TAP CH PHAT TRIEN KH&CN, TAP 12, SO 04 - 2009

KHO ST KH NNG THU NHN V C NH ENZYM GLUCOAMYLASE T Asp.niger V Asp.awamori TRN CHT MANG KAOLIN
Ng Minh Nh (1), ng Th Thanh Thu (2) (1)Trng i hc Bch Khoa, HQG-HCM (2)Trng i hc Khoa Hc T Nhin, HQG-HCM
(Bi nhn ngy11 thng 8 nm 2008, hon chnh sa cha ngy 04 thng 12 nm 2009)

TM TT: Ngy nay enzym c nh c s dng rt rng ri trong cc ngnh cng nghip nh vo kh nng ti s dng v kh nng iu khin qu trnh sn xut bng enzym. Nghin cu ny kho st kh nng thu nhn v c nh enzym glucoamylase t Asp.niger v Asp.awamori trn cht mang Kaolin. Hot ca ch phm enzym (CPE) glucoamylase thu nhn t Asp.niger l 143325,62 UI/g-CPE v Asp.awamori l 133418,20 UI/g-CPE. Thi gian c nh enzym glucoamylase t Asp.niger t hiu sut c nh cao nht sau 50 pht v i vi Asp.awamori l 40 pht. Khi lng cht mang cho hiu sut c nh enzym glucoamylase cao nht l 1g/0,1g enzym. Kh nng ti s dng ca enzym glucoamylase t Asp.niger sau khi c c nh trn kaolin cao hn kh nng ti s dng ca enzym glucoamylase t Asp.awamori sau khi c c nh trn kaolin. 1. M U Glucoamylase l mt enzym quan trng thuc h enzym amylase [7]. Enzym ny ang thu ht s quan tm ca nhiu nh sn xut nh vo kh nng thy phn lin kt -1,4 glycoside v -1,6 glycoside thm ch c lin kt -1,3 glycoside trong mch amylose v amylopectin thu c sn phm trit l glucose [1]. Nh vo c tnh ny, glucoamylase c s dng trong cng nghip sn xut ng glucose v mt tinh bt. Hin nay gi thnh ca enzym kh cao, nn vic c nh enzym rt c ch trng. Nhm phn no p ng nhu cu trn, chng ti thc hin nghin cu: Kho st kh nng c nh v thu nhn enzym glucoamylase t Asp.niger v Asp.awamori trn cht mang Kaolin. Mc ch ca nghin cu ny l tm hiu kh nng c nh enzym glucoamylase ca Kaolin. Ni dung nghin cu gm mt s vn sau: Kho st hot ca glucoamylase thu nhn t Asp.niger v Asp.awamori Kho st mt s yu t nh hng n s c nh CPE glucoamylase. Hiu sut c nh enzym trn cht mang Kaolin v kh nng ti s dng. 2. NGUYN LIU V PHNG PHP NGHIN CU 2.1. Nguyn liu Chng nm mc Asp.niger v Asp.awamori do phng th nghim Sinh ha trng H Khoa hc T nhin Tp.HCM cung cp Mi trng nui cy (Mi trng bn rn) [2] Cm 70% Tru 25% B sn 5%

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Science & Technology Development, Vol 12, No.04 - 2009 Dung dch khong Czapeck b sung vo mi trng to m 50% Kh trng mi trng nui cy 1210C, 20 pht Cc ha cht v cht mang Kaolin s dng trong nghin cu ny u do Trung Quc sn xut. 2.2. Phng php nghin cu Phng php thu nhn CPE glucoamylase t Asp.niger v Asp.awamori:[1], [4] Sau thi gian nui mc 60 h qu trnh tch chit thu nhn CPE bn tinh khit c thc hin bng cch sau: Canh trng nui nm mc c khuy trn vi nc (t l nc ct v canh trng l 3:1), lc qua vi lc ly dch lc, thu c phn dch cha enzym. Lm lnh nhanh dch chit enzym xung cn 3-50C, kt ta enzym bng cn theo t l 3 cn : 1 dch enzym (cn cng c lm lnh 3-50C). Ly tm dch ta trong 15 pht tc 5000 vng/pht. Thu ta, sy nhit 35-400C cho n khi m ca ta t 11-14%, bo qun nhit lnh. Cc phng php phn tch Xc nh hm lng protein theo phng php Lowry [6] Xc nh hot enzym glucoamylase theo phng php so mu vi DNS [3]
Hiu sut thu enzym (%) = Trng lng CPE Trng lng canh trng x 100 (%)

