lu b le and Polym eric P ronase 30 C 50 C 70 c S oluble In s o lu b le S o lu b le In s o lu b le S o lu b le In so lu b le Hr % O rig in a l A c tiv ity 0 100 100 100 100 100 100 1 105.1 97.8 10U.6 83.6 0 7U.8 2 99.0 88.6 9 0.3 8 h.l 0 51.0 3 99.0 82.14 8 7.8 7 2.6 0 65.3 h 96.9 83.7 69. U 79.7 0 31.5 5 9 9.0 82.0 6 6.8 85.7 0 hk.9 6 90.7 80.8 6 5.3 7 9.2 0 36.7 S olu b le pro n ase h e ld a t 30 C f o r 6 hours l o s t only 10% o f i t s o r ig in a l enzym atic a c t i v i t y w h ile polym eric p ro n ase l o s t ap p ro x im ately 20% o f i t s o r ig i n a l b io lo g ic a l a c t i v i t y. For th e f i r s t th r e e hours a t 50 C th e re was only a 10% lo s s in s o lu b le pro n ase a c t i v i t y, b u t f u r th e r h e a t tre a tm e n t caused an approxim ate 35% lo s s in c a t a l y t i c power. On th e o th e r h an d, in s o lu b iliz e d p ro n ase r e ta in e d 00% o f i t s a c t i v i t y a f t e r 6 hours exposure t o 50 C. When th e te m p e ra tu re was r a is e d to 70 C, so lu b le pron ase l o s t a l l i t s c a t a l y t i c a t x l i t y by th e end o f th e f i r s t h o u r. However, th e im m obilized enzyme r e ta in e d 65% o f i t s

Ill a c t i v i t y a f t e r 3 h o u rs and 3 6.7% a f t e r 6 h o u rs. In c o n c lu sio n, th e s e r e s u l t s show t h a t th e im m obilized enzyme is much more s ta b le a t e le v a te d te m p e ra tu res w ith th e s o lu b le pronase b e in g more s ta b le a t tem p eratu re s around 30 C. For prolonged u s e, te m p e ra tu re s above 50 C ap p ear t o be im p ra c tic a l. E ffe c t o f Flow B ate on A c tiv ity o f Polym eric P ronase O p tim izatio n o f th e o p e ra tin g c o n d itio n s f o r polym eric pron ase in a packed bed r e a c to r re q u ire s knowledge o f th e e f f e c t o f flow r a te on enzym atic a c t i v i t y. Flow r a t e w i l l a f f e c t th e degree of tu rb u le n c e in th e column, th e boundary la y e r a t th e g la s s su p p o rt in te r f a c e and th e d iff u s io n o f th e s u b s tr a te th ro u g h th e f lu i d medium and th e porous g la s s. A s e r ie s o f experim ents were conducted t o determ ine th e e f f e c t of flow r a t e on enzym atic a c t i v i t y. P re lim in a ry in v e s tig a tio n s were conducted w ith packed bed columns o f polym eric pronase u sin g a l l fo u r m ilk p ro te in s u b s tr a te s. S u b stra te was pumped through th e column c o n tin u o u sly a t given flow r a te s a t a tem p e ra tu re o f 30 C, ph 7.1 and an io n ic s tre n g th o f 0.0 1. A nalyses were made a t v a rio u s tim es a f t e r i n i t i a t i o n of flow. I t became ap p a re n t th a t a p e rio d o f tim e was re q u ire d to reach a maximum r a te o f c a t a l y s is a t a g iv en flow r a te and th a t th e tim e re q u ire d t o reach a maximum v e lo c ity v a r ie d as a fu n c tio n o f flow r a t e. R esidence tim e (min) which = f io v r a t ^ T ml/mitit was used 3X1 expr e s s io n of th e c o n ta c t tim e in th e column. T o p ical d a ta a re p re se n te d in F igure 26 fo r 0 -la c to g lo b u lin C0.1i7g/100 ml) a t flow r a te s ran g in g from 0.17 nil t o 1.57 ml/rain and

112 F ig u re 26 : E ffe c t o f flow r a te on th e tim e re q u ire d to come t o a stead y r e a c tio n v e lo c ity f o r th e polym eric pronase B -la c to - g lo b u lin r e a c tio n (0.^7 Srams p ro te in /1 0 0 ml in 0.05 N T ris b u f f e r ; 1; tem p eratu re 50 C +_ 1 C ; c o n tro l fs -lacto g lo b u lin n o t t r e a t e d enzyme). 1. F lov r a te 1.57 m l/m inute R esidence tim e 1.U6 m inutes 2. Flow r a te 0.89 m l/m inute R esidence tim e 2.36 m inutes 3. Flow r a te 0.65 m l/m inute R esidence tim e 3.2 3 m inutes h. Flow r a te 0.57 m l/m inute R esidence tim e 3.68 m inutes 5. Flow r a te 0.31 m l/m inute R esidence tim e 6.77 m inutes 6. Flow r a te 0.17 m l/m inute R esidence tim e 13 m inutes

