TP CH PHT TRIN KH&CN, TP 10, S 07 - 2007

THU NHN ENZYME CELLULASE CA TRICHODERMA REESEI TRN MI TRNG BN RN

Trn Thnh Phong, Hong Quc Khnh, V Th Hnh, L Bch Phng, Nguyn Duy Long, L Tn Hng, Trng Th Hng Vn Vin Sinh hc Nhit i Vin Khoa hc v Cng ngh Vit Nam
(Bi nhn ngy 26 thng 06 nm 2006, hon chnh sa cha ngy 25 thng 06 nm 2007)

TM TT: Mc tiu ca bi bo l nghin cu thu nhn cellulase Trichoderma reesei VTT-D-80133 sinh trng trn mi trng bn rn vi c cht b ma kt hp vi cm m. T l BM:CM (7:3), 8 ln nng dinh dng, m ban u 60%, thi gian nui cy 7 ngy l ti u cho T. reesei VTT-D-80133 sinh tng hp cellulase trn mi trng ln men bn rn.Hot tnh v hiu sut sinh tng hp cellulase iu kin trong bnh tam gic l CMCase (Carboxymethyl cellulase) 280,64 IU/g v FPU (Filter Paper Unit) 5 IU/g; thp hn 3,2 v 37 ln so vi ch phm Amano T (cellulase c sn xut t T. reesei) ca Hng AMANO. Ngoi cellulase, canh trng cn cha: -amylase 368,75 UI/g, protease 12,43 UI/g v xylanase 10073,25 BXU/g. Cellulase thu nhn c c kh nng ng ha 21% giy in qua s dng (10%) v qua phn tch trn gel polyacrylamide c cc vch protein c trng lng phn t bng vi cc vch protein c trong ch phm Amano T. T kha: Cellulase, Trichoderma reesei, b ma, cm m, ln men bn rn 1. GII THIU Nhiu loi nm si c kh nng sinh ra mt lng ln cellulase thuc ging Alternaria, Trichoderma, Aspergillus, Pinicillium,Trong Trichoderma v Aspergillus c nhiu nh khoa hc nghin cu sn xut cellulase (Bothast & Saha, 1997). Cellulase l enzym a cu t gm: exoglucanase hay C1 (EC 3.2.1.91), endoglucanase hay Cx (EC 3.2.1.4) v -glucosidase (EC 3.2.1.21) hot ng phi hp thy phn cellulose thnh glucose. Cellulase c ng dng ci thin ga tr dinh dng ca thc n gia sc, gia cm; ch bin thc phm; trch ly cc cht t thc vt, t cy thuc; ng ha cc ph liu giu cellulose sn xut ethanol. Vit Nam c lng ph ph liu nng nghip thi ra rt di do, trong lng b ma thi ra t cc nh my ng chim khong 20% ma nguyn liu, trong b ma c hm lng cellulose khong 50% v hemicellulose khong 25% nn c th s dng nh ngun carbon cm ng nm si sinh tng hp cellulase. Mc tiu ca bi bo ny l thu nhn enzym cellulase ca T. reesei VTT-D-80133 sinh trng trn c cht b ma kt hp vi cm m trong qa trnh ln men bn rn nhm tn dng b ma thu nhn enzym cellulase. nh hng ca cc yu t (t l b ma/cm m (BM:CM), m ban u, nng dinh dng v thi gian nui cy) n s sinh ra cellulase c nghin cu bng phng php qui hoch thc nghim. 2. VT LIU V PHNG PHP Nm si: Chng T. reesei VTT-D-80133 nhn c t bo tng ging Roal Oy, Phn Lan. C cht: B ma v cm m.

