Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 7 i hc Nng nm 2010

KHO ST CC IU KIN NUI CY V PHNG PHP TCH CHIT ENZYME CELLULASE T VI KHUN BACILLUS SUBTILIS
INVESTIGATION OF CULTURING AND EXTRACTING CELLULASE OF BACILLUS SUBTILIS SVTH: Trn Th nh Tuyt, Trng Quc Huy
Lp 05SH, Khoa Ha, Trng i Hc Bch khoa

GVHD: PGS.T.S Trn Th X, ThS. Ng Thi Bcch Vn, KS. Phm Trn Vnh Ph
Khoa Ha, Trng i Hc Bch khoa
TM TT Enzyme cellulase c tng hp nhiu Bacillus subtilis v c nhiu ng dng quan trng. Trong ti ny, chng ti tin hnh kho st cc iu kin nui cy Bacillus subtilis v phng php tch chit thu nhn enzyme cellulase. Kt qu thu c cho thy nhit o 40 C, pH=7,0 v thi gian nui 30h, lng enzyme cellulase sinh ra l ln nht . Phng php tch chit cellulase bng dung mi aceton cho hiu qu cao hn dung mi ethanol. ABSTRACT Cellulase, which produced in a great quantity by Bacillus subtilis, has many important applications. In this project, we investigated cultivation conditions and methods to obtain cellulase 0 from the culture of Bacillus subtilis. The results show that at 40 C, pH=7,0 and 30 hour of cultivation, cellulase production is highest. That extracting celluase by using aceton is more effective than using ethanol.

1. M u Chng Bacillus subtilis c pht hin bi Ferdinand Cohn vo nm 1872.[1] Bacillus subtilis l trc khun, Gram dng, kch thc (3- 5) 0.6m, c kh nng sinh bo t v l loi sinh vt t dng, hiu kh hoc k kh ty nghi.[2] H enzyme ca Bacillus subtilis rt phong ph v a dng gm protease,[6] amylase,[7] glucoamylase, glucanase, cellulase, dextranase, pectinase [5]. Bacillus subtilis c ng dng kh nhiu trong cc ngnh cng nghip c bit l cng nghip sn xut enzyme nh protease, amylase....[1] c nhiu cng trnh nghin cu v ng dng ca Bacillus subtilis trong ch bin thc phm, trch ly cc cht t thc vt, t cy thuc...[3] Cng vi Bacillus subtilis, h enzyme cellulase cng c nghin cu t lu. [4]Enzyme ny l mt phc h gm nhiu enzyme tham gia vo qu trnh chuyn ha cellulose[14][15]. Enzyme exoglucanase hay C1 Enzyme endogluconase hay Cx Enzyme -1,4-glucosidase Cellulase c sn xut vi qui m cng nghip v ng dng rng ri trong lnh vc ch bin thc phm; trong nng nghip, cellulase c dng trong x l t tng mu m ; trong cng nghip, cellulase c ng dng trong cng ngh sn xut ethanol [4]

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Vi mc tiu thu nhn cellulase t chng Bacillus subtilis, trc tin chng ti tin hnh kho st nh hng ca nhit , pH, thi gian nui n s sinh cellulase ca Bacillus subtilis. Tip n chng ti tin hnh nh gi hiu qu chit tch enzyme bng hai dung mi axeton v ethanol. 2. i tng v phng php nghin cu 2.1. i tng Chng Bacillus subtilis (V5) c nui cy v bo qun ti phng Th nghim Cng ngh Sinh hc, khoa Ha, trng i hc Bch Khoa Nng. 2.2. Phng php nghin cu 2.2.1. Phng php hot ha chng Bacillus subtilis Chng Bacillus subtilis c hot ha trn mi trng vi thnh phn: NaCl 0,5%, pepton 1 %, Cao nm 1 %, Cao tht 0,3 %, Agar 0,7 %, pH = 7.[3] 2.2.2. Phng php nh tnh kh nng sinh enzyme cellulase Chng ti s dng phng php cy im trn mi trng hot ha c b sung CMC 1 %, nhit 300C trong 24h. Sau nhum mu bng thuc th Lugol v o ng knh vng thy phn. 2.2.3. Phng php thu nhn dch chit enzyme th Nui cy chng Bacillus subtilis trong mi trng lng vi thnh phn CMC 1 %, Glucose 0,1 %, Pepton 5 %, Cao nm 2,5 % cc thi im, nhit v pH khc nhau. Sau ly tm tc 8000 vng/pht, thu dch ni enzyme.[3] 2.2.4. Phng php xc nh hot enzyme Phng php c l thch: Mi trng xc nh hot tnh enzyme cellulase l CMC 1%, Agar 2%. ng knh thy phn c xc nh tng t nh phn 2.2.3 Phng php ng kh Bernfeld : Nguyn tc phng php ny da vo phn ng mu ca nhm C=O vi 3, 5-dinitrosalicilic acid (DNS), gi tr mt quang o c bc sng 540 nm phn nh lng ng c to thnh t qu trnh phn ct phn t cellulose ca enzyme cellulase.[8][10][13] Phng php chit tch enzyme S dng hai dung mi hu c : aceton v etanol 900. Tin hnh kt ta dch chit enzyme th theo t l 1 :4 (Vdch chit :Vdung mi), hn hp -200C trong 60 pht, ly tm 13000 vng/pht, thu cn. So snh hiu qu chit tch ca aceton v etanol 900 bng phng php c l thch (xem phn 2.2.5) 3. Kt qu 3.1. Kt qu hot ha v nh tnh kh nng sinh enzyme cellulase ca chng V5 kim tra kh nng sng st ca chng sau qu trnh bo qun - 200C, chng ti tin hnh hot ha trn mi trng ti thiu (Hnh 1.a), ng thi xc nh kh nng sinh enzyme cellulase (Hnh 1.b). Kt qu hot ha cho thy khun lc ln rt mnh, t gh gh, mu trng, c hnh rng ca.
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3.2. Kt qu kho