Phng php c nh enzym [1], [5] - Ha tan mt lng enzym v kaolin vo mt th tch dung dch m nht nh (50ml). Khuy nh cho enzym c kt dnh ln b mt cht mang, to dng enzym khng tan (enzym c nh). - Sau mt thi gian, lc hn hp trn bng giy lc, ra hn hp li bng dung dch m loi b lng enzym cha c c nh cn li trong hn hp. Thu lng enzym c nh trn kaolin, sy nhit 35-400C, bo qun nhit lnh. Cng thc tnh ton [3] - Mt n v hot (UI) ca glucoamylase l lng enzym xc tc thy phn tinh bt gii phng ra 1g glucose trong 1 pht iu kin nhit v pH thch hp. - Hot ring ca enzym l s n v hot enzym c tnh trn 1mg protein ca CPE - Hiu sut c nh enzym l t l gia tng n v hot enzym sau khi c c nh v tng n v hot enzym ca mu trc khi c nh. 3. KT QU V BN LUN 3.1. Kho st hot ca glucoamylase thu nhn t Asp.niger v Asp.awamori - CPE sau khi trch ly t Asp.niger v Asp.awamori c ha tan vo dung dch m c pH=5 v xc nh hot theo phng php so mu vi DNS. Kt qu thc nghim c trnh by bng 3.1. Bng 3.1.Hot glucoamylase thu nhn t Asp.niger v Asp.awamori
Ging vi sinh vt Asp.niger Asp.awamori Hot (UI/g-CPE) 143325,62 133418,20

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TAP CH PHAT TRIEN KH&CN, TAP 12, SO 04 - 2009 Nhn xt: Hot ca CPE glucoamylase thu t Asp.niger cao hn CPE t Asp.awamori. Hot ca enzym glucoamylase t Asp.niger l 143325,62 U/g-CPE v t Asp.awamori l 133418,20 U/g-CPE. Nh vy c th thy trong cng iu kin nui cy, kh nng sinh enzym glucoamylase ca 2 loi Aspergillus l khc nhau v Asp.niger c th sinh enzym glucoamylase c hot tnh cao hn enzym glucoamylase t Asp.awamori - S dng cn l tc nhn gy ta enzym, hiu sut thu nhn CPE c trnh by bng 3.2. Bng 3.2.Hiu sut thu nhn CPE glucoamylase
Ging vi sinh vt Asp.niger Asp.awamori Trng lng canh trng (g) 50 50 Trng lng CPE thu c (g) 3,56 2,92 Hiu sut thu enzym (%) 7,12 5,83

Nhn xt: Vi tc nhn ta l ethanol 96% th hiu sut thu hi CPE t canh trng Asp.niger l 7,12% cao hn so vi hiu sut thu hi CPE t Asp.awamori 5,83%. Khi cho dung mi hu c vo dung dch enzym s tch trit lp phn t nc bao ly xung quanh phn t protein, lm gim tnh tan ca protein, tng kh nng kt ta ca chng. Chng ti s dng cng mt phng php thu CPE th, nhng hiu sut thu nhn enzym glucoamylase t 2 loi nm mc trn li khc nhau. C th do cu trc glucoamylase khc nhau gia cc loi nm mc nn khi tip xc vi tc nhn gy ta to nn lng kt ta chnh lch 1,22 ln. - Hot ring ca CPE glucoamylase c trnh by bng 3.3. Bng 3.3.Hot ring ca CPE glucoamylase
Ging vi sinh vt Asp.niger Asp.awamori Hot (UI/g-CPE) 143325,62 133418,20 Hm lng protein (mg/g-CPE) 37,11 36,67 Hot ring (UI/mg protein-E) 3862,18 3638,34

Nhn xt: Hot ring ca CPE glucoamylase t Asp.niger l 3862,18 UI/mg protein cao hn hot ring ca glucoamylase t Asp.awamori l 3638,34 UI/mg protein. V hot ring c trng cho mc tinh sch ca enzym, iu chng t tinh sch ca enzym glucoamylase t Asp.niger cao hn enzym t Asp.awamori. 3.2. Kho st mt s yu t nh hng n s c nh CPE glucoamylase 3.2.1.Kho st thi gian c nh enzym ln cht mang - Ha tan 0,1g enzym glucoamylase v 1g kaolin trong 50ml dung dch m acetat pH=5. Thc hin phng php hp ph c nh enzym vi thi gian c nh khc nhau 20 pht, 30 pht, 40 pht, 50 pht v 60 pht. Kt qu thc nghim c trnh by hnh 3.1

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Science & Technology Development, Vol 12, No.04 - 2009

UI
8000 7000 6118.83 6000 5000 4000 3000 2000 1000 0
1 2 3 4 5

6986.88

7287.81 5814.04 5187.31 4341.82

5756.75

4471.45 4814.04 4185.57

20
Asp.niger

30
Asp.awamori

40

50 60 Thi gian (pht)

Hnh 3.1. S bin i s n v hot CPE glucoamylase t Asp.niger v Asp.awamori c c nh theo thi gian

Nhn xt: Kaolin l mt khong vt st thng gp trong t nhin, trng mn hoc ng vng. N c cu to thnh tng lp, mi lp nh vy c mt tm t din SiO4-4 v mt tm bt din Al(OH)6-3. Tm t din quay nh chung v pha tm bt din. v tr ca nh chung t din v bt din th ion OH- ca bt din c thay th bng ion O2- ca t din. O-2 trong trng hp ny l nhng ion c kh nng lin kt tng i bn vng vi cc cht b hp ph nh cc phn t enzym [8]. i vi enzym glucoamylase t Asp.niger tng n v hot enzym c c nh tng dn v cao nht sau thi gian khuy l 50 pht l 7287,81 UI v hiu sut c nh t 50,85%, sau thi gian 50 pht th hot gim dn v thi gian khuy qu lu s lm cho enzym b tch ra khi cht mang. Cn vi enzym glucoamylase t Asp.awamori th thi gian khuy ti u l 40 pht, tng n v hot enzym c c nh l 5756,75 UI v t hiu sut c nh 43,15%, sau thi gian khuy 40 pht th tng n v hot enzym c nh cng gim dn. 3.2.2. Kho st nh hng ca khi lng cht mang n kh nng c nh ca enzym - Ha tan 0,1g enzym glucoamylase trong 50ml dung dch m acetat c pH=5 v khi lng kaolin s dng ln lt l 0,5g; 1g; 1,5g; 2g. Tin hnh c nh enzym, kt qu thc nghim c trnh by hnh 3.2
UI

9000 8000 7000 6000 5000 4000 3000 2000 1000 0

1 Asp.niger Asp.awamori

7964.04 6799.23 5652.01 6314.04 6071.45 5087.19 4297.38 3670.52

0.5

1.5

2 KL Kaolin (g)

Hnh 3.2.S bin i tng s n v hot ca CPE glucoamylase t Asp.niger v Asp.awamori c nh theo khi lng kaolin

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TAP CH PHAT TRIEN KH&CN, TAP 12, SO 04 - 2009 Nhn xt: S thay i khi lng kaolin c nh hng r rt n tng s n v hot enzym glucoamylase c nh. i vi CPE glucoamylase t 2 loi nm mc khi tng khi lng kaolin t 0,5g ln 1g th s n v hot ca enzym cng tng theo. Cn khi tng khi lng cht mang t 1g n 2g th tng n v hot enzym b gim i. V kaolin c cu to lp v phn tn cao, nn chng c tnh do c bit, c kh nng to kt dnh, v vy khi tng khi lng cht mang, cc ht kaolin kt dnh vo nhau lm cho din tch tip xc vi enzym gim i v gim hiu sut c nh enzym. S n v hot enzym glucoamylase t 2 loi Aspergillus c nh t gi tr cao nht khi s dng t l khi lng enzym: kaolin = 1:10. S n v hot enzym glucoamylase ca Asp.niger c nh l 7964,04 UI (hiu sut c nh 55,56%) v s n v hot enzym glucoamylase ca Asp.awamori c nh l 6314,04 UI (hiu sut c nh 47,32%) T nhng kt qu nghin cu thu c trn, chng ti nhn thy t l khi lng cht mang 1g/0,1g enzym c hiu sut c nh enzym tt nht. i vi enzym glucoamylase t Asp.niger thi gian c nh enzym hiu qu nht l 50 pht v thi gian c nh tt nht cho enzym glucoamylase t Asp.awamori l 40 pht 3.2.3. Kho st kh nng ti s dng ca enzym c nh Xc nh kh nng ti s dng ca enzym glucoamylase c nh trn Kaolin qua s thy phn tinh bt v s bin i tng n v hot UI . Kt qu thc nghim c trnh by hnh 3.3.
UI
8000 7000 6000 5000 4000 3000 2000 1000 0 1
Asp.niger

6966.51

6573.53 5899.88 5381.52

4489.20

4329.32 3958.80 3443.60 3229.86 2663.58 2441.90 2325.62 1468.94

Asp.awamori

S ln s dng (ln)

Hnh 3.3: So snh kh nng ti s dng ca CPE glucoamylase t Asp.niger v Asp.awamori c nh qua s bin i UI

Nhn xt: Sau khi c c nh trn kaolin, enzym glucoamylase t Asp.niger c th ti s dng n 7 ln. Sau 7 ln s dng s n v hot enzym ch cn 2441,90 UI tng ng 35,05% so vi tng UI c nh ban u. Cn i vi enzym glucoamylase t Asp.awamori, sau 6 ln ti s dng s n v hot l 1468,94 UI tng ng 32,72% so vi tng UI c nh ban u. C th ni enzym glucoamylase t Asp.niger c nh trn kaolin c kh nng ti s dng cao hn enzym t Asp.awamori.

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Science & Technology Development, Vol 12, No.04 - 2009 4. KT LUN Qua cc thc nghim, chng ti i n nhng kt lun nh sau: Hot ca CPE glucoamylase thu nhn t Asp.niger l 143325,62 UI/g-CPE v t Asp.awamori l 133418,20 UI/g-CPE. Thi gian c nh enzym glucoamylase t Asp.niger t hiu sut c nh cao nht l 50,85% sau 50 pht .Thi gian c nh enzym glucoamylase t Asp.awamori t hiu sut c nh cao nht l 43,15% sau 40 pht . Khi lng cht mang cho hiu sut c nh enzym glucoamylase cao nht l 1g/0,1g enzym. Hiu sut c nh i vi CPE t Asp.niger l 55,56% v hiu sut c nh CPE t Asp.awamori l 47,32%. Kh nng ti s dng ca enzym glucoamylase t Asp.niger sau khi c c nh trn kaolin cao hn kh nng ti s dng ca enzym glucoamylase t Asp.awamori sau khi c c nh trn kaolin. Enzym glucoamylase c hai loi nm mc trn sau khi c nh c th ti s dng trn 6 ln. Cc s liu th nghim trong nghin cu ny c tnh ton, x l theo phng php ANOVA

STUDYING THE EXTRACTION AND THE IMMOBILIZATION GLUCOAMYLASE FROM Aspergillus niger AND Aspergillus awamori ON KAOLIN
Ngo Minh Nha (1), ong Thi Thanh Thu (2) (1)University of Technology, VNU-HCM (2)University of Natural Sciences, VNU-HCM ABSTRACT: Nowadays the immobilized enzyme used universally in industrials thanked to capability re-use and industrial process control capability by enzyme. This research studying the receiving and the immobilization glucoamylase from Aspergillus niger and Aspergillus awamori on kaolin. Activity of enzyme glucoamylase extract from Aspergillus niger is 143325, 62 UI/ g-product enzyme and Aspergillus awamori 133418, 20 UI/ g-enzyme product. Examination the time of support-enzyme, activity of enzyme glucoamylase extract from Aspergillus niger is highest at 50 and from Aspergillus awamori is 40 minutes. The suitable mass of kaolin is 1g/0,1g enzymes .The capability re-use of the immobilized enzyme glucoamylase from Aspergillus niger is higher than the immobilized enzyme from Aspergillus awamori. Keyword: enzyme glucoamylase, immobilization, kaolin, Aspergillus niger, Aspergillus awamori.

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TAP CH PHAT TRIEN KH&CN, TAP 12, SO 04 - 2009 TI LIU THAM KHO [1]. Nguyn c Lng (ch bin), v cng s, Cng ngh enzym, NXB i hc Quc gia Tp.HCM, trang 60, 160 (2004) [2]. Nguyn c Lng (ch bin), Phan Th Huyn, Nguyn nh Tuyt, Th nghim Cng ngh Sinh hc Tp 2, NXB i hc Quc gia Tp.HCM trang 108-109 (2006) [3]. V Vit Phi, Nghin cu thu nhn glucoamylase t Asp. Kawasaki, Chuyn ngnh Khoa hc v Cng ngh Thc Phm, i hc Bch Khoa TP.HCM trang V-VI (2005) [4]. ng Th Thanh Thu, Sinh ha ng dng, NXB i hc Quc gia Tp.HCM, trang 214-218 (2003) [5]. ng Th Thanh Thu, S c nh enzym v ng dng, Gio trnh i hc Khoa hc T nhin TP.HCM,trang 30-38 (2001) [6]. Lowry.O.H, N.J.Rosebrough, Protein measurement with the Folin-phenol reagents, J. Biol.Chem.193, trang 265-275 (1965) [7]. Wolfgang Aehle, Enzymes in Industry, Wiley_VCH Verlag GmbH and Co.KgaA, Weinheim, trang 203-204 (2004) [8]. http://www.patentstorm.us/patents/5614401/description.html

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