Imm obilization of Pen ic illin G Acylase on Calc ined Layered D ouble Hydrox ides 3

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1 CH EM. R ES. CH IN ESE U. 2003, 19 (3), Imm obilization of Pen ic illin G Acylase on Calc ined Layered D ouble Hydrox ides 3 R EN L ing2ling, H E J ing, Evan s D. G. and DUAN Xue 3 3 (K ey L abora tory of S cience & T echnology of Con trollable Chem ica l R eactions, M in istry of E d uca tion, B eij ing U n iversity of Chem ica l T echnology, B eij ing , P. R. Ch ina) R eceived M ay 26, 2002 A hydro talcite2like M g 2+ ga l 3+ layered double hydroxide (LDH ) m aterial w as p repared by m eans of a modified cop recip itation m ethod invo lving a rap id m ixing step fo llow ed by a separate aging p rocess. LDH cal2 cined at 500, deno ted as CLDH, w as characterized by XRD, IR and BET surface area m easurem ents. CLDH has a poo r crystalline M go 2like structure w ith a h igh surface area and po ro sity. CLDH w as used as a suppo rt fo r the immobilization of penicillin G acylase (PGA ). T he effect of varying the immobilization condi2 tions, such as ph, contact tim e and the ratio of enzym e to suppo rt, on the activity of the immobilized enzym e in the hydro lysis of penicillin G has been studied. It w as found that the activity of the immobilized enzym e de2 creased sligh tly w ith decreasing ph and reached a m axim um after a contact tim e of 24 h. T he activity of the immobilized enzym e increased w ith increasing the ratio of enzym e to suppo rṫ It w as found that the adso rp2 tion of PGA inh ibited the expected reaction of CLDH w ith an aqueous m edium to regenerate a LDH phase. Its o riginal activity (36% ) after 15 cycles of reuse of the immobilized enzym e w as retained, but no further lo ss in the activity w as observed. Ke yw o rds Immobilized enzym e, Penicillin G acylase, L ayered double hydroxide, Calcination A rtic le ID (2003) In troduction Β2L actam an tib io tics can be syn thesized by ei2 ther chem ical o r enzym atic hydro lysis. Enzym atic hydro lysis has becom e a m ajo r alternative to chem i2 cal hydro lysis becau se of the imp rovem en ts in en2 zym e imm ob iliza t ion techn iques and the cho ice of suppo rts. Pen icillin G acylase (E. C ) (ab2 b reviated as PGA ) catalyzes the deacylation of p en icillin G to affo rd 62am inop en icillan ic acid ( 62 A PA ), w h ich is an impo rtan t in term ediate in the m anufactu re of sem isyn thet ic p en icillin s such a s amoxycillin and amp icillin [1, 2 ]. PGA has also been emp loyed in indu strial p rocesses as a catalyst in the sub sequen t step in the syn thesis of these sem isyn2 thetic Β2lactam an tib io tics, w h ich is the N 2acyla2 tion of 62A PA o r o ther an tib io tic p recu rso rs w ith carboxylic acid derivatives [3 ]. T he free enzym e po s2 sesses a lim ited therm a l stab ility and a very low to lerance to o rgan ic so lven ts [4 ]. T h is has stim u lat2 ed fu rther the researches in to the immob ilization of PGA. T he enzym e can be immob ilized on differen t suppo rts, such as po lym er beads, modified po ly2 acrylic fibers and po rou s glass [5, 6 ]. T hough the cu r2 ren t enzym atic p rocess fo r the p roduction of 62A PA is econom ically viab le, there is m uch in terest in the immob ilization of PGA on new m aterials in o rder to imp rove the catalytic efficiency and reduce the h igh co st of the suppo rts cu rren tly u sed. L ayered doub le hydrox ides (LDH ) are a class of syn thetic an ion ic clays w ho se structu res can be described as tho se con tain ing b rucitem g (OH ) 22like layers in w h ich som e of the divalen t cation s are re2 p laced by trivalen t ion s, giving po sitively2charged sheets [7 9 ]. T hese charges are balanced by the in2 terca la t ion of an ion s in the hyd ra ted in terlayers. T hey can thu s be con sidered as the comp lem en tary to a lum ino silica te clayṡ LDH can be rep resen ted by the general fo rm u la [M g 1- xm g x (OH ) 2 ] x + (A n- ) x gn yh 2O. T he iden tities of the di2 and tri2valen t cation s (M g and M g respectively) and the in terlayer an ion (A n- ) together w ith the value of the sto ich io2 m etric coefficien t ( x ) m ay be varied over a w ide range, giving rise to a large class of iso structu ral m aterial. T he m atrix of th is class is the natu ral m ineral hydro talcite w h ich has a fo rm u la M g6a l2 (OH ) 16CO 3 4H 2O and LDH is con sequen t2 ly also know n as hydro talcites o r, mo re accu rately, 3 Suppo rted by the N ational N atural Science Foundation of Ch ina (N o ). 3 3 To w hom co rrespondence should be addressed.