(a) (b) Hnh 1. Kt qu hot ha (Hnh a) v nh tnh kh nng sinh b)sinh enzyme cellulase st s tng trng enzyme v kh(Hnh nng

theo thi gian

3.2.1. Kt qu ng cong tng trng Chng ti tin hnh nui mu cc thi im 0h, 4h, 6h, 12h, 18h, 24h, 30h, 32h, 34h v 36h, nhit 300C xc nh gi tr mt quang bc sng 600nm. Kt qu ng cong tng trng ca Bacillus subtilis (V5) cc thi im khc nhau c biu din biu hnh 2.

Hnh 2. th biu din s tng trng ca vi khun Bacillus subtilis theo thi gian

Da vo th chng ti nhn thy chng V5 bt u tng trng sau 6h nui cy, t thi im 18h - 30h chng i vo pha n nh v thi im 32-36h l giai on suy tn. 3.2.2. Kt qu ng knh thy phn ng thi vi vic o gi tr o OD600, chng ti tin hnh thu dch enzyme th v kho st kh nng sinh cellulase theo thi gian ca chng V5. Kt qu phn gii CMC 1 % trn mi trng thch rn cho thy hot ca cellulase tng tuyn tnh theo thi gian v t gi tr ln nht thi im 30h.

Hnh 3. Kt qu ng knh thy phn theo thi gian

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3.2.3. Kt qu xc nh nng glucose sinh ra Ngoi phng php khuch tn a, chng ti nh lng hot cellulase bng phng php ng kh a. Kt qu tng quan gia nng Glucose chun v gi tr OD540nm

Hnh 4. th th hin tng quan gia nng glucose chun v gi tr mt quang 540nm

b. Nng Glucose sinh ra trong phn ng thy phn CMC 1% ca cellulase

Hnh 5. th biu din nng Glucose gii phng theo thi gian

Trc tin chng ti tin hnh dng th tng quan gia nng Glucose chun v gi tr OD540 (Hnh 4). Nng Glucose gii phng theo thi gian c ngoi suy t ng chun. Kt qu cho thy ti thi im 32h v 34h nng Glucose l ln nht. Nh vy, t kt qu ca ng knh thy phn v nng Glucose gii phng, chng ti nhn thy hot ca enzyme cellulase mnh nht trong pha n nh ca ng cong sinh trng. 3.3. Kt qu kho st kh nng sinh enzyme cellulase theo nhit Chng ti tin hnh nui mu nhit 300C, 400C, 500C v 600C trong thi gian 30h. Sau xc nh hot enzyme da vo nng Glucose sinh ra trong phn ng thy phn CMC 1%.

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Hnh 6 . th biu din nng Glucose gii phng theo nhit

Da vo th chng ti nhn thy ti nhit 500C nng Glucose sinh ra l ln nht. Kt qu thu c cho php kt lun chng Bacillus subtilis c kh nng chu nhit cao. Sau 30 gi nui cy, 500C l khong nhit tt nht cho qu trnh sinh tng hp enzyme cellulase. 3.4. Kt qu kho st kh nng sinh enzyme cellulase theo pH Chng ti tin hnh nui cy chng Bacillus subtilis trong mi trng c tnh axit (pH=5 v pH=6), mi trng trung tnh (pH=7) v mi trng kim (pH=8 v pH=9) nhit 300C, trong thi gian 30h. Sau xc nh hot enzyme da vo nng Glucose sinh ra trong phn ng thy phn CMC 1 %.

