Science & Technology Development, Vol 11, No.

08 - 2008

THU NHN ENZYME PECTINASE T ASP.NIGER - TINH SCH BNG

PHNG PHP LC GEL & LC MNG

Hunh Ngc Oanh, Trn Ngc Hng

Trng i hc Bch khoa, HQG -HCM
(Bi nhn ngy 10 thng 01 nm 2008, hon chnh sa cha ngy 12 thng 05 nm 2008)

TM TT: Ch phm enzyme pectinase c sn xut t chng nm mc Aspergillus

niger trn mi trng c cha cht cm ng bt c rt sau 48 gi t hot cao nht
30,01(UI/g). Phn tch ch phm enzyme bng phng php lc gel s dng ct Bio Gel P 30
thu c mt peak c hot pectinase v vi tc chy qua ct 15ml/gi c kh nng phn
tch tp cht tt hn tc 30 ml/gi.
Tinh sch ch phm enzyme bng phng php lc mng (cross flow membrane) vi tc
bm u vo 150rpm th tinh sch (tng 3,1 ln trc khi lc) cao hn tc 200rmp
c tinh sch (ch tng 2,2 ln so vi trc khi lc).

1.GII THIU
Enzyme pectinase l enzyme xc tc s thy phn pectin (polysaccharide d th).
Pectinase c sn xut ch yu bi nm mc Aspergillus sp., Botrytis cinerea, Fusarium
moniliforme, Rhizopus stolonifer, Trichoderma sp., Neurospora crassa
Enzyme pectinase thng c dng trong cng nghip thc phm v dc phm c bit
trong cng ngh sn xut cc loi nc qu v ru vang. Ngi ta s dng enzyme pectinase
kh pectin, trnh hin tng b c v lng cn trong qu trnh sn xut v bo qun [3,5].
Trong bi bo co ny, chng ti trnh by kt qu nghin cu iu kin thch hp thu
nhn v tinh sch enzyme pectinase bng lc gel v lc mng.

2. NGUYN LIU & PHNG PHP

2.1. Nguyn liu thu nhn enzyme pectinase
Nm mc Aspergillus niger c gi ging trn mi trng Czapek. Bt c rt: c rt xay
nhuyn, sy nhit 60oC cho n khi m c rt t 4% (ngun pectin cht cm ng)
Thnh phn mi trng nui cy thu nhn enzyme pectinase gm: cm go 65,5%, tru
21,5%, bt c rt 11%, (NH4)2SO4 2%, m 55%. Kh trng 121oC trong 30 pht.
2.2. Phng php nui cy thu nhn enzyme pectinase t phng php nui cy b
mt [2]
2.3. Xc nh hot enzyme pectinase [4]
Enzyme pectinase th c xc nh hot bng phng php o nht vi nht k
Borosil: ht 2ml dung dch enzyme th 5% (w/v) phn ng vi 18ml dung dch pectin 1%
pH 4,5 v nhit 40oC. Mt n v hot pectinase (UI) l lng enzyme cn thit lm
gim 10% nht ca hn hp cha 180mg pectin di nhng iu kin nh trn.
2.4. Hm lng protein ca ch phm enzyme c xc nh bng phng php
Lowry[1]
2.5. Phn tch ch phm enzyme bng phng php lc gel Bio Gel P 30

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 TP CH PHT TRIN KH&CN, TP 11, S 08 - 2008