2 N o. 3 R EN L ing2ling et al. 325 hydro talcite2like m aterials. T he p roducts of the therm al decompo sition of LDH at are of con siderab le in terest in their ow n righ t [10, 11 ]. T hese poo r crystalline calcined layered doub le hy2 drox ides (CLDH ) w h ich have also been described as sp inel2like m ixed ox ides o r, som ew hat m isleading2 ly, as layered doub le ox ides regenerate LDH by re2 hydration. T he calcination and rehydration p ro2 cesses have been studied in detail by tim e2reso lved in situ X2ray diffraction [12 ], 27 A lm A S NM R [13 ] and M g and A l K2edge XA FS [14 ] m ethods. CLDH have h igh ly po rou s structu res, large specific su rface areas and abundan t basic sites and have been u sed as catalysts and catalyst suppo rts fo r a variety of reaction s including aldo l conden sation s [15 ] and o lefin isom erization [16 ]. Since CLDH are p roduced by calcination of LDH, it is likely that the mo rpho2 logical p roperties of the LDH p recu rso r can influ2 ence the mo rpho logical p roperties of CLDH and thu s affect its ab ility to act as a suppo rt fo r PGA. LDH m aterials are u sually syn thesized by co2 p recip itation in one of the tw o w ays [17 ]. O ne w ay (A ) is that a m ix tu re of m etal salts is slow ly added to a so lu tion of sodium hydrox ide and sodium car2 bonate w ith a certain ratio, w h ich m akes the ph value variab le. T he o ther w ay (B ) is carried ou t by sim u ltaneou s dropw ise addition of m ixed salts and base so lu tion s to a reaction vessel at such a rate that the ph rem ain s con stan ṫ O nce addition is comp leted in either case, the resu lted su spen sion is then aged at a given temperatu re. Since the addi2 tion p rocess takes a certain period of tim e in each ca se, there is a m uch longer t im e fo r the nuclei fo rm ed at the beginn ing of the addition p rocess to undergo crystal grow th than tho se fo rm ed at the end of the addition p rocess. A fter aging a w ide dis2 persion of crystallite sizes m ay be ob tained. W e [18 ] have developed a new p rocess fo r the syn thesis of ca rbona te2p illa red LDH w ith a na rrow range of particle sizes. T h is p rocess invo lves a very rap id m ix ing and nucleation p rocess, fo llow ed by a sepa2 rate aging p rocesṡ T he m ix inggnucleation p rocess is carried ou t in a co llo id m ill [19 ] w h ich con sists of a ro to r at a speed of 3000 rgm in and a stato r. T he so lu tion s are added to the co llo id m ill th rough a specially designed feed device and are m ixed under the cond it ion s of h igh sp eed flu id sheer and h igh p ressu re and friction betw een the stato r and the ro2 to r w ith the m ix tu re being also sub jected to in ten2 sive vib ration. In th is w ay, the m ix ing and the nu2 cleation of a large vo lum e of a reactan t so lu tion m ay be comp leted in a sho rt tim e. T he resu lted m ix tu re of the crysta llite nuclei w ith a na rrow range of particle size is then aged in a separate p ro2 cedu re, resu lting in w ell2fo rm ed crystallites w ith a sim ilarly narrow range of diam eters. T h is p rocess has been successfu lly scaled up to the p ilo t p lan t scale, allow ing p roduction of the suppo rt p recu rso r w ith very econom ical starting m aterials. W e have been therefo re in terested in exp lo ring the po ten tial ab ility of CLDH to act as suppo rts fo r immob iliza2 tion of enzym es such