Hnh 7. th biu din nng Glucose theo pH

Kt qu cho thy trong mi trng trung tnh v mi trng c tnh kim, Bacillus subtilis sinh tng hp enzyme cellulase tt hn mi trng c tnh axit, th hin qua nng Glucose c gii phng (Hnh 7). 3.5. Kt qu kt ta enzyme cellulase t dch nui lc Bacillus subtilis 300C trong 30 gi. Chng ti tin hnh ta dch nui cy lc Bacillus subtilis thu nhn enzyme cellulase bng hai loi dung mi l aceton v etanol 900 (xem phn 2.2.6). So snh hiu qu chit tch ca 2 dung mi da vo ng knh thy phn CMC 1 % ca dch ta.
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Bng1. Kt qu ng knh vng thy phn CMC 1 % ca enzyme cellulase trc v sau khi kt ta bng aceton v bng etanol 90 0

Dung mi hu c Aceton Etanol 900

Kt qu ng knh thy phn trc ta (cm) 2,2 2,2

Kt qu ng knh thy phn sau ta (cm) 2,4 2,3

Hnh 8. Kt qu ng knh thy phn ca enyme trc v sau khi kt ta bng acetone v etanol 900

Nh vy, phng php ta s dng dung mi hu c aceton v etanol 90 0 u lm tng hot ca enzyme cellulase. Trong , kt ta enzyme bng aceton lm tng 9,1 % v kt ta bng etanol 900 lm tng 4,5% hot enzyme ban u. 4. Kt lun T nhng kt qu thu c, chng ti kt lun: Khi nui cy 300C, s tng trng ca chng Bacillus subtilis n nh trong khong thi gian 18-30 gi v sau thi im ny (t 32 -34 gi), lng enzyme cellulase sinh ra l ln nht. Mi trng trung tnh (pH=7) l mi trng thch hp nht cho qu trnh tng hp cellulase ca chng Bacillus subtilis khi nui cy 300C. Chng Bacillus subtilis s dng trong ti ny c kh nng chu nhit cao, t 300 n 500C. Phng php ta enzyme cellulase t dch nui cy lc bng dung mi aceton t ra c hiu qu hn dung mi etanol 900 TI LIU THAM KHO [1]. Nguyn Th Xun Hin, Nghin cu kh nng chu nhit ca bo t v enzym protease ca Bacillus subtilis, kha lun tt nghip, i hc Bch Khoa Nng. [2]. L Xun Phng , Vi sinh vt hc cng nghip (2001), NXB Xy dng H Ni.

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[3]. Phm Thi Hng Thm, Nghin cu phn lp v bc u kho st mt s vi sinh vt phn hu cellulose t v la sn, kha lun tt nghip, i hc Bch Khoa Nng. [4]. Trn Xun Ngch, Cng ngh enzyme, (2005), i hc Bch Khoa Nng. [5]. D.R.Kashyap, S.Chandra, A.Kaul, R.Tewari, Production, purification and characterization of pectinase from a Bacillus sp. DT7, World Journal of Microbiology & Biotechnology Vol 16, (2000), tr. 277-282. [6]. Harry P. Rappaport, A Bacillus Subtilis Proteinase, The Journal of Biological Chemistry, (1965), Vol. 240, No. 1, [7]. Konsoula Z., Kyriakides M.L, -amylase production in aqueous two-phase systems by Bacillus subtilis, FEBS Journal 272, (2005), tr. 2. [8]. Kwong-Yu Chan, K. S. Au, Studies on cellulase production by a Bacillus subtilis. Antonie van Leeuwenhoek 53, (1987), tr 125-136 [9]. Maria Y. Yang v cng s, Cloning of the Neutral Protease Gene of Bacillus subtilis and the use of the Cloned Gene to Create an In Vitro Derived Deletion Mutation, Journal of Bacteriology, tr. 15-21. [10]. Po-Jui Chen, Tao-Chun Wei, Yao-Tsung Chang, Lng-Ping lin, Carboxymethyl xenlulaza, CBhote.n [11]. J. Perez J. Munoz-Dorado, Biodegradation and biological treatments of cellulose, hemicellulose and lignin, Dd Microbio,( 2002) [12]. Zhi W., Song J., Bi J. and Ouyang F., Partial purification of -amylase from culture supernatant of Bacillus subtillis in aqueous two-phase systems, Bioprocess Biosyst Eng.27, (2004), tr. 3-7. [13]. http://www.springerlink.com/content/x111666875j6lhw8/ [14]. http://www.fao.org/docrep/w7241e/w7241e08.htm [15]. http://www.worthington-biochem.com/CEL/default.html

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