1 0 113 0*9 0*8 0-7 A Abs 0*4 0 3 0*2 0*1 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time in Minutes

n il co rresp o n d in g re sid e n c e tim es ran g in g from 13 m inutes to l.h6 m in u tes. As in d ic a te d in th e F ig u re, th e tim e re q u ire d t o come to a stead y r e a c tio n v e lo c ity in c re a se d as th e flow r a t e d ecreased w ith th e maximum v e lo c ity achieved a ls o d e c re a sin g as th e flow r a t e d ecrease d. G e n e ra lly, b e t t e r h y d ro ly s is o f a l l s u b s tr a te s was observed a t th e f a s t flow r a te s u t i l i z e d. However, a t e x c e ssiv e ly hig h flow r a t e s, in th e range o f 15 t o 30 ml p er m in u te, th e enzym atic re a c tio n r a t e dropped t o v alu e s below 0.03 absorbancy u n its /m in. The r e la tio n s h ip o f flow r a t e (re sid e n c e tim e) on th e tim e re q u ire d to ach iev e a c o n sta n t v e lo c ity a t a g iv en flow r a t e was determ ined f o r each s u b s tr a te a t th re e d i f f e r e n t c o n c e n tra tio n s and a t l e a s t 5 d i f f e r e n t flow r a t e s. The s t a t i s t i c a l a n a ly s is o f th e l i n e a r re g re s s io n a n a ly s is o f th e d a ta is p re se n te d in Table 11. C o rre la tio n c o e f f ic ie n ts were s ig n if ic a n t in a l l c a se s. The slo p e o f th e lin e a r re g re s s io n lin e in d ic a te d iffe re n c e s in th e e f f e c t of v a r ia tio n o f re sid e n c e tim e on th e tim e re q u ire d t o achieve a stea d y r e a c tio n v e lo c ity a t a c o n sta n t flow r a t e. B -la c to g lo b u lin was a f fe c te d to th e g r e a te s t e x t e n t, follow ed by a -c a s e in and w ith a -la c ta lb u m in and whole c a se in e x h ib it th e l e a s t a f f e c te d. S u b s tra te c o n c e n tra tio n was a f a c to r only in th e case o f B -la c to g lo b u lin. The in te r c e p t v a lu e s cannot be given any s ig n if ic a n c e, sin ce th e r e a c tio n v e lo c ity drops t o 0 a t very h ig h flow r a t e s, The tim e re q u ire d fo r th e a c t i v i t y o f p ro n ase t o reach a c o n sta n t v a lu e a t d i f f e r e n t given flow r a te s is shown in F ig u re 27 f o r B -la c to g lo b u lin and a -la c ta lb u m in. At flow r a te below 0.5 ml/min (30 m l/h r ),

115 Table 11 L in e a r R egression A n aly sis o f R esidence Time and Time R equired to Reach a C o n stan t Enzymatic R eactio n V e lo c ity f o r Polym eric P ronase A cting on M ilk P ro te in s S u b s tra te C o n ce n tratio n g/150 ml C o rre la tio n C o e fic ie n t* L in ea r R egression Line slo p e in te r c e p t t min ) B -la c to g lo b u lin 0.27.96 9.6-3.1* 0.30.9U 3.1 1*.5 0.37.98 ll*.2-6.7 0.1*7.90 8.3-0.5 a -la c ta lb u m in 0.35.91 3.6 3.8 0.1*0.95 3.1-0.37 0.1*7.89 2.1(6 1.6 a -c a s e in 0,19.91* 9.2-1 0.6 0.27.73 6.9-6.5 o.uo.9h 6.9-0.16 c a se in 0.25.83 1.9 5.8 0.30.70 2.1 0.6 0.35.91 3.1 -.28 R e la tio n s h ip betw een re sid e n c e tim e (min) (X) and tim e re q u ire d to re ac h a c o n s ta n t re a c tio n v e lo c ity o f enzyme c a t y l s i s (Y).

F ig u re 27: Time re q u ire d f o r th e a c t i v i t y o f polym eric p ro n ase t o reach a c o n sta n t v alu e a t d i f f e r e n t flow r a te s fo r 8 - la c to g lo b u lin and a -la c ta lb u m in (0.21 grams a -la c ta lb u m in and 0.6*t grams 6 - la c to g lo b u lin p er 100 ml 0.05 N T ris b u f f e r ; ph T.l te m p e ratu re 50 C +_ 1 C; c o n tr o l d e sig n a te d p r o te in not t r e a t e d w ith enzyme).