Ha tan ch phm enzyme th trong m Mc Ilvaine pH 4,5 ly tm b cn thu c dung

dch enzyme sau ht 0,5ml cho chy qua ct Bio Gel P 30 (0,8 x 30cm) (hng Biorad M)
c cn bng vi m Mc Ilvaine pH 4,5. Tc chy qua ct ln lt l 30ml/gi v
15ml/gi. Mi phn on thu 2ml v 1,5ml. o hp thu A ca tng phn on bc sng
280nm. V th ghi nhn cc peak to thnh v xc nh hiu sut thu hi enzyme v hot
tnh tng ng.
2.6. Tinh sch enzyme pectinase bng phng php lc mng (cross flow membrane)
Tin hnh ta enzyme t 500ml dch chit thu c bi ethanol 96o, nhit ta 4oC, thi
gian ta l 1 gi. Sau , ly tm 5000 vng/pht trong 15 pht thu enzyme th. Ha tan
enzyme th ny trong 500ml dung dch m Mc Ilvaine pH 4,5. Tin hnh lc 500ml dung
dch enzyme va ha tan trn bng h thng lc QuixStand Benchtop Systems (hng
Amersham Biosciences) vi b lc mng (membrane) c khong phn on 50kDa. Tc
bm mu u vo ln lt l 150rpm & 200rpm v iu chnh sao cho p sut trn b mt
mng khng qu 5 Psi. Thu dch qua lc v xc nh hiu sut thu hi enzyme v hot
tng ng.

3. KT QU & BN LUN
3.1. Hot chung v hot ring ca ch phm pectinase th (CP E)

Hnh 1. th tng quan gia thi gian nui cy v hot ca enzyme th

Qu trnh sinh tng hp pectinase trn mi trng c cht cm ng l pectin ca bt c rt
bi chng Aspergillus niger c hot cao nht sau 48 gi (30,01UI/g CP E).

Hnh 2. th tng quan gia thi gian nui cy v hot ring ca ch phm enzyme th

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Science & Technology Development, Vol 11, No.08 - 2008

Qu trnh sinh tng hp pectinase trn mi trng c cht cm ng ca chng Aspergillus

niger c hot ring cao nht sau 72 gi t 8,97.10-2 (UI/mg protein).
Da vo 2 th ta thy, hot chung gia thi gian nui cy 48 gi v 72 gi chnh
lch nhau kh nhiu (30,01 & 25,02UI/g CP E). Trong khi , hot ring gia thi gian
nui cy 48 gi v 72 gi chnh lch nhau khng nhiu (8,26.10-2 & 8,97.10-2 UI/mg
protein). Do , chng ti quyt nh thu nhn enzyme pectinase sau 48 gi nui cy v sau
em tin hnh tinh sch.
3.2. Phn tch ch phm enzyme pectinase bng phng php lc gel Bio Gel P 30
Dung dch enzyme x l theo mc 2.5, kt qu sau khi lc gel vi tc chy qua ct
30ml/gi thu c 2 peak chnh (hnh 3a). Trong , hot pectinase tp trung ch yu
peak I, peak II khng c hot . Hiu sut thu hi enzyme t 71,96% v hot cao hn 1,1
ln so vi trc khi lc gel.
Khi gim tc chy qua ct xung 15ml/gi kt qu thu c 4 peak (hnh 3b). Hot
pectinase tp trung hu ht peak I. Hiu sut thu nhn hi enzyme sau khi gim tc lc
t 59,02% v hot cao hn trc khi lc gel 1,65 ln.

a-tc : 30ml/gi b-tc : 15ml/gi

Hnh 3. th tng quan gia gi tr OD 280nm vi cc phn on ca dung dch enzyme sau khi lc
gel
Khi gim tc chy xung 15ml/h th cc phn t protein nm trong ht gel b ra gii
chm hn. Khi , cc phn t khc khng nm trong ht gel s b ra gii ra khi ct nhanh
hn v dn n hm lng protein c hot pectinase tng. Hiu sut thu hi enzyme khi lc
vi tc 15ml/gi thp hn hiu sut thu hi enzyme qua lc vi tc 30ml/gi do kh
nng tch protein tp khi lc vi tc thp th tt hn.
3.3. Tinh sch ch phm enzyme pectinase bng phng php lc mng
Chng ti s dng b lc membrane c kch thc phn on rng 50kDa cho kt qu
nh sau:
Sau khi lc dung dch enzyme nh mc 2.6, kt qu thu c khi lc vi tc bm
150rpm thu dch qua lc. Kt qu thu c hiu sut thu hi enzyme v hot t 27,87% v
87,98%, tinh sch tng 3,1 ln. Khi tng tc bm ln 200rpm th hiu sut thu hi
enzyme v hiu sut hot t 33,33% v 73,65%, tinh sch ch tng 2,2 ln.
Nguyn nhn l khi tng tc bm 200 rmp th p sut u vo tng (5,2Psi) lm cho
dng chy trong b lc chuyn ng ri, cc phn t s va p mnh vo nhau v y chng
vo thnh b lc kt qu hnh thnh mt mng mng xung quanh thnh b lc (hay cn gi l
lp tr lc). Mt khc di tc dng ca lc ly tm trong dng chy s y mt s phn t