as PGA [20 ] and found that the immob ilization of PGA on CLDH p roduced by the calcination of a carbonate2ldh p recu rso r w ith a M gga l mo lar ratio of 2 at 500 affo rded an im 2 mob ilized enzym e w h ich exh ib ited a m ax im um en2 zym e activity. In th is w o rk an attemp t w as m ade to op tim ize the activity and the operational stab ility of PGA imm ob ilized on th is suppo rt by va rying the immob ilization condition s. Exper im en ta l 1 M a ter ia ls Pen icillin G po ta ssium sa lt and p en icillin G acyla se w ere supp lied by the H uabei M ed icine Group. 2 M ethod T he M gga l2ldh con tain ing carbonate as the in terlayer an ion w a s syn thesized by m ean s of a modified cop recip itation m ethod as described else2 w here [18 ]. T yp ically a so lu tion con tain ing M g (NO 3) 2 6H 2O ( 6116 g, 0124 mo l ) and A l(no 3) 3 9H 2O (4512 g, 0112 mo l) in 150 ml of distilled w ater and a so lu tion con tain ing N aoh ( 2312 g, 0158 mo l) and N a2co 3 ( 2514 g, 0124 mo l) in 150 ml of distilled w ater w ere rap idly (over a period of 2 m in) added to a co llo id m ill [19 ]. T he resu ltan t gel w a s then aged fo r 5 h under reflux w ith stirring. T he w h ite so lid w as sub sequen tly iso lated by filtration under suction, w ashed w ith distilled w ater un til the ph of the w ash ings w as be2 low 8 and then dried at 70 fo r 24 h. E lem en tal analysis: M g 2017%, A l 1016%. CLDH w as sub2 sequen tly p roduced by calcination of LDH at 500 fo r 3 h. A pho sphate buffer so lu tion of ph 5187 w as added to 013 g of CLDH under N 2 p ro tection un til a specified ph value w as ob tained. PGA of 3 ml (133 IU gml ) w as added after 3 m in (1 IU co rresponds to the amoun t of enzym e w h ich cleaves 1 Λmo l of pen icillin G to 62A PA per m inu te at ph 716 and 37

3 3 26 CH EM. R ES. CH IN ESE U. V o l. 19 ). T he su spen sion w as shaken fo r a certain tim e at 30. T he immob ilized PGA w as recovered by cen trifugation, w ashed and dried at am b ien t tem 2 peratu re. A p ink so lid w as ob tained. T he activities exp ressed in the hydro lysis of a pen icillin G so lu tion (4%, m ass fraction) in mo lgl pho sphate buffer of ph 719 w ere determ ined in a stirred reacto r w ith au tom atic ph co rrection at 37 over a period of 12 m in [5 ]. T he X2R ay pow der diffraction (XRD ) data w ere co llected on a Siem en s D 5005 pow der diffrac2 tom eter w ith Cu K Α radiation (Κ= nm ) by scann ing betw een 3 and 70 at a step of 0102 gṡ T he n itrogen adso rp tiongdeso rp tion iso therm s at liqu id n itrogen temperau re w ere ob tained on a Car2 lo E rba So rp tom atic 1990 in strum en ṫ Fo r su rface area m easu rem en ts and po re size distribu tion m ea2 su rem en ts CLDH w as evacuated at 300 fo r 4 h and the immob ilized enzym e w as evacuated at room temperatu re fo r 6 h. T he BET su rface area w as de2 term ined from the low p ressu re reg ion of the ad2 so rp tion iso therm. M esopo re vo lum es and po re size distribu tion s w ere ob tained from the BJH model. Results and D iscuss ion 1 Character iza tion 1. 