3.o 25 \ 0-loctoglobulin V \\ i 2 0 ' 15 o (C 5 10 u. \ \ N \ \ A X N.. V v > a-lactolbumin \ M x w» 3 O H*5 d 3 te -3 a 05 \ 4 6 5. «D 01 > y. O 0 J T2 T5 Z? 30" K 3 6 Time in minutes to reach steady state

118 i t to o k more th a n 20 m inutes t o achieve a maximum r e a c tio n v e lo c ity a t a c o n s ta n t flow r a t e. The e f f e c t o f re sid e n c e tim e on th e maximum v e lo c ity a c h ie v a b le a t a g iv en flow r a t e, w ith a c o n sta n t s u b s tr a te c o n c e n tra tio n, is shown in F ig u re 28. The r e s u l t s show t h a t re sid e n c e tim es lo n g er th an s ix m inutes r e s u lte d in l i t t l e change in th e maximum enzym atic v e lo c ity a c h ie v a b le whereas s h o rt re sid e n c e tim e s r e s u lte d in la r g e r maximum v e l o c i t i e s. Long re s id e n c e tim es r e q u ir e very slow flow r a t e s, which in c re a s e th e th ic k n e ss o f th e f lu i d boundary la y e r surro u n d in g th e p a r t i c l e. T h e re fo re, th e maximum r e a c tio n v e lo c ity a tta in a b le becomes d if f u s io n lim itin g. On th e o th e r h an d, s h o rt re sid e n c e tim es re q u ire f a s t e r flow r a te s which in c re a s e tu rb u le n c e, d ecrease th e f lu i d boundary la y e r th ic k n e s s, th ere b y in c re a s in g maximum enzym atic v e lo c ity. A ttem pts were made t o determ ine th e e f f e c t o f r e te n tio n tim e on th e r e a c tio n r a t e. This was p o s s ib le only fo r B -la c to g lo b u lin, w ith th e d a ta a v a ila b le from th e s e ex p erim en ts. R e c ip ric a l p lo ts shown in F ig u re 29 a re based on l i n e a r unw eighted r e g re s s io n a n a ly s is o f d a ta fo r $ -la c to g lo b u lin a t v a rio u s s u b s tr a te c o n c e n tra tio n s as a fu n c tio n o f flow r a t e. As th e r e te n tio n tim e in c re a s e d, th e maximum v e lo c ity d ecreased and K a ls o d e c re a se d, in d ic a tin g th e e f f e c ts o f d iff u s io n in on th e glass-enzym e system. E ffe c t o f Pronase P re tre a tm e n t o f Skimmilk on B io lo g ic a l O x id a tio n : The prem ise in i n i t i a t i n g th e s e in v e s tig a tio n s, which n e c e s s ita te d th e c h a r a c te r iz a tio n o f polym eric pronase a g a in st m ilk p r o te in s, was t h a t p re d e g ra d a tio n o f m ilk p ro te in s would e lim in a te problem s in th e

119 F ig u re 28: E ffe c t o f re s id e n c e tim e on th e maximum v e lo c ity a c h iev ab le a t a g iv en flow r a t e, w ith a c o n sta n t s u b s tr a te c o n c e n tra t i o n, f o r polym eric pronase r e a c tin g w ith fi-la c to g lo b u lin and a -la c ta lb u m in (0.21 grains a -la c ta lb u m in and 0,6*+ grams 3 -la c to g lo b u lin p e r 100 ml 0.05 N T r is b u f f e r ; ph 7.1 ; te m p e ra tu re 50 C +_ 1 C; c o n tr o l d e sig n a te d p r o te in not t r e a te d w ith enzyme).

120 of Reaction lactoglobulin X o a-lactalbumin Residence Time in Minutes

121 F ig u re 29: L in ev eav er-b u rk e p lo t f o r th e p olym eric p ro n ase - B -la c to g lo b u lin r e a c tio n based upon v a lu e s o b ta in e d from l i n e a r u nw eighted r e g r e s s io n a n a ly s is o f d a ta f o r 8 - la c to g lo b u lin a t v a rio u s s u b s t r a te c o n c e n tr a tio n s.