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Science & Technology Development, Vol 10, No.07 - 2007 Ln men bn rn: xc nh thnh phn mi trng v cc iu kin ti u cho s sinh trng v sinh tng hp enzym, nm si c nui trn mi trng c t l b ma v cm m khc nhau (7:3, 6:4, 5:5, 4:6 v 3:7), m mi trng (t 50-70%), nng dinh dng (da theo mi trng Mandel, x1-x8 hay 1-8 ln) v thi gian nui cy t 2-12 ngy. Trch ly enzym: Cho 45 ml dung dch m Na-acetate 50 mM pH 5 vo 5 g canh trng, lc trn my lc vi tc 150 vng/pht trong 5 pht, lc thu dch. em ta dch lc bng cn 960 c lm lnh trc. Thu kt ta v ha li vi cng th tch dung dch m. Xc nh hot tnh cc enzym: CMCase theo phng php ca Cng ty Shin Nihon Nht Bn vi c cht l CMC 1%; FPU theo phng php ca Hng Biopract GmbH - c, vi c cht l giy lc Whatman no. 1; Xylanase theo phng php ca t chc EDC (Enzym Development Corporation, M), vi c cht l xylan 1%; -amylase theo phng php ca Hng Amano - Nht Bn, vi c cht l tinh bt 1%; Protease theo phng php ca Cng Ty Amano - Nht Bn, vi c cht l casein 1,5%. Xc nh hm lng protein: Theo phng php Bradford s dng bovine serum albumin nh protein chun. Thy phn giy: cho dch enzym cellulase (5 FPU/ml) vo giy xay nh (10%) 500C, pH 5 trong 24 gi. Hiu sut (%) = lng ng kh (g)*0,9*(100/lng giy in (g)) in di protein: Sodium Dodecyl Sulfate Polyacrylamide gel (SDS-PAGE) c thc hin trn gel ng cha 10% (w/v) polyacrylamide. Ti u ha thnh phn mi trng bng phng php quy hoch thc nghim: T l BM:CM, m ban u, nng dung dch dinh dng v thi gian nui cy l 4 yu t c nh hng r rt n kh nng sinh tng hp cellulase ca T. reesei VTT-D-80133 nn c chn ti u ha theo phng php quy hoch thc nghim. 3. KT QA V THO LUN 3.1. Kt qa ti u ha thnh phn mi trng v cc iu kin nui cy. Cc kt qa th thim trc y, chng ti xc nh c thnh phn mi trng c s cho chng T. reesei VTT-D-80133 sinh ra cellulase theo phng php ti u ha c in: t l BM:CM (4:6), m ban u 54%, 5 ln nng dinh dng, thi gian nui 7 ngy, t l ging 6x106 bo t/g mi trng, hot tnh cellulase t c l 251,43 IU/g. Tuy nhin, thnh phn mi trng v cc iu kin nui cy mi ch c nghin cu nh hng mc ring r. Trong nm yu t trn th bn yu t l t l BM:CM, m ban u, nng dinh dng v thi gian nui cy c nh hng ng k n kh nng sinh tng hp cellulase ca T. reesei VTT-D-80133 nn c chn nghin cu ti u ha theo phng php qui hoch thc nghim. Qui hoch c thc hin vi ma trn y vi s th nghim N = 24 = 16 Bng 1. M ha cc bin s Cc bin s X1: T l BM:CM X2: Nng dinh dng (ln) X3: m ban u (%) X4: Thi gian nui cy (gi) Mc di (-) 2:8 x2 50 3 Mc trung bnh (0) 4:6 x5 54 7 Mc trn (+) 6:4 x8 58 11

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TP CH PHT TRIN KH&CN, TP 10, S 07 - 2007

T bng 1, tin hnh nui cy T. reesei trong cc mi trng m c cc yu t kho st trn hai mc. Kt qa xc nh hot tnh cellulase c ghi nhn trong bng 2.

Bng 2. Hot lc CMCase t T. reesei theo thc nghim v theo phng trnh hi qui.
TTN 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X1 2:8 6:4 2:8 6:4 2:8 6:4 2:8 6:4 2:8 6:4 2:8 6:4 2:8 6:4 2:8 6:4 4:6 4:6 4:6 X2 x2 x2 x8 x8 x2 x2 x8 x8 x2 x2 x8 x8 x2 x2 x8 x8 x5 x5 x5 X3 50 50 50 50 58 58 58 58 50 50 50 50 58 58 58 58 54 54 54 X4 3 3 3 3 3 3 3 3 11 11 11 11 11 11 11 11 7 7 7 y 28,67 0,15 1,24 2,57 144,48 18,07 5,62 99,18 130,64 58,36 188,73 55,23 51,94 219,5 129,45 262,94 154,03 155,07 156,1 y^ 28,92 0,4 1,48 2,8 143,72 17,82 5,38 98,94 130,4 58,12 188,48 55 52,72 219,74 129,7 263,18

Cc h s trong phng trnh hi qui c xc nh nh sau:

- Tnh b0:

y
i =1

16

16
16 i =1 ji

, b0 = 87,30

- Tnh bi:

bj =

x y
16

b1 = 2,2; b2 = 5,82, b3 = 29,1, b4 = 49,8

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Science & Technology Development, Vol 10, No.07 - 2007

- Tnh bij:

jl

(x x ) y
16 i =1 j l i

16

b12 = 9,66, b23 = 2,08, b13 = 31,32, b14 = 9,71, b24 = 16,17, b34 = -0,24, b123 = 13,58, b124 = - 21,27, b134 = 32,03, b234 = 6,17, b1234 = -10,19. H s c ngha phi tha mn iu kin:

t
Vi

=
lt

Sbj

t lt ,

: p = 0,05, bc t do f = n-1 = 2

t ( 0,05; 2 ) = 4,3 (Tra bng Student)

Vi y: Hot tnh CMCase (UI/g) theo thc nghim; y^: hot tnh CMCase (UI/g) theo phng trnh hi qui
Sbj phng sai ca cc h s:

S bj =

th

1,03 = 0,26 (vi N = 16, 4

S th =

y y
0

i =1

n 1

= 1,07 (vi n = 3)

S th = 1,03))
So snh cc h s tj vi tlt ta thy, cc h s ca phng trnh u c ngha, ngoi tr cc h s t34. Vy hm mc tiu c dng:

y = 87,3 + 2,2x1 + 5,82x2 + 29,1x3 + 49,8x4 + 9,66x1x2 + 31,32x1x3 + 9,71x1x4 + 2,08x2x3

+16,17x2x4 + 13,18x1x2x3 21,57x1x2x4 + 32,03x1x3x4 + 6,17x2x3x4 10,19x1x2x3x4 T phng trnh hi qui thu c, ta thy rng thi gian nui , m ban u v nng dinh dng c nh hng ng k n hot tnh cellulase hn t l b ma:cm m. Kim tra s tng thch ca m hnh nh gi s sai lch gia thc nghim v m hnh ta da vo tiu chun Fisher. lt , iu kin m hnh tng thch l: tn

F F

Flt: gi tr chun Fisher mc p = 0,05; f1 = N l; f2 = n-1; trong N = 16, l: s h s c ngha = 15, n =3), ( 0 , 05;1; 2 ) = 18,5 = lt (Tra bng tiu chun Fisher)

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TP CH PHT TRIN KH&CN, TP 10, S 07 - 2007

^ y y 2 i i i =1 = ). N l 16

S = 2,00075 = 1,87 ( F : F = S 1,07 S Ta c: F F (v 1,87 < 18,5)

tn
tn tt 2 th

2 tt

tn

lt

Vy: Phng trnh hi qui thu c tng thch vi thc nghim Ti u ha thc nghim theo k hoch leo dc Bng 3.Mi trng nui cy T. reesei VTT-D-80133 theo k hoch leo dc
Mi trng 1 2 3 4 5 6 7 X1 4:6 5:5 6:4 7:3 8:2 9:1 10:0 X2 x5 x6 x7 x8 x9 x10 x11 X3 54 56 58 60 62 64 66 y 265.7233 275.1800 276.2700 280.6400 280.0900 233.1800 140.4600

Kt qu cho thy, mi tng 4 cho hot tnh CMCase cao nht (280,64 IU/g). y c l l mi trng tng i thch hp cho T. reesei VTT-D-80133 sinh trng v to ra cellulase. Vy, gi tr ln cn ti u ca cc yu t nh hng n kh nng sinh tng hp cellulase ca T. reesei VTT-D-80133 l: t l BM:CM (7:3), 8 ln nng dinh dng, m ban u 60%, thi gian nui cy 7 ngy. Kt qa kim chng li hot tnh cellulase v cc enzym khc trong canh trng nh sau: CMCase 280,63 UI/g, FPU 5 UI/g thp hn 3,2 v 37 ln so vi ch phm Amano T. Ngoi cellulase, canh trng cn cha: -amylase 368,75 UI/g, protease 12,43 UI/g v xylanase 10073,25 BXU/g Canh trng nui T. reesei VTT-D-80133 thi im 0 gi v sau 7 ngy, c pH tng i n nh (pH 5,4 5,05); hm lng ng t do t 66,77 mg/g gim cn 35,05 mg/g do nm si s dng lng ng t do c sn trong mi trng nui , lng ng t do sinh ra trong qa trnh sinh trng khng ng k; hm lng protein t 0,79 tng ln 14,45 mg/g; trng lng kh ca mi trng t 8,01 g gim cn 6,17 g cho thy mt lng c cht trong mi trng c s dng. 3.2. Kt qa ng ha giy in qua s dng Dch chit enzym cellulase (5 FPU/ml) ng ha khong 20% giy in qua s dng (10%) trong 24 gi thy phn 500C, pH 5,0; dch ng ha cha 23,62 mg ng kh/ml c th c s dng ln men ethanol hoc ln men sn xut cc sn phm c gi tr. 3.3 Kt qa phn tch h cellulase trn gel SDS-PAGE Kt qu in di trn gel SDS-PAGE ca cc dch enzym cellulase ghi nhn c nh hnh bn. Ging 1: Dch enzym t T. reesei VTT-D-80133.

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Science & Technology Development, Vol 10, No.07 - 2007 Ging 2: Dch enzym t ch phm Amano T Ging 3: Thang phn t lng nh Kt qu cho thy, ging 1 c cc vch protein rt ging vi ging 2. Kt qu ny c th c gii thch nh sau: theo Barnett (1991), h cellulase t T. reesei gm CBHI, CBHII, EGI, EGII, EGIII, EGV v mt -glucosidase; theo bo co gn ay th T. reesei sinh ra hai loi glucosidase l Bgl1 v Bgl2 (Alinda A. Hasper et al., 2001). Da vo cc d liu trn, ta c th kt lun rng canh trng nui cy T. reesei VTT-D-80133 c y cc tiu phn ca h cellulase 1 2 3 kDa

66

45 36 29 24 20 4. KT LUN Mi trng ti u cho T. reesei VTT-D-80133 sinh ra cellulase l: t l BM:CM (7:3), 8 ln nng dinh dng, m ban u 60 %, thi gian nui cy 7 ngy. Hot tnh CMCase v FPU tng ng l: 280,63 IU/g v 5 FPU/g; thp hn 3,2 v 37 ln so vi Amano T. Ngoi cellulase, canh trng cn cha -amylase 368,75 UI/g, protease 12,43 UI/g v xylanase 10073,25 BXU/g Qua phn tch trn gel polyacrylamide, cellulase thu nhn c c cc vch protein vi trng lng phn t bng vi cc vch protein ca ch phm AmanoT. Dch enzym cellulase ca T. reesei VTT-D-80133 vi hot lc 5 FPU/ml, c kh nng ng ha khong 20% giy in qua s dng; dch ng ha cha 23,62 mg ng kh/ml c th c s dng ln men ethanol hoc ln men sn xut cc sn phm c gi tr.

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TP CH PHT TRIN KH&CN, TP 10, S 07 - 2007

OPTIMIZATION OF CELLULASE PRODUCTION BY TRICHODERMA REESEI ON SOLID SUBSTRATE MEDIUM

Tran Thanh Phong, Hoang Quoc Khanh,Vo Thi Hanh, Le Bich Phuong, Nguyen Duy Long, Le Tan Hung, Truong Thi Hong Van Institute of Tropical Biology, Vietnamese Academy of Science and Technology ABSTRACT: Sugarcane baggase/wheat bran ratio, initial moisture content, concentration of basal medium and cultivation time were optimized in solid state fermentation (SSF) for the production of cellulase by an Trichoderma reesei VTT-D-80133 mutant using experiment optimization. Sugarcane baggase/wheat bran ratio (7/3), initial moisture content of 60%, 8 times concentration of basal medium and cultivation time of 7 days were optimum for cellulase production in SSF. The activity and productivity of cellullase obtained after 7 days of fermentation were 280,64 IU/g and 5 FPU/g of fermented medium; These activities are lower than 3,2 and 37 times compared with commercial cellulase (Amano T) of AMANO Company (Japan). Besides cellulase, fermented substrate contain activities of -amylase 268,75 UI/g, protease 12,43 UI/g and xylanase 10073,25 BXU/g. The cellulase extract was shown to have some bands that their molecular weights are equivalent to commercial cellulase (Amano T) by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). At the filter paper activity of cellulase was 5 FPU/ml, the yield of enzymatic hydrolysis of paper waste was 21%. The sugars (23,62 mg/ml) converted from paper waste by cellulase could be used further in the fermentation of valuable products.

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Science & Technology Development, Vol 10, No.07 - 2007 TI LIU THAM KHO [1]. Nguyn Cnh, Qui hoch thc nghim, Trng HBK Tp. H Ch Minh (1993). [2]. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, Enzym vi sinh vt, tp 2, Nh xut bn khoa hc v k thut, H Ni (1982). [3]. Chahal D. S., Solid-state fermentation with Trichoderma reesei for cellulase production, Applied and Environmental Microbiology, Vol. 49, No. 1, p. 205-210 (1985). [4]. Jeffries T. W., Production and applications of cellulase laboratory procedures, Forest Products Laboratory, Madison, Wisconsin (1987). [5]. Liming Xia, Xueliang Shen, High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue, Bioresource Technology 91, pp. 259-262 (2004). [6]. Mary Bigelow and Charles E. Wyman, Cellulase production on bagasse pretreated with hot water, Applied Biochemistry and Biotechnology, pp. 98-100 (2002). [7]. Smits J.P., Solid-state fermentation, modelling fungal growth and activity, The Doctor Thesis (1998). [8]. Uhlig Helmut, Elfriede M. Linsmaier-Bednar, Industrial enzymes and their applications, Awiley-Interscience Publication, John Wiley & Sons, Inc (1998).

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