Optimization of Protease Enzyme Production Using Bacillus Sp. Isolated from Different Wastes

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1 Botny Rsrh Intrntionl 2 (2): 83-87, 2009 ISSN IDOSI Pulitions, 2009 Optimiztion of Prots Enzym Proution Using Billus Sp. Isolt from Diffrnt Wsts 1 1 Uni Boominhn, Rjnrn Rjkumr, 2 2 Plnivl Krpg Vinyg Sivkumr n Mnohrn Mlvin Jo 1 Dprtmnt of Miroiology, Fulty of Sin, Annmli Univrsity, Ini 2 Dprtmnt of Miroiology, Fulty of Agriultur, Annmli Univrsity, Ini Astrt: Th tril isolts inluing Billus sutilis, Billus myloliqufins, Billus mgtrium n Billus lihniformis isolt from iffrnt wsts wr srn for prots nzym proution. Among th iffrnt sours gluos ws foun to st ron sour for ll th four Billus isolts. Yst xtrt ws foun to th optimum nitrogn sour for prots nzym proution y ll th tst isolts. A tmprtur of 50 C ws foun to optimum for ll th isolts for nzym proution. Soy k foun to th st sustrt for nzym proution mong iffrnt gro s wsts for ll th four tst isolts grown unr sumrg frmnttion onitions. Ky wors: Billus sp Prots Pross Optimiztion INTRODUCTION MATERIALS AND METHODS Commril protss ount for nrly 60% of th Cultur n Growth Conition: Th tril totl inustril nzym mrkt. This protolyti nzym ulturs wr isolt from iffrnt sours, fins numrous pplitions in phrmutil, foo suh s slughtrhous wst, iry inustry n trgnt inustris [1]. Protss of ommril fflunt, mrkt wst n swg wst n th importn r prou from miroil, niml n plnt iniviul tril olonis wr srn for sours [2]. Miroil protss r prou from protolyti nzym proution on Skim gr mium. high yiling strins inluing spis of Billus sp., Th inoult plts wr tkn inoult t 37 C Alligns flis, Psuomons fluorsns n for 48 hrs n osrv for zons of lrn, whih Aromons hyrophili grown unr sumrg ultur init protolyti tivity. onitions. Among ths, Billus sp is th most Bs on morphology n iohmil stuis, importnt group of tri tht r involv in th th isolts wr intifi s Billus sutilis DL-1, nzym inustry n this trium is lso known to B. lihniformis SH-2, B. mgthrium MW-1 n prou protolyti nzyms quit fftivly. Dspit B. lihniformis SW2. Th tril ulturs wr this, only fw stuis hv n on on protolyti purifi n mintin on nutrint gr slnts hving nzyms from Billus sp. Furthrmor stuis hv ph 8.5 t 35±2 C slnts. show tht nutritionl ftors inluing sours of ron n nitrogn n influn prots nzym Assy for Prots: Th nzym ws ssy in th proution. Bsis this nutritionl ftor, physil rtion mixtur ontining 2.0 ml of 0.5 pr nt sin ftors suh s inoulum onntrtion[3], tmprtur, solution in 0.1 M CO3-HCO 3 uffr (ph 9.5) n 1 ml ph [4] n inution tim [5] n lso signifintly fft nzym solution in totl volum of 3.0 ml. Aftr prots proution. inoultion t 30 C for fiv minuts, th rtion ws Th prsnt invstigtion ws im t stopp y ition of 3.0 ml of 10 pr nt i ol optimiztion of growth onitions of iffrnt Billus TCA n ntrifug t rpm for fiv minuts. sp. isolt iffrnt sours to nhn th prots Protin in th suprntnt ws stimt y th mtho nzym proution. of Lowry t l. [6]. Corrsponing Author: Uni Boominhn, Dprtmnt of Miroiology, Fulty of Sin, Annmli Univrsity, Ini 83