Res. Time 2*.2 \( Res. Time 2 0 2 3 4 5

u t i l i z a t i o n o f miix p r o te in s d u rin g b io lo g ic a l o x id a tio n tre a tm e n t of d a ir y food p la n t w astes and improve th e e f f ic ie n c y o f b io lo g ic a l o x i 123 d a tio n. To t h i s end so lu b le pronase was s e le c te d f o r i n i t i a l tre a tm e n t o f skim m ilk t o be used in a la b o ra to ry a c tiv a te d slu d g e system o f th e extended a e r a tio n ty p e. R e su lts o f t h i s stu d y a re p re se n te d in T able 12. From t h i s T able th e r e appears t o be l i t t l e change in th e b io lo g ic a l oxygen demand, lo a d in g, biom ass c o n c e n tra tio n, food-biom ass r a t i o, o r e f fic ie n c y o f th e b io lo g ic a l oxygen demand rem oval w ith e i t h e r th e pronase t r e a t e d skim m ilk o r th e c o n tr o l sample over th e th r e e day p e rio d. However, th e re i s a s ig n i f i c a n t d e cre ase in th e e f f ic ie n c y o f th e chem ical oxygen demand rem oval from 37-7# fo r day one to 23.6# f o r day two (compared t o U3.6$ f o r th e c o n tro l) w ith no s i g n i f i c a n t change n o tic e a b le in p erc en t c e l l n itro g e n (1.6 6 and 1.60 fo r th e r e s p e c tiv e d a y s). A lso, t o t a l n itro g e n in th e e f f lu e n t in c re a s e d from 0.223$ N/ml fo r day one to 0. 2595? N/ml f o r day two w ith a c o n tr o l v alu e o f 0.000$ N/ml. Since about 5# la c to s e i s p re s e n t in skim m ilk w ith th e 3.2$ p r o te in, i t appears t h a t la c to s e is b ein g u t i l i z e d p r e f e r e n t i a l l y to th e amino a c id s, acco u n tin g f o r th e 50$ b io lo g ic a l oxygen demand e f fic ie n c y. High v o lta g e e le c tr o p h o re s is o f samples c o lle c te d over th e th r e e day p e rio d confirm ed t h a t p e p tid e s and amino ac id s were not b ein g u t i l i z e d by th e biom ass (F ig u re 3 0 ), e x p la in in g th e in c re a se d t o t a l n itro g e n in th e e f f lu e n t w ith no in c re a se in p e rc e n t c e l l n itro g e n.

Table 12 Biomass Performance Data Experimental Conditions mg B.O.D, lo ad in g / day Biomass Conc e n tra tio n (g/1.5 1) Food/Biomass Ratio % E fficien cy C.O.D. Removal % E fficien cy B.O.D. Removal T otal N itrogen in E fflu en t C ell Nitroj % Loading Day C alculated 3000 mg/ 1 6395 1*0.1+0 1.78 37.7 55.1* 0.22335 N/ml 1.66 B B.O.D. Pronase 2 6079 1*2.30 1.62 23.6 1*7.2 0.25935 N/ml 1.60 Treated Skimmilk 3 615** 1*3.1*0 1.69 22.1* 1*7.0 0. 26555 N/ml 1.56 3000 mg/ 3 5796 _ 1*3.6 50,0 0. 080/S N/ml 1.30 L B.0.D. Restored w ith Skimmilk ro tr

125 F ig u re 3 0 t High v o lta g e e le c tr o p h o re s is o f sam ples o b ta in e d from a b io fe rm e n te r given pronase t r e a te d skim m ilk over a th re e day p e rio d. 100 ml sam ples a p p lie d t o p a p e r, e le c tro p h o re s e d a t 2500 V f o r 1.5 hours a t 150 ma in ph 3.^ p y rid in e : a c e tic a c id : w ater m ixture (5 :1 5 :8 5 ). From l e f t t o r ig h t : Sample 1 Skimmilk 3000 B.O.D. Sample 2 N e u tra l and a c id ic amino acid s Sample 3 Mixed liq u o r 3000 B.O.D. skimmilk Sample b Skimmilk 3000 B.O.D. pronase t r e a te d Sample 5 Mixed liq u o r 1 st day Sample 6 C e ll f r e e e f f lu e n t 1 s t day Sample 7 Mixed liq u o r 2nd day Sample 8 C e ll f r e e e f f lu e n t 2nd day Sample 9 Mixed liq u o r 3rd day Sample 10 C e ll F ree e f f lu e n t 3rd day Sample 11 T o ta l e f f lu e n t 2nd day

D ire c tio n o f m ig ratio n N eu tral and a c id ic amino acid s base lin e Samples 1 2 3 1 + 5 6 7 8 9 10 11