1 X RD D if f raction M easu rem en ts A s show n in F ig11a, the LDH p recu rso r has the characteristic X2ray pow der diffraction (XRD ) pattern of a hydro talcite2like m aterial sim ilar to ( 003) and ( 110) reflection ṡ T he fo rm er co rre2 spond s to th ree t im es the layer2to 2layer d istance and the latter co rresponds to the average m etal ion2 m etal ion distance w ith in the layer. Since A l 3+ ion s are sm aller than M g 2+ ion s, the latter value is sen2 sitive to the M g 2+ ga l 3+ mo lar ratio. A value of a= 0130 nm co rresponds to a M g 2+ ga l 3+ mo lar ratio of abou t 2, sim ilar to the ratio determ ined by elem en2 tal analysiṡ A fter calcination at 500, the lay2 ered structu re has been lo st and the XRD pattern (F ig11b) is sim ilar to that of poo r crystalline m ag2 nesium ox ide as repo rted elsew here [12, 13 ]. If CLDH is reacted in air w ith an aqueou s so lu tion of pho s2 phate buffer at 37 fo r 40 h, a poo r crystalline LDH m aterial can be regenerated as show n by the XRD pattern in F ig11c. T h is phenom enon has been w idely docum en ted [12 ]. T he basal spacing of the re2 generated LDH is almo st iden tical to that of the LDH carbonate p recu rso ṙ If pho sphate ion s have been in tercalated in the in terlayer, a sign ifican t in2 crease of the basal spacing w ou ld be expected [22 ]. A s show n in F ig12, the IR spectra of the m a2 terial show s the p resence of carbonate ion s (bands at 1365 and 780 cm - 1 ) bu t the ab sence of any bands can be assigned to pho sphate ion s [22 ]. A lthough, in p rincip le, a pho sphate2ion in tercalated LDH phase is expected, in fact, these data confirm that a car2 bonate ion in tercalated phase m igh t have been p ro2 duced by the competitive reaction w ith atmo spheric carbon diox ide. It is w ell know n that LDH m ateri2 als have a very h igh selectivity fo r carbonate ion s in the p resence of o ther ion s un less the regeneration is carried ou t under a n itrogen atmo sphere, carbonate phases often fo rm [12 ]. In terestingly w hen CLDH is reacted w ith an aqueou s pho sphate buffer so lu tion con ta in ing PGA under the sam e cond it ion s, the LDH st ructu re is no t regenera ted. A s show n in F ig11 X-ray powder diffraction pattern s of layered LD H (a ), LD H af ter calc ination (b), poor crystall ine LD H regenerated in the presence of phosphate buffer (c) and LD H immobilized PGA (d). that repo rted in the literatu re [12 ]. A series of strong (00l) reflection s at the low er angle and a series of w eaker reflection s at the h igher angle are the char2 acteristic of a layered m aterial. O n the basis of as2 sum ing a 3R stack ing of the layers [21 ], the lattice param eters of the un it cell (c = 2130 nm and a = 0130 nm ) can be calcu lated from the d2values of the F ig12 IR spectra of LD H af ter calc ination (a) and LD H immobilized PGA (b). F ig11d, the XRD p a t tern is sim ila r to tha t of

4 N o. 3 R EN L ing2ling et al. 327 CLDH it self, suggest ing tha t the ad so rp t ion of PGA on the su rface of CLDH p reven ts the regener2 ation of LDH. Second, even po ssessing mo re poo r crystalline, the phase is also p resen t, the iden tity of w h ich is no t clear S u rf ace A rea and P orosity M easu rem en ts T he calcination of LDH is know n to resu lt in a la rge increa se in su rface a rea a ssocia ted w ith the fo rm ation of a h igh ly po rou s structu re as the steam and carbon diox ide are expelled in a som ew hat ex2 p lo sive fash ion [16 ]. T he resu lts of BET fo r CLDH and the immob ilized enzym e are show n in T ab le 1. T he specific su rface area of CLDH w as found to be 206 m 2 g, and the specific po re vo lum e w as 016 cm 3 g, sim ilar to the values repo rted in the litera2 tu re [16 ]. A fter PGA, w ho se size is abou t 2 4 nm, w a s imm ob ilized on CLDH, the sp ecific su rface area w as decreased, by 83%, to 37 m 2 g, and the specific po re vo lum e to 013 cm 3 g. T he po re size distribu tion s fo r CLDH and CLDH 2immob ilized PGA are show n in F ig13 (a and b) respectively. A l2 though the po re size distribu tion of CLDH 2immob i2 lized PGA is w ider than that of CLDH, the value is no t changed apparen tly ( as show n in T ab le 1). T hese resu lts indicate that the enzym e has already been immob ilized on the su rface of the po res, thu s leading to a sign ifican t decrease in the specific su r2 face area and the po re vo lum e of the suppo rṫ Table 1 Results of BET for support(cld H) and immobilized enzyme ( IM E) Samp les Specific surface areasg(m 2 g - 1 ) Specific surface vo lum eg(m 3 g - 1 ) M ean po re diam etergnm CLDH IM E Activ ity of Imm obilized Enzym e 2. 1 B lank E xp erim en ts T he activity of the enzym e fo r the hydro lysis reaction w as determ ined by au tom atic titration (w ith aqueou s sodium hydrox ide ) of the phenyl2 acetic acid (PAA ) p roduced along w ith 62A PA in the hydro lysis of pen icillin G. B lank experim en ts, w h ich w ere carried ou t to determ ine the accu racy amoun t of the alkali con sum ed in the assay, w ere perfo rm ed w ith sub strate (Pen icillin G) and immo2 b ilized enzym e resp ect ively. It w a s found tha t in each case a negligib le quan tity of sodium hydrox ide w as con sum ed to m ain tain con stan t ph, confirm ing that the alkali con sum ed in the assay is an accu rate amoun t w h ich can be u sed to determ ine the amoun t of phenylacetic acid p roduced in the hydro lysis re2 act ion Ef f ect of Im m obiliz a tion T im e on the A ctiv ity of IM E T he effect of immob ilization tim e on the activi2 ty of the immob ilized enzym e ( IM E) w as investigat2 ed by adding a mo lgl pho sphate buffer of ph 5187 to CLDH to g ive an in it ia l ph of 618, fo l2 low ed by add it ion of the enzym e ( 1330 IU of en2 zym e per g of suppo rt). A s show n in T ab le 2, the amoun t of enzym e adso rbed increased w ith increas2 ing immob ilization tim e, bu t the exp ressed activity of immob ilized PGA reached a m ax im um after 24 h and then decreased. T hese resu lts suggest that ei2 ther the po res are b locked by excess enzym e due to F ig. 3 Pore size distr ibution of CLD H (a) and the immob ilization fo r a longer tim e, inh ib iting the CLD H- immobilized PGA (b). access of the sub strate to the enzym e o r the enzym e w as denatu red due to p ro longed con tact w ith the Table 2 Effect of immobilization time on the activ ity of the immobilized PGA Immobilization tim egh F ree enzym e loadedg Enzym e adso rbed a g (% ) 1st cycle IM E assay 2nd cycle A ctivityg Exp ressed b (% ) A ctivityg Exp ressed b (% ) a. D eterm ined by assay of the residual activity of the immobilization so lution; b. IM Eg ]g[enzym e adso rbedg ] 100%. based on the activity of the bound enzym e: [A ctivity of