2 Bot. Rs. Intl., 2 (2): 83-87, 2009 To fifty ml quntitis of stnr growth roth Influn of Diffrnt Agro Bs Wst s Sustrts ontining 1 pr nt sin, vrious ron sours on th Prots Proution: To fin out th suitility viz., strh, gluos, frutos, mltos, suros n of gro-s wst s sustrt for prots lluloios t 0.5 pr nt lvls wr in sprt proution, iffrnt sustrts viz., grounnut k, 100 ml Erlnmyr flsks without ing ron sour. oonut k, rwry spnt grin, soy k n wht Thy wr striliz, inoult n inut t 30 C rn (Agro s inustril wsts) wr tkn y for 3 ys n t th n of inution, th ll fr filtrt rpling sin in th growth mi unr sumrg srv s th nzym sour. onition (ph 10). Th nzym tivity ws msur t 3 ys intrvl for prots proution. Efft of Diffrnt Nitrogn Sours: To fifty ml qulitis of stnr roth ontining on pr nt sin, vrious RESULTS AND DISCUSSION orgni nitrogn sours viz., f xtrt, yst xtrt, ppton n inorgni nitrogn sours viz., mmonium Protss, rprsnt out 60% of totl nzyms nitrt, Ammonium ront n ur t onntrtion mrkt sls [7]. At prsnt, th lrgst prt of th of 0.5 pr nt ws in sprt 100 ml Erlnmyr hyrolyti nzym mrkt is oupi y th lkli flsks without ing nitrogn sour. Thy wr protss. striliz, inoult n inut t 30 C for 48 hours In th prsnt stuy, gluos ws foun to th n t th n of inution th ll fr xtrt srv s optimum ron sour for prots tivity y ll th th nzym sour. four Billus isolts follow y suros, frutos, mltos, strh n llulos. Gluos ws th st Efft of ph: Erlnmyr flsks ontining 50 ml ron sour for prots proution y Billus quntitis of moifi stnr growth roth ontining sutilis [8], strh ws foun to st ron sour for th optimum sin onntrtion n ron sours prots proution y B. lihniformis. This rsults r wr tkn n th ph of th roth ws just to in onformity with our prsnt finings. 7.0, 8.0, 9.0, 10.0 n 11.0 in iffrnt flsk using ph mtr In th prsnt stuy, Bf xtrt ws foun to th with 1 N HCl or 1 N KOH n striliz. Th tril st orgni nitrogn sour for ugmnting th prots ulturs wr inoult n inut t 30 C for 3 ys. nzym proution for th Billus sp. Among th At th n of inution prio th ll fr ultur filtrt iffrnt inorgni nitrogn stui mmonium ront ws otin n us s nzym sour. ws foun to th st for th Billus isolts. Th rsults of our prsnt stuy r in lin with th finings of Efft of Inution Tmprtur: Fifty ml quntitis of [9]; f xtrt mong th iffrnt orgni nitrogn moifi stnr growth roth wr ispns in 100 ml sours n mmonium ront mong th iffrnt Erlnmyr flsks. Th ph just to 10 y ing inorgni nitrogn sours l to high protolyti 0.1 N NOH. Th flsks wr striliz, inoult n tivity y Billus sp., t 48 hrs of inution prio. inut t iffrnt tmprturs viz., 35, 40, 45, 50, 55 In th prsnt stuy, mong th iffrnt ph lvls n 60 C for 3 ys. At th n of inoultion prio, th tst, ph 8 ws foun optimum for ll th tst isolts. ll fr ultur filtrt ws otin n us for th Mnjt Kur t l. [3] rport tht vn though ph nzym ssy. initilly rs, it rh lklinity whn sonry Tl 1: Efft of iffrnt ron sour on th protolyti tivity of th Billus sp. Enzym tivity (U/mL) Csin onntrtion Sl. No. in pr nt B. sutilis B. myloliqufins B. mgtrium B. lihniformis 1. Gluos ± ± ± ± Frutos ± ± ± ± Suros ± ± ± ± Mltos ± ± ± ± Strh ± ± ± ± 1.48 f f f f 6. Cllulos ± ± ± ± 1.58 On unit of nzym tivity is th mount of nzym tht rls 1 µg of tyrosin/ml/min Vlus r mn of thr-rplition ± SD. Vlus follow y iffrnt lttrs r signifintly iffr t P vlu of 0.05 oring to DMRT 84

3 Bot. Rs. Intl., 2 (2): 83-87, 2009 Tl 2: Efft of orgni n inorgni nitrogn sour on th protolyti tivity of th Billus sp Enzym tivity (U/mL) Csin onntrtion Sl. No. in pr nt B. sutilis B. mgtrium B. myloliqufins B. lihniformis Orgni N sour 1. Bf xtrt ± ± ± ± Yst xtrt ± ± ± ± Ppton ± ± ± ± 0.84 Inorgni N sour 4. Ammonium nitrt ± ± ± ± Ammonium ront ± ± ± ± Ur ± ± ± ± 1.12 Prots tivity ws trmin y th prour. On unit of nzym tivity is th mount of nzym tht rls 1 µg of tyrosin/ml/min Vlus r mn of thr-rplition ± SD. Vlus follow y iffrnt lttrs r signifintly iffr t P vlu of 0.05 oring to DMRT Tl 3: Pross optimiztion of Billus sp. for prots proution using iffrnt protin rih sustrts Enzym tivity (U/mL) Sl. No. Sustrts B. sutilis B. mgtrium B. myloliqufins B. lihniformis 1. Soy k ± ± ± ± Coonut k ± ± ± ± Wht rn ± ± ± ± Grounnut k ± ± ± ± Brwry spnt grin ± ± ± ± 1.23 On unit of nzym tivity is th mount of nzym tht rls 1 µg of tyrosin/ml/min Vlus r mn of thr-rplition ± SD. Vlus follow y iffrnt lttrs r signifintly iffr t P vlu of 0.05 oring to DMRT Fig. 1: Efft of ph on th protolyti tivity of th Billus sp. Vlus r mn of thr-rplition ± SD. Vlus follow y iffrnt lttrs r signifintly iffr t P vlu of 0.05 oring to DMRT 85