DISCUSSION P ronase i s a complex o f enzymes o b ta in e d from Streptom yces g ris e u s K -l t h a t e x h ib its broad s p e c i f i c i t y under a wide range o f environm ental c o n d itio n s. The p re p a ra tio n of in s o lu b le d e r iv a tiv e s o f t h i s enzyme by d iaz o co u p lin g o f pron ase t o arylam ine g la s s p ro duced a s t a b l e, a c tiv e enzym e-glass d e r iv a tiv e. C h a ra c te riz a tio n of t h i s enzym e-glass d e r iv a tiv e by fo u r m ilk p r o te in s p o sse ssin g d if f e r e n t c o n fig u ra tio n s and m o lecu lar w eig h ts was achieved under v ario u s en v iro n m en tal c o n d itio n s o f ph, te m p e ra tu re, io n ic s tr e n g th, and subs t r a t e c o n c e n tra tio n. In s e v e ra l in s ta n c e s, com parisons were made w ith s o lu b le pronase under i d e n t i c a l c o n d itio n s t o a s c e r ta in th e e f f e c ts o f im m o b ilizatio n on th e h y d ro ly s is o f m ilk p r o te in s. The r e s u l t s o f t h i s stu d y showed t h a t in s o lu b iliz e d pronase could be re u sed many tim es w ith o u t lo s s o f i t s. b i o l o g i c a l a c t i v i t y as long as i t was kept m o ist and s to re d under r e f r i g e r a te d c o n d itio n s. Im m obilized pro n ase proved to be le s s cap ab le th an s o lu b le pronase in degrading l a r g e, s o lu b le m ilk p r o te in m o lecu les, b u t more s ta b le to th e rm a l in a c tiv a tio n a t e le v a te d te m p e ra tu res. Q u a n tif ic a tio n of th e a c t i v i t y o f im m obilized pronase was determ ined by d e p le tio n a n a ly s is, u t i l i z i n g benzoyl a rg in in e e th y l e s te r as th e s y n th e tic s u b s t r a te, and was th e r e fo r e n o t n e c e s s a r ily a r e f l e c t i o n o f a c tiv e enzyme b u t r a th e r o f t o t a l enzyme bound. For 10 b in d in g experim ents an average o f 9.6 mg of bound enzyme/g o f g la s s was o b tain ed w hich i s com parable t o r e s u l t s f o r o th e r polym eric enzyme 127

analogues (1^9, 152) bound t o in o rg a n ic su p p o rts. 128 The r a t e o f d eg rad atio n o f v a rio u s m ilk p r o te in s was u t i l i z e d as an index o f enzym atic a c t i v i t y f o r b o th s o lu b le and in s o lu b le p ro n ase. However, in d isc u s s in g th e s e r e s u l t s s e v e ra l f a c to r s need t o be m entioned. F i r s t, an enzyme u s u a lly h y d ro ly zes on ly c e r ta in chem ical b o nds, th e kind o f which are dependent upon th e prim ary amino a c id sequence o f th e p r o te in s u b s t r a te, and th e se w i l l vary w ith d i f f e r e n t p r o te in s. A lso, secondary and t e r t i a r y fo ld in g o f th e prim ary s tr u c tu r e as w e ll as d is u l f i d e and hydrophobic bonding a l l may lim it th e a v a i l a b i l i t y of s u s c e p tib le bonds t o enzym atic a c tio n. T h ird, p r o te in m olecule ag g re g a tio n a lso can impede enzym atic c a ta ly s is by c r e a tin g an u n fav o rab le stere o c h e m ica l r e la tio n s h ip betw een th e subs t r a t e and th e enzym e-support m a te r ia l. These f a c to r s in co n ju n ctio n w ith th e m icroenvironm ental c o n d itio n s around th e s u b s tr a te and enzyme support m a te r ia ls can produce s ig n if ic a n t changes in th e v e lo c ity o f th e enzym atic r e a c tio n w ith o u t cau sin g enzyme d e n a tu r a tio n. Thermal p r o f i l e s a re s im ila r f o r s o lu b le and polym eric pronase r e a c tin g w ith a - and whole c a se in to 60 C. S ince l i t t l e q u a lita tiv e o r q u a n tita tiv e d iff e r e n c e s e x is t c o m p o s itio n a lly, t h i s would be expected because th e c a se in s e x i s t in randomly c o ile d c o n fig u ra tio n s due to th e high hydrophobic c o n te n t and d i s t r i b u t i o n o f a p o la r amino a c id s in th e prim ary s tr u c tu r e. With B -la c to g lo b u lin s im ila r th erm al p r o f i l e s r e s u lte d w ith s o lu b le and polym eric p ro n a se, alth o u g h a g r e a te r in c re a s e in re a c tio n v e lo c ity o ccu rred w ith polym eric pronase

betw een 30 and 50 C. However, s u b s ta n tia lly d i f f e r e n t th erm a l p ro f i l e s e x i s t f o r th e a c tio n o f s o lu b le and polym eric p ro n ase on 129 a -la c ta lb u m in. These d iff e r e n c e s may be a t t r i b u t e d to th e e f f e c t s o f lo n g e r tim e exposure on th e prim ary amino a c id s t r u c t u r e, r e s u ltin g in more random c o i l fo rm atio n w ith subsequent s u s c e p tib le bond form a t i o n f o r s o lu b le p ro n ase. S ince th e h e a t tre a tm e n t was o f s h o rte r d u ra tio n fo r polym eric p ro n a s e, a more g ra d u a l u n fo ld in g o f th e a -la c ta lb u m in m olecule would occur as te m p eratu re in c re a se d. This could e x p la in th e lin e a r in c re a s e in r e a c tio n v e lo c ity w ith in c re a s in g te m p e ra tu re. G e n e ra lly, c o v ale n t b in d in g o f enzymes to su p p o rt m a te ria ls le a d s to a d e c re a se in th erm a l s t a b i l i t y (U l), However, in c re a s e s in th erm al s t a b i l i t y have been re p o rte d f o r enzymes bound to g la s s (1 5 1 ). Immobiliz e d p ro n ase was much more s ta b le th an th e s o lu b le p re p a ra tio n a t te m p era tu res o f 50 C o r h ig h e r ; th e s o lu b le p re p a ra tio n b ein g more s ta b le a t tem p eratu res around 30 C, U su a lly, d ecrease s in th e th erm al s t a b i l i t y o f a bound enzyme r e s u l t from th e i n a b i l i t y t o assume th e th re e -d im e n s io n a l c o n fig u ra t i o n o f th e so lu b le enzyme in s o lu tio n. P ro n ase, how ever, i s not a s in g le enzyme, b u t a m ix tu re o f a t l e a s t 13 enzymes. M oreover, i t is n o t known w ith c e r ta in ty which of th e se enzymes p o sse ss th e c a p a b ility o f d eg rad in g m ilk p r o te in s o r how th e se enzymes are bound to th e g la s s. One e x p la n a tio n, which acco u n ts fo r th e observed r e s u l t s, a t t r i b u t e s th e in c re a s e in th erm al s t a b i l i t y o f im m obilized p ro n ase to m inim al a l t e r a t i o n s in th e c o n fig u ra tio n o f th o se enzymes a s s o c ia te d w ith

130 p r o te in "breakdown. S in ce th e enzymes are a tta c h e d to a s o lid su p p o rt, enzyme a u to ly s is cannot occur as i t does w ith s o lu b le p ro n a se. Likew is e, c o n fig u ra tio n changes in th e enzyme m o le c u le s, b ro u g h t about by in c re a s e s in te m p e ra tu re, may o ccu r more e a s i ly w ith s o lu b le pronase th a n w ith th e su p p o rted m a te r ia l, r e s u ltin g in a d ecrease in enzym atic v e lo c ity as te m p e ra tu re in c r e a s e s. This e x p la n a tio n o f bound enzyme th e rm a l s t a b i l i t y s a t i s f a c t o r i l y e x p la in s th e d a ta o b tain ed in th i s stu d y. Royer (125) has re p o rte d t h a t when sm all m olecules a re used as s u b s t r a te s, a s i m i l a r i t y in th e ph optim a fo r s o lu b le and polym eric p ro n ase occurs (ph 8.0 ) w ith c o n sid e ra b le b roadening o f th e ph optimum tow ard h ig h e r a lk a lin e ph 's n o ted f o r th e in s o lu b le enzyme. With la rg e p r o te in m o le c u le s, th e ph a c t i v i t y p r o f i l e of im m obilized pronase depends upon th e summation o f th e e f f e c ts o f ph on th e p k 's o f th e io n iz a b le groups p re se n t on th e enzyme and s u b s tr a te m olecules and th e a l t e r a t io n s in c o n fig u ra tio n of th e enzyme and s u b s tr a te m olecules b ro u g h t about by th e s e e f f e c t s. R esu lts showed t h a t im m obilized p ro n ase d id e x h ib it g r e a te r r e a c tio n v e l o c i t i e s a t a lk a lin e ph 's fo r th e fo u r m ilk p r o te in s examined w ith ph optim a d if f e r in g f o r th e v a rio u s p r o te in s. These r e s u l t s support th e u t i l i z a t i o n o f im m obilized p ro n ase as a p re tre a tm e n t p ro c ess fo r d a iry w astes sin ce th e a c tio n of im m obilized pronase on m ilk p r o te in s was op tim al a t th e optimum ph found in a c tiv a te d slu d g e. The r e a c tio n p r o f i l e s f o r so lu b le and in s o lu b le p ro n ase in d ic a te d t h a t th e m ilk p r o te in s were hy d ro ly zed a t more th an one p la c e in t h e i r amino a c id chain and a t d i f f e r e n t r a t e s. The high K v a lu e s fo r