5 3 28 CH EM. R ES. CH IN ESE U. V o l. 19 suppo rṫ A fter immob ilization fo r a longer tim e, how ever, IM E show ed a sm aller decrease in activi2 ty w hen sub sequen tly filtered, w ashed and reu sed in a second react ion, suggest ing tha t the enzym e w as mo re strongly bound to the suppo rt after a longer con tact tim e, thu s enhancing its operational stab ility Ef f ect of ph V a lue of the Im m obiliz a tion suppo rt). T he immob ilization m ix tu re w as sub se2 quen tly shaken at 30 fo r 40 h. A s show n in Sec2 t ion 2. 2, the exp ressed act ivity reaches a m ax i2 m um after 24 h bu t the op era t iona l stab ility is h igher after 48 h. In these experim en ts a comp ro2 m ise tim e of 40 h w as cho sen fo r the immob iliza2 tion. T he data listed in T ab le 3 show that no en2 zym e w as immob ilized w hen the amoun t of pho s2 phate buffer so lu tion w as sm all, w h ich m ay be be2 cau se the final ph of the so lu tion w as too h igh (1019). How ever, w hen the amoun t of the pho s2 phate buffer so lu tion w as large enough to give a low er ph value, the suppo rt show ed a strong affin2 ity fo r the enzym e, w h ich decrea sed on ly very sligh t ly w ith increa sing the am oun t of the buffer so lu tion. T he exp ressed activity w as negligib ly varied w ith the in itial ph of the immob ilization so2 lu tion. Table 3 Effect of ph value on the activ ity of the immobilized PGA M ix tu re on the A ctiv ity of the Im m obiliz ed E nzym e T he activity of PGA is related to the ph val2 ue, w h ich is h igher w hen ph value is betw een 7 and 8, o therw ise the activity w ill decrease. T he ef2 fect of the ph value of the immob ilization m ix tu re on the activity of the immob ilized enzym e ( IM E ) w as investigated by adding differen t vo lum es of a mo lgl pho sphate buffer so lu tion of ph 5187 (see T ab le 3) to 013 g of CLDH fo llow ed by addi2 tion of the enzym e (1330 IU of enzym e per gram of V o lum e ph of supernatant F ree enzym e Enzym e adso rbed a IM E assay of bufferg ml Befo re addition of enzym e A fter immobilization of enzym e fo r 40 h loadedg A ctivityg A dso rbed (% ) A ctivity N one N one Exp ressed b a and b are the sam e as tho se in T able Ef f ect of A m oun t of PGA L oad ed on S upp ort m ix tu re increa sed, the fract ion of the ad so rbed T he effect of the amoun t of PGA added to the PGA decreased as the percen tage exp ressed activity suppo rt w a s stud ied by add ing a m o lgl did. T h is suggests that the addition of excess en2 pho sphate buffer of ph 5187 to CLDH to give an zym e leads to the b lock ing of the po res so that the in itial ph of 618, fo llow ed by addition of the en2 diffu sion lim itation p reven ts the sub strate from zym e. T he m ix tu re w as shaken at 30 fo r 40 h. reach ing the p a rt of the imm ob ilized enzym e. A A s show n in T ab le 4, sm all amoun ts of PGA can be almo st comp letely adso rbed by the suppo rt. A s the amoun t of PGA added to the immob ilization loading of 1330 IU g gives a reasonab le balance be2 tw een the act ivity of the imm ob ilized enzym e and the efficien t u tilization of the enzym e. Table 4 Effect of amoun t of PGA loaded on the support on the activ ity of IM E V (PGA ) adso rbed a gml F ree enzym e loadedg Enzym e IM E assay A ctivityg A dso rbed a (% ) A ctivityg Exp ressed b (% ) a and b are the sam e as tho se in T able 2. 3 Opera tiona l Stability T he operational stab ility in the repeated reac2 tion s of the immob ilized enzym e w as determ ined by m ean s of reaction s carried ou t in a discon tinuou s batch reacto ṙ T he immob ilized enzym e w as p re2 pared by adding a mo lgl pho sphate buffer (% ) so lu tion of ph 5187 to CLDH to give an in itial ph of 618, fo llow ed by addition of the enzym e. T he m ix tu re w as then shaken at 30 fo r 40 h. A s show n in F ig14, after 15 cycles the imm ob ilized enzym e disp layed 36% activity reten tion. Betw een 15 and 20 cycles there w as no fu rther lo ss in the

6 N o. 3 R EN L ing2ling et al. 329 act ivity, w ith the exp ressed act ivity rem a in ing h igher than 100 IU g. T h is suggest s tha t PGA w as m u lti2layered adso rbed on CLDH, w h ile the enzym e on ou ter layer is on ly w eak ly adso rbed on CLDH and readily lo st after a few reaction cycles. How ever, the rem a ined in ter fract ion of the en2 zym e on the suppo rt is strongly adso rbed and is no t easily lo st from the suppo rt, even after 20 cycles of reu se. F ig14 Operational stability of CLD H- immobilized enzym e. Conclus ion s Calcined layered doub le hydrox ides are effec2 tive suppo rts fo r pen icillin G acylase. T he adso rp2 tion of PGA inh ib its the expected reaction of the CLDH phase w ith an aqueou s m edium to regener2 ate an LDH m aterial. T he cho ice of op tim um im 2 mob ilization condition s invo lves a comp rom ise be2 tw een m ax im izing the act ivity of the imm ob ilized enzym e and the efficiency of the enzym e u t iliza2 tion. T he op tim um immob ilization condition s w ere found as fo llow s: in itial ph 618; immob ilization tim e 40 h; enzym e loading 1330 IU of enzym e per g of suppo rt. A fter 15 cycles the immob ilized enzym e show ed 36% activity reten tion. Betw een 15 and 20 cycles there w a s no fu rther lo ss in the act ivity, w ith the exp ressed activity rem ain ing h igher than 100 IU g. Re fe re nce s [ 1 ] B ruggink A., Roo s E. C., D e V room E., O rg. P roc. R eṡ D ev., 1998, (2), 128 [ 2 ] Shew ale J. G., Sudhakaran V. K., E nzym e M icrob. T ech2 nol., 1997, 20, 402 [ 3 ] Cao L., van Rantw ijk F., Sheldon R. A., O rg. L etṫ, 2000, 2, 1361 [ 4 ] Ro sell C. M., Fernandez2L afuente R., Guisan J. M., B io2 catal. B iotranṡ, 1995, (12), 67 [ 5 ] B ianch i D., Go lini R., Bo rto lo R., et al., E nzym e M icrob. T echnol., 1996, 18, 592 [ 6 ] J iang W. P., L i H. Z., Sun W. R., Ch in. J. B iotechnol., 1985, (1), 48 [ 7 ] Cavani F., T rifiro F., V accari A., Catal. T od ay, 1991, (11), 173 [ 8 ] V accari A., Catal. T od ay, 1998, 41, 53 [ 9 ] V accari A., A pp. C lay S ci., 1999, 14, 161 [ 10 ] Reich le W. T., Kang S. Y., Everhardt D. S., J. Catal., 1986, 101, 352 [ 11 ] H ibino T., Yam ash ita Y., Ko suge K., et al., C lay s C lay M iner., 1995, 43, 427 [ 12 ] M illange F., W alton R. İ, O H are D., J. M ater. Chem., 2000, 10, 1713 [ 13 ] Rocha J., del A rco M., R ives V., et al., J. M ater. Chem., 1999, 9, 2499 [ 14 ] van Bokhoven J. A., Roelofs J. C. A. A., de Jong K. P., Chem. E ur. J., 2001, 7, 1258 [ 15 ] Rao K. K., Gravelle M., V alente J. S., et al., J. Catal., 1998, 173, 115 [ 16 ] Reich le W., J. Catal., 1985, 94, 547 [ 17 ] Yun S. K., P innavaia T. J., Chem. M ater., 1995, 7, 348 [ 18 ] D uan X., L i L., J iao Q. Z., Ch inese P atent, CN , 1999 [ 19 ] Perry R. H., Green D. W., M aloney J. O., Eds., P erry s Chem ical E ng ineers H and book, 6th Edn., M cgraw H ill, N ew Yo rk, 1984 [ 20 ] Ren L. L., H e J., Evans D. G., J. M ol. Catal. B, E nzy 2 m atic, 2001, 16, 65 [ 21 ] Book in A. S., D rits A., C lay s C lay M iner., 1993, 41, 551 [ 22 ] L egrouri A., Badreddine M., Barroug A., et al., J. M ater. S ci. L etṫ, 1999, 18, 1077