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 TP CH PHT TRIN KH&CN, TP 11, S 08 - 2008

trong mng mng theo dch qua lc ra pha ngoi. V th, chng ti nhn thy vi tc bm
u vo 150rpm s thc hin qu trnh tinh sch tt hn.

Hnh 4. So snh hot ring ca dung dch enzyme trc v sau khi lc mng

4. KT LUN
Ch phm enzyme pectinase c sn xut t chng nm mc Aspergillus niger trn mi
trng c cha cht cm ng l pectin-bt c rt bng phng php nui cy b mt sau 48
gi t hot chung cao nht 30,01(UI/g).
Phn tch ch phm enzyme bng phng php lc gel s dng ct Bio Gel P 30 (0,8 x
30cm) thu c mt phn on c hot enzyme pectinase v vi tc chy qua ct
15ml/gi tch tp cht tt hn 30 ml/gi.
Phng php lc mng vi tc bm 150rpm c th ng dng tinh sch enzyme
pectinase thu nhn t Asp. niger ni ring v enzyme ni chung vi k thut n gin v hiu
qu.
phn tch v tinh sch c hiu sut cao hn nn chn ct lc gel v kch thc mng
lc ph hp vi tng loi enzyme.

PURIFICATION OF PECTINASE FROM ASP.NIGER BY THE

TECHNIQUES OF GEL FILTRATION AND CROSS-FLOW MEMBRANE

Huynh Ngoc Oanh, Tran Ngoc Hung

University of Technology, VNU-HCM

ABSTRACT: Pectinase from Aspergillus niger by solid state medium containing

powered carot as an inducer obtained the highest activity after 48 hours. Separating enzyme
on a gel filtration column packed with Bio gel P 30, which showed that one peak contained
activity of pectinase, and with column flow rate of 15 ml/h the separation was better than 30
ml/h.
Using cross flow membrane with speed-pump of 150 rpm at the input, the purification of
enzyme (3.1 times before filtration) was higher than at rate of 200rmp (2,2 times before
filtration).

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Science & Technology Development, Vol 11, No.08 - 2008

TI LIU THAM KHO

[1]. Lm Th Kim Chu, Vn c Chn, Ng i Nghip. Thc tp ln Sinh ha, Nh

Xut Bn i hc Quc gia Tp. HCM.(2004).
[2]. H Xun Hng. Thu nhn v s dng enzyme pectinase trong sn xut nc da,
Lun n cao hc i hc Bch khoa Tp. HCM.(2002).
[3]. Nguyn Quang Tm, L Th Hng Nga, ng Th Thanh Thu, Nguyn Th Tuyt
Thanh. Tinh sch v c nh enzyme pectinase thu nhn t mt s chng nm mc.
Bo co Hi ngh khoa hcTrng i hc Khoa Hc T Nhin, p.187-193.(2002).
[4]. Favela-Torres, E.; Volke-Seplveda, T.; Viniegra-Gonzlez, G. Production of
hydrolytic depolymerase pectinase, Food Technol. Biotechnol, 44:221-227. (2006).
[5]. Patil, S.R.; Dayanand, A. Exploration of regional agrowaste for the production of
pectinase by Aspergillus niger, Food Technol. Biotechnol, 44:289-292, (2006).

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