b o th enzyme system s on a -la e ta lb u m in in d ic a te d t h a t t h i s p r o te in had th e low est a f f i n i t y o f th e fo u r s u b s tr a te s f o r pro n ase w h ile fi-la c to - 131 g lo b u lin had th e h ig h e s t a f f i n i t y f o r p ro n ase. The much la r g e r V m flix v a lu e s f o r polym eric pronase may in d ic a te th e p re se n c e and a c t i v i t y o f a much la r g e r amount o f enzyme th a n was used in th e so lu b le p ro n a se - s u b s tr a te ex p erim en ts. However, th e 20 d iffe re n c e in tem p eratu re betw een th e s e two experim ents w i l l a f f e c t th e maximum v e lo c ity o f th e enzyme system and account fo r s u b s t a n t i a l p a r t o f th e in c re a se d v e lo c i t y o f polym eric p ro n ase. The la r g e d iscrep an cy in ap p aren t v a lu e s f o r th e s o lu b le and polym eric pronase a -la c ta lb u m in and a -c a s e in r e a c tio n s su g g est t h a t flow r a te may be a f f e c tin g th e d e te r m in atio n. R e su lts o f in v e s tig a tio n s i n t o th e e f f e c ts o f flow r a t e on th e a c t i v i t y of polym eric pronase in d ic a te d : (a) t h a t low flow r a te s d ecreased th e maximum enzym atic v e lo c ity a t t a i n a b l e ; (b) th a t as th e tim e t o come t o a ste a d y r e a c tio n v e lo c ity in c re a s e d, flow r a te d e c re a se d ; (c) t h a t enzym atic r e a c tio n r a te dropped to n e ar zero a t e x c e s siv e ly high flow r a t e s. Low flow ra te s w i l l d e c re a se th e tu rb u le n c e in th e column, th e re b y in c re a sin g th e f l u i d d iffu s io n boundary la y e r a t th e g la s s su p p o rt in te rf a c e and th e d iff u s io n o f s u b s tr a te t o and p ro d u ct from th e enzym e-glass su p p o rt. This r e s u l t s in a d ec re ase in th e maximum enzym atic v e lo c ity a t t a i n a b l e. S ince a c o n c e n tra tio n g ra d ie n t o f s u b s tr a te w i l l be e s ta b lis h e d acro ss th e u n s tir r e d boundary la y e r durin g th e course o f th e enzym atic r e a c tio n, th e tim e to reach a stead y r e a c tio n v e lo c ity w i l l in c re a s e. At e x c e s siv e ly h ig h flow r a t e s, tu rb u le n c e w ill be g r e a tly in c re a se d and

132 th e c o n ta c t tim e betw een s u b s tr a te and enzyme may be to o s h o rt to p e rm it b in d in g t o o ccu r. This r e s u l t s in an alm ost z e ro enzym atic r e a c tio n v e lo c ity. A p p lic a tio n of s o lu b le p ro n ase to p r e t r e a t skim m ilk f o r su b sequent u t i l i z a t i o n in a b io lo g ic a l ferm en ter r e s u lte d in a ^0!6 d e c rea se in C.O.D. v a lu e s over a th re e day p e rio d. F u rth e r in v e s tig a tio n s showed t h a t th e amino a c id s and p e p tid e s were no t b ein g u t i l i z e d by th e biom ass alth o u g h s u f f i c i e n t q u a n titie s of oxygen were b ein g s u p p lie d. S e v eral e x p la n a tio n s f o r t h i s e f f e c t may be su g g e sted. S ince th e c e l l s are no t u t i l i z i n g th e p e p tid e s and amino a c id s as a n itro g e n so u rc e, th e s e components could be com plexing t o th e biom ass and s e t t l i n g o u t. T his would e x p la in why th e r e is no s ig n if ic a n t change in th e biom ass c o n c e n tra tio n over th e th re e day p e rio d. The a c tio n o f e x t r a c e l l u l a r p ro te a se s a lre a d y p re s e n t in th e medium would f u r th e r degrade th e p e p tid e s t o amino a c id s, th e re b y c o n trib u tin g to B.O.D. e f f ic ie n c y. S ince such la rg e q u a n titie s o f degraded p ro te in s were p re se n t in t h i s ex p erim en t, c a ta b o lic feedback r e p re s s io n a lso may be lim itin g tr a n s p o r t o f th e amino a cid s and p e p tid e s in to th e c e l l. In a d d itio n th e high le v e ls o f p e p tid e s p re s e n t in th e f lu i d medium may p o s s ib ly i n h i b i t th e fo rm atio n o f e x t r a c e l l u l a r p ro te a s e s.

SUMMARY I n v e s tig a tio n s were conducted t o c h a r a c te r iz e th e r e a c tio n o f polym eric p ro n ase w ith fo u r m ilk p r o te in s under v a rio u s c o n d itio n s in o rd e r t o o p tim ize c o n d itio n s f o r p o s s ib le u t i l i z a t i o n o f t h i s bound enzyme as a p re tre a tra e n t method fo r d a iry food p la n t w a stes. Pronase i s a complex m ix tu re of enzymes o b tain ed from S tre p to - myces g ris e u s K -l by means of ammonium s u lp h a te p r e c i p i ta ti o n. B inding o f t h i s enzyme system t o porous arylam ine g la s s beads i s achieved th ro u g h th e tr y o s in e re s id u e s not e s s e n t i a l f o r b io lo g ic a l a c t i v i t y. An average o f 9.5 8 rag enzyme bound/mg g la s s was o b tain e d f o r te n b in d in g experim ents u t i l i z i n g d e p le tio n a n a ly s is w ith b e n z o y l-a rg in in e e th y l e s t e r as th e s y n th e tic s u b s tr a te. I n v e s tig a tio n s showed th a t BAEE d e te r io r a te s upon prolonged s to ra g e. P o ss ib le a p p lic a tio n o f im m obilized pronase to d a iry food p la n t w aste tre a tm e n t n e c e s s ita te d th e c h a r a c te r iz a tio n of th e in s o lu b iliz e d p ro n a se. A th e rm a l p r o f i l e o f polym eric pronase showed s im ila r r e s u l t s fo r a - c a s e in, whole c a s e in, and a -la c ta lb u m in from 30 to 50 C. Large changes in th e r e a c tio n v e lo c ity of polym eric pronase on B -lact g lo b u lin were r e l a t e d t o l i t e r a t u r e re p o rts o f d is s o c ia tio n o f th e dimer to th e monomer form, th ere b y f a c i l i t a t i n g i t s h y d ro ly s is. Throughout th e t r i a l s, im m obilized pronase d id not appear to d e n a tu re, even a f t e r exposure t o 70 C, 133

The p H -a c tiv ity r e la tio n s h ip f o r polym eric pronase showed an 13U optimum ph h ig h e r th a n 8 f o r a l l m ilk p r o te in s examined. The magnitu d e o f th e e f f e c t o f ph on th e a c t i v i t y a g a in s t d i f f e r e n t s u b s tr a te s v a rie d in th e fo llo w in g d e c re a sin g o rd e r: B -la c to g lo b u lin, whole c a s e in, a -la c ta lb u m in, a - c a s e in. Polym eric pronase dem onstrated a s im ila r r e a c tio n p a tte r n f o r a - c a s e in and whole c a s e in w ith a d e crea se in a c t i v i t y o c c u rrin g a t a 0.30 M calcium c h lo rid e c o n c e n tra tio n. Changes in calcium c h lo rid e c o n c e n tra tio n on e i t h e r s id e o f t h i s v alu e in c re a se d th e enzym atic v e lo c ity. In s o lu b iliz e d pronase gave th e g r e a te s t b io lo g ic a l a c t i v i t y w ith a - la c ta lb u m in, w ith th e g -la c to g lo b u lin curve s im ila r t o th e a -1 actalb u m in c u rv e, b u t d isp la c e d f a r t h e r t o th e r i g h t. K in e tic s tu d ie s on im m obilized pro n ase w ith fo u r m ilk p r o te in s in d ic a te d th a t pronase was h y d ro ly zin g th e s u b s tr a te s a t more th an one s i t e in th e p o ly p e p tid e c h ain. Comparison o f th e polym eric p ro n ase ap p aren t f o r th e v a rio u s p r o te in s showed t h a t a -la c ta lb u m in was th e most d i f f i c u l t p r o te in to degrade and a -c a s e in was th e e a s ie s t p r o te in d egraded. With two o f th e fo u r p r o te in s, th e d iffe re n c e s in apparent K ^'s f o r th e so lu b le and polym eric pronase were s u b s ta n tia l. Experim ents u n d ertak en w ith polym eric pronase t o show th e e f f e c ts o f d if f u s io n on enzym atic v e lo c ity in d ic a te d t h a t a t low flow r a t e s, th e maximum a tta in a b le enzym atic v e lo c ity was reduced and th e tim e to reach a ste a d y v e lo c ity was in c re a se d. These e f f e c ts were a t t r i b u t e d t o th e fo rm atio n and th ic k n e ss o f a f lu i d boundary la y e r w ith th e th ic k n e s s o f th e la y e r c o n tr o lle d by flow r a t e.

135 A p p lic a tio n o f a s o lu b le pronase tre a tm e n t to skim railk su b seq u en tly used in a b io -fe rm e n te r y ie ld e d d is a p p o in tin g r e s u l t s. A lthough b io lo g ic a l oxygen demand e f f ic ie n c y rem ained r e l a t i v e l y c o n s ta n t over th e th re e day p e rio d, chem ical oxygen demand e f fic ie n c y d e crea se d by I4O3S. R e su lts from high v o lta g e e le c tr o p h o re s is o f samples c o lle c te d over th e th re e day p e rio d showed t h a t p e p tid e s and amino acid s were n o t u t i l i z e d by th e b io m ass, b u t rem ained in th e e f f lu e n t. t

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