4 Bot. Rs. Intl., 2 (2): 83-87, 2009 Fig. 2: Efft of tmprtur on th protolyti tivity of th Billus sp. Vlus r mn of thr-rplition ± SD. Vlus follow y iffrnt lttrs r signifintly iffr t P vlu of 0.05 oring to DMRT mtolits wr rls n om onstnt. Furthr 2. Ptl, P.R., Enzym isoltion n stuis y B. lihniformis S-40 ws l to grow in ph purifition. In: Biothnology: Applitions n rng of 7-12 with ttr prots proution in lklin Rsrh. Thnomi Pulishing Co. In., USA, rng [10]. pp: Th influn of inution tmprtur on prots 3. Kur, M., S. Dhillon, K. Chuhry n R. Singh, proution y tri ws stui y svrl workrs Proution, purifition n hrtriztion of [11-13]. Among th iffrnt inution tmprturs thrmostl lklin prots from Billus tst for prots proution 50 C ws foun to polymyx. Inin J. Miroiol., 38: optimum for ll tst isolts. Th optimum tmprtur for 4. To, S., Y. Ngoh, T. Wtn n T. Mukiym. prots proution for B. lihniformis ws 45 C [10] Btriolyti tivity of trgnt prots n n for B. sutilis 50 C. But intiv t C, thy its nhnmnt y trgnts mtrils. J. Olo Si., furthr rport tht th optimum tmprtur for prots 54: tivity ws 50 C. 5. Yossn, Q., Y. Png, X. Li, H. Wng n Y. Zhng. Th fft of gro s yprouts s ltrntiv Purifition n hrtriztion of n sustrt on tril prots proution unr xtrllulr lklin srin prots with hiring sumrg frmnttion ws stui y svrl Funtion from Billus pumilus. Curr. Miroiol., workrs[3]. In th prsnt stuy, soy k ws foun to 46: th st inur of prots nzym proution y ll 6. Lowry, t l., Miroil protinss. Av. th four Billus isolts. Biohm. Eng. Biothnol., 36: REFERENCES 7. Gupt, A., I. Roy, R.K. Ptl, S.P. Singh, S.K. Khr n M.N. Gupt On stp purifition n hrtriztion of n lklin prots from 1. Moon, S.H. n S.J. Prulkr A prmtri hlolkliphili Billus sp. J. Chromtogr., stuy of prots proution in th n f-th 1075: ulturs of Billus firmus. Biothnol. Biongg., 8. Gomm, M.A., M.M. Mostf, M.M. Aouzi n 37: M. El-Hshy Egyptin. J. Foo Si., 16: 9. 86

5 Bot. Rs. Intl., 2 (2): 83-87, Shf, N., Syngku Norriti Aris, Rj Noor zlih 12. Vortul, J., J. Pzlrov, M. Dvorkov, L. Vohov, A Rhmn, Mhirn Bsri n Bkr Sllh, M. Strnov, H. Kurov, V. Vintr, R. Zouin J. Appl. Si. Rs., 1(1): 1-8. n J. Chloupk, Extrnl ftors 10. Sn, S. n T. Stynryn, Optimiztion of involv in th rgultion of synthsis of n lklin prots proution y thrmophili xtrllulr protins in Billus mgtrium Billus lihniformis S-40. Inin J. Miroiol., fft of tmprtur. Appl. Miroiol. Biothnol., 33: : Shwimmr, S., Enzym: Purifition, 13. Tskliou, E., R. Mstsiou, I.V. Brgh, nrihmnt n isoltion. In: Sour ook of foo J.V. Bumn n G. Klntzopoulos, Cll-wll nzymology. Th AVI Pulishing Co. In., USA. oun protins of Ltoillus lrukii pp: 107. su sp. ltis ACA-DC 178: hrtriztion n spifiity for µ-sin. Appl. Miroiol. Biothnol., 36: