Effect of Incubation Period (with Static and Shaking Condition) on -Amylase Production from Aspergillus flavus

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1 Avns in Biologil Rsrh 9 (1): 01-06, 2015 ISSN IDOSI Pulitions, 2015 DOI: /iosi.r Efft of Inution Prio (wi Stti n Shking Conition) on -Amyls Proution from Asprgillus flvus B.M. Lll, Ji Shnkr Pul n S.K. Jhv 1 Dprtmnt of Botny, Govt. DB Girls PG Collg, Ripur (C.G.) Ini 2 Shool of Stuis in Biothnology, Pt. Rvishnkr Shukl Univrsity, Ripur (C.G.) Ini Astrt: Enzyms r t s iotlyst for wi vrity of hmil rtions. Amylss r xtrllulr nzyms whih n hyrolys strh to gluos units. Miroil mylss hv immns pplitions in vrious fils in worl mrkt us of ir wi pplition. In prsnt stuy, Asprgillus flvus fungl spis isolt from soli wst ump sit soil ws us for myls proution to osrv suitl r inution prio (3 y, 5 y, 7 y, 9 y, 11 y n 13 y) n nlys fft of shking n stti onition for myls proution. Th rsult init t ftr 11 y of inution t ph 5.5, tmprtur 27 ±1 C in shking onition Mximum ±1.01 U/ml of -Amyls ws prou n in stti ±0.80 U/ml of -Amyls ws prou. Compr to o, in shking onition proution of myls ws mximum. It ws lrly osrv t inution prio n onition ply n importnt rol in proution of myls nzym. Ky wors: Amyls Proution Asprgillus flvus Stti n Shking Conition INTRODUCTION proution. Amyls nzyms ount for out 30 % of worl s nzym proution [5-7]. Th worl Amylss r nzyms whih hyrolys strh mrkt for nzyms rmins in xss of $4500 million moluls to giv ivrs prouts inluing xtrin n n inrs y 4% nnully [6, 8]. Wi ontinuously progrssivly smll polymrs ompos of gluos unit. inrs in morniztion n popultion, us of Th history of myls gn in 1811 whn first strh nzym is lso inrss. But proution rt is not s pr gring nzyms ws isovr y Kirhhoff. This n of nzym. So in prsnt stuy, fungl spis ws follow y svrl rports of igstiv myls n isolt from soil ws us for myls proution n mlt mylss. Ohisson 1930 suggst lssifition osrv suitl inution prio n nlys of strh igstiv nzym in mlt n mylss fft of shking n stti onition for myls oring to nomri typ of sugrs prou y proution. nzym rtion. In 1831, Erhr Fririh Luhs, sri hyrolysis of strh y sliv, u to MATERIALS AND METHODS prsn of n nzym in sliv, "ptylin", n myls [1]. Th morn history of nzyms gn in 1833, whn Enzyms r t s iotlysts n -myls is on Frnh hmists Anslm Pyn n Jn-Frnçois of most usl nzym in inustris. Now most of Prsoz isolt n myls omplx from grminting myls nzym is prou from miroorgnism. Fungi rly n nm it "ists [2]. Alph-Amyls r mjor prour of myls nzym. So in is work (EC ) lso nm s 4- -D-glun fungus ws us for myls proution in iffrnt glunohyrols, hs foun its pplition in rng of inution onition. inustris inluing foo, rwing, istilling inustry, txtil, ppr phrmutil n ioonvrsion of soli Miroorgnism: Asprgillus flvus ws us in is wst t [3, 4]. Lrg rng of pplitions is stuy for myls proution, whih ws isolt from triggring ftor for inustriliztion of lph myls soil smpl of soli wst ump sit, ollt from Govt. Corrsponing Auor: S.K. Jhv, Shool of Stuis in Biothnology, Pt. Rvishnkr Shukl Univrsity, Ripur (C.G.) Ini. Tl:

2 DB Girls PG Collg Cmpus of Ripur, Chhttisgrh, prform y nlysis of vrin (ANOVA). All Ini rough sril ilution mo. Fungus ws xprimnts wr prform in triplits n rpt mintin in potto xtros gr (PDA) slnt t 4 C r tims. Th smpls ollt from h rplit [9, 10]. wr tst for myls tivity. Mn vlus wr nlyz oring to Dunn s Multipl Rng (DMR) Srning for Amyls Proution: -Amyls Tst ompr t 5 % lvl of signifint iffrn. prouing fungi ws srn y strh hyrolysis tst. Fungus ws inoult n grown in Strh RESULTS Agr Mium (SAM) t 27 ±1 C for 3 to 4 ys. Aftr inution strh hyrolysis tst ws prform y Wi ontinuously inrs in morniztion n ioin solution (0.3 % I 2 n 1 % KI). Ioin solution ws popultion, us of nzym is lso inrss. But floo to SAM plt fungl ultur n positiv proution rt is not s pr n of nzym. Still mny rsult ws show in lr zon in SAM mium n rsrhrs r trying to fin out nw thniqus for ngtiv rsult hving no zon [9, 11]. lrg proution of myls nzym. So in is stuy iffrnt inution prio n onition ws tri to Frmnttion n Cultur Conition: Proution of - inrs proution of myls nzym. Amyls from fungus ws rri out in moifi Proution Mium [12, 13]. Fungus ws firstly grown in Miroorgnism n Srning of -Amyls: Potto Bro (PB) for furr us t propr onition. Asprgillus flvus fungus ws us in is stuy for Aftr propr grow of fungus two rops of Twn proution of myls. Asprgillus flvus shows rpi 80 solution ws into PB ultur n shk grow, oliv to lim grn in front wi rm olour in ultur flsk. Thn 100 µl of ultur ro ws rvrs, in PDA mium (Fig. 1 n 2) inoult in Erlnmyr flsk ontining 50 ml of Amyls proution ws srn y strh Proution Mium (g/l), Strh - 5 g, Yst xtrt - 5 g, hyrolysis tst wi ioin rgnt. Apprn lr K2HPO g, MgSO 4. 7H2O g, CCl 2. 2H2O - 0.1g. zon inits proution of myls n hyrolysis Thn flsks wr inut t 27 ±1 C t ph 5.5 for of strh in t r n ngtiv rsults shows lu r iffrnt inution prio (3 y, 5 y, 7 y, 9 y, olour in ntir SAM mium [9, 11] (Fig. 3). 11 y n 13 y) [10, 14, 15] in stti n shking onition for myls proution. -Amyls Proution: -Amyls proution ws prform in two ultur onition Stti n Shking, t -Amyls Assy: Amyls nzym ws ssy y ph 5.5 n Tmprtur 27 ±1 C t iffrnt inution moifi ioin ssy mo [12, 16, 17]. In 0.5 ml of 1 % r prio (3 y, 5 y, 7 y, 9 y, 11 y n 13 strh n 0.3 ml of 0.1 M Soium tt uffr wi 1 ml y) wi Asprgillus flvus n stimt in Unit/ml of istill wtr mixtur ws inut t 55 C for (U/ml). Proution of myls ws inrs wi 10 minuts for quilirium. Thn 0.5 ml of ru nzym inrss inution prio up to 11 y n xtrt ws n llow to rtion for 5 minuts. rss on furr inution in o stti n Thn to stop rtion 0.5 ml of 1 M HCl to shking onition. Proution of myls ws mximum rtion mixtur n ilut mixtur volum up to in 11 y of shking onition ±1.01 U/ml 15 ml y istill wtr wi ing 0.1 ml of ioin ompr to stti ±0.80 U/ml. It ws lrly rgnt. Thn sorn ws tkn t 610 nm wi show t inution prio n onition ply n hlp of Sptrophotomtr. On unit of -myls is importnt rol in proution of myls nzym. fin s mount of nzym whih hyrolyzs 0.1 mg (Tl 1 n Fig. 4). of strh in 10 min t 55 C in ssy onition. Protin Conntrtion: Protin onntrtion of o Protin Conntrtion: Conntrtion of protin stti n shking ru nzym xtrt of iffrnt trmin y lowry s mo [18] using ovin srum inution prio ws stimt y Lowry s mo lumin (BSA) s stnr. using BSA s stnr. Amount of nzym ws rlt to onntrtion of protin. Wi inrss in Sttistil Anlysis: -myls proution ws sujt onntrtion of protin, unit mount of nzym ws to tst for signifint squntil mols, whih ws lso inrs. But inution onition ws fft 2

3 Fig. 1: Colony morphology of Asprgillus flvus (Front n Bk viw in PDA Mium) Fig. 2: Mirosopi photogrphs of Asprgillus flvus Fig. 3: -Amyls srning positiv tst of Asprgillus flvus Fig. 4: Proution of -Amyls y Asprgillus flvus n onntrtion of protin in stti n shking onition 3

4 Fig. 5: Ativity prntg of myls nzym in iffrnt inution onition Tl 1: Proution of -Amyls y Asprgillus flvus Inution 27 ±1 C, ph Conition S. no. Prio Stti Mn ±SE U/ml Shking Mn ±SE U/ml 1. r 3 y ±0.12 f ± y ± ± y ± ± y ± ± y ± ± y ± ±0.60 * ANOVA Stti f = 5, f = , p < 0.000, LSD (5%) = * ANOVA Shking f = 5, f = , p < 0.000, LSD (5%) = *Mn follow y similr suprsript lttrs (,,,, n f) o not iffr signifintly t 5% lvl y Dunn s Multipl Rng Tst. Tl 2: Estimtion of protin onntrtion on stti n shking onition Inution 27 ±1 C, ph Conition S. no. Prio Stti Mn ±SE µg/ml Shking Mn ±SE µg/ml 1. r 3 y ± ± y ± ± y ± ± y ± ± y ± ± y ± ± * ANOVA Stti f = 5, f = , p < 0.000, LSD (5%) = 77.1 * ANOVA Shking f = 5, f = , p < 0.000, LSD (5%) = *Mn follow y similr suprsript lttrs (,,, n ) o not iffr signifintly t 5% lvl y Dunn s Multipl Rng Tst. Tl 3: Ativity prntg (%) of -Amyls t iffrnt inution prio in stti n shking onition Prio Conition r 3 y 5 y 7 y 9 y 11 y 13 y Stti 21.95% 53.05% 59.61% 62.83% 100% 77.59% Shking 15.46% 63.54% 72.79% 78.95% 100% 87.15% onntrtion of protin s ompr to unit mount proution of nzym in stti n shking onition. In is stuy onntrtion of protin ws show mximum in stti onition. (Tl 2 n Fig. 4). Prntg Ativity: In is work prntg tivity of myls nzym in stti n shking onition t iffrnt inution prio ws lult, highr nzym mount ws tkn s hunr prntg of rltiv tivity. In o onition t 11 y of inution rltiv tivity ws mximum. Wi inrs n rs of inution prio from 11 y, rltiv tivity ws vri. It ws osrv t inution prio n onition fft tivity of myls nzym (Tl 3 n Fig. 5). DISCUSSION Som or rsrhrs r lso work wi Asprgillus spis, Akpn t l. [19] stui on proution of lph myls y Asprgillus nigr in hp soli mium using ri rn n griulturl mtrils. Shrm n Shukl [20] work on Asprgillus fumigtus n Chtomium gloosum isolt from soil smpl. Akpn n Alj [21] work wi Asprgillus oryz for proution of myls from ri rn soli mium. Figuir n Hirook [22] prou myls from fungi Fusrium moniliform n Asprgillus flvus. Thy foun mximum myls proution from Asprgillus flvus. In proution of myls, r r som or workrs r lso foun som rlvnt rsults in myls nzym proution wi som fungl sp., Vlprl [23] foun mximum myls tivity in Miz strh sustrt of U/ml n lowst tivity in Tpio strh sustrt of 4.66 U/ml t 30 C in shking onition from Armonium sporosultum. Sinhu t l. [10] work on 4

5 iffrnt -myls proution prmtrs, y t ftr 11 y of inution t ph 5.5, tmprtur foun t t ph 5 proution of myls ws 27 ±1 C in shking onition proution of -Amyls mximum. Thy lso work on inution prio up to ws mximum ompr to stti onition. 7 ys n tmprtur 30 to 55 C, t 4 y of inution t 30 C proution of myls ws mximum wi ACKNOWLEDGMENT Pniillium jninllum NCIM Shrm n Shukl [20]work on Asprgillus fumigtus n Chtomium Auors r grtly knowlg to prtmnt of gloosum isolt from soil smpl. Thy foun mximum otny, Govt. D.B. Girls P.G. Collg, Ripur (C.G.) for myls nzym tivity of Asprgillus fumigtus in 9 to proviing l filitis n or rquirmnts for is 11 ys of inution. Ygr t l. [24] foun optimum rsrh work. ph n tmprtur ws 5.0 n 40 C rsptivly from Asprgillus slrotiorum. Blkn n Ertn (2007) REFERENCES prou -Amyls from Pniillium hrysognum y iffrnt griultur wst prouts, in ir work 1. Erhr Fririh Luhs, Efft of sliv on mximum proution of myls from Wht Brn (WB) strh, Poggnorff's Annln r Physik un 160 U/ml [16]. Figuir n Hirook [22] foun highst Chmi., 22: 623. (morn ittion: Annln r myls proution, whih ws t 10 y of Physik, 98(8): 623). frmnttion, from Fusrium moniliform n Asprgillus 2. Anslm, Pyn n Jn-Frnçois Prsoz, flvus. Thy foun mximum myls proution from Mmoir on ists, prinipl prouts of its Asprgillus flvus. rtions n ir pplitions to inustril Tiwri t l. [25] foun 3.07 mg/ml xtrllulr n rts)". Annls Chimi t Physiqu 2 sris, onntrtion of protin from Pniillium rugulosum. 53: Vlprl [23] foun mximum mount of protin from 3. Gupt, R., P. Gigrs, H. Mohptr, V.K. Goswmi n Miz strh ws 34 mg y Armonium sporosultum. B. Chuhn, Miroil - mylss: Amu t l. [26] foun mg/ml of protin t iothnologil prsptiv. Pross Biohm., sttionry phs from Billus sutilis KCX : Bkri t l. [27] stui in rltiv tivity fft 4. Pny, A., P. Nigm, C.R. Sool, V.T. Sool, of ph, tmprtur n inution tim on nzym D. Singh n R. Mohn, Avns in miroil tivity. Thy foun highst prntg of tivity in mylss. Biothnol. Appl. Biohm., 31: ph 6 n inution tim of nzym is 60 C n t 5. Akn, N., High Lvl Proution of 50 C 87 % of tivity ws trmin, y foun suitl Extrllulr -Amyls from Billus lihniformis tmprtur for tivity is twn 40 C to 60 C. Amu ATCC, in Sumrg Frmnttion. Romnin t l. [26] foun mximum tivity in 10 min. t 50 C. Ygr Biothnol. Ltt., 16(6): t l. [24] work on fft of ph n tmprtur on 6. D, P., S.A. Tlukr, K. Mohsin, P.K. Srkr n prntg tivity of nzym. Thy foun highst S.M.A. Sym, Proution n prtil prntg of nzym tivity in ph 5 n tmprtur hrtriztion of xtrllulr myls nzym from 40 C. Billus myloliqufins P-001. Springr Plus, CONCLUSION 7. 2(154): Mll, D., J. Pirim, L.C. Huw, I. Rhmwti n W. Purowsito, Isoltion n Intifition of In is work fft of inution prio in two A Thrmostl Amyls-Prouing Btrium from onitions ws nlys n it ws foun t inution Htus Hotspring. Miroiol Inonsi., 6(2): prio n onition plys n importnt rol for 8. Sivkumr, T., T. Shnkr, P. Vijyskr, proution of myls. Asprgillus flvus ws us for J. Muukumr n E. Ngnrknnn, proution of -Amyls t iffrnt inution Amyls Proution Using Billus rus Isolt prio in two onition stti n shking t ph 5.5, from Vrmiompost Sit. Int. J. Miroiol. Rs., tmprtur 27 ±1 C. In o onition proution of 3(2): myls ws inrs wi inrss in inution 9. Prsott, L.M. n J.P. Hrly, Lortory prio. In 11 Dy of inution, proution of myls Exriss in Miroiology. Fif ition, TMH ws mximum in o onition, it ws lrly show Compnis. 5

6 10. Sinhu, R., G.N. Suprh n S. Shshihr, Akpn, I., M.O. Bnkol n A.M. Asmovo, Optimiztion of pross prmtrs for Proution of lph myls y Asprgillus nigr in proution of - myls from Pniillium hp soli mium using ri rn n griulturl jninllum (NCIM 4960) unr soli stt mtrils. Trop Si., 39: frmnttion. Afrin J. Miroiol. Rs., 3(9): Shrm, D. n A.K. Shukl, Strh hyrolysis 11. Hg, S.V., A. Rmsh n C. Srinvs, n -Amyls tivity of Asprgillus n Optimiztion of myls proution from n Chtomium. Asin J. Biohm., 3(5): nophyti fungi Disosi sp. isolt from 21. Akpn, I. n F.A. Alj, Proution n Clophyllum inophyllum. J. Agri. Thnol., stiliztion of myls prprtions from ri rn 7(3): soli mium. Worl J. Miroiol. & Biothnol., 12. Gogoi, B.K., R.L. Bzruh, K.R. Pilli n 20: J.N. Bruh, Proution, purifition n 22. Figuir, E.L.Z. n E.Y. Hirook, hrtriztion of n -myls prou y Cultur Mium for Amyls Proution y Shromyopsis fiigr. J. Appl. Btriol., Toxigni Fungi. Brzilin Arhivs of Bio n 63: Thnol., 43(5): Viylkshmi, R., R. Prnmn n J. Inhumi, 23. Vlprl, V.K., Purifition n Proprtis of Amyls Proution on Sumrg Thrmostl -Amyls y Armonium Frmnttion y Billus spp. Worl J. Chm., sporosultum. Int J. Biothnol. n Biohm., 4(1): (1): Snkrlingm, S., T. Shnkr, R. Rmsuuryn, 24. Ygr, H., F. Ertn n B. Blkn, Comprison S. Prksh n C. Kumr, Optimiztion of of Som Proprtis of Fr n Immoiliz Cultur Conitions for Proution of Amyls -Amyls y Asprgillus slrotiorum in Clium from Billus lihniformis on Sumrg Algint Gl Bs. Prp Biohm & Biothnol., Frmnttion. Amrin-Eursin J. Agri. & Environ. 38: Si., 12(11): Tiwri, K.L., S.K. Jhv n A. Ftim, Cultur 15. Sivkumr, T., T. Shnkr, P. Vijyskr, onition for proution of rmostl myls J. Muukumr n E. Ngnrknnn, y Pniillium rugulosum. Glo J. Biothnol. & Amyls Proution Using Billus rus Isolt Biohm., 2(1): from Vrmiompost Sit. Int J. Miroiol. Rs., 26. Amu, K. n K.J. Priy, Efft of ph, 3(2): tmprtur n mtl ions on myls tivity from 16. Blkn, B. n F. Ertn, Proution of Billus sutilis KCX 006. Int J. Phrm. n Bio Si., -Amyls from Pniillium hrysognum unr 2(2): B407-B413. Soli-Stt Frmnttion y using som Agriulturl 27. Bkri, Y., H. Ammounh, S. El-Khouri, M. Hr n By-Prouts. Foo Thnol Biothnol., P. Thonrt, Isoltion n intifition of nw 45(4): Billus strin for myls proution. Rs in 17. Rshmi, R., G. Snjy n S. Sugunn, Biothnol., 3(6): Enhn tivity n stility of -myls immoiliz on lumin. Ctl Commun., 7: Lowry, O.H., N.J. Rosrough, A.L. Frr n R.S. Rnll, Protin Msurmnt Wi Folin Phnol rgnt. Th J. Biol. Chm., 193:

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

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