HOl NGHI KHOA HOC C O N G N G H E SINH HQ :
T 6 I U'U H 6 A CAC DI&U KIEN MOI TRUCXNG A N H HUONG D ^ N KHA NANG^
SINH T 6 N G nap CHITINASE CCIA CHONG NAM PENICILLIUM SP.M4 PHAN LAP TO* RUQNG MIA
VQ Thj Thanh, Vu Van H^nh, Nghlinn Ngpc Minh, Quyfin Dinh Thi Viin C6ng ngfi^ sinh hpc, Vifn Hin tim Khoa hgc vi Cdng nghi W$t Nam
T 6 M TAT
Nim ki sinh con trimg thuong tac dong dfin nhirng lofii mo nhat djnh nhu m5 mo va cic mo khfic bi phan huy boi cic nzymc (chitinase, protease, lipase) ciia nim. D|tc bi?t, hoat tinh chitinase cing mimh thi t6c dO di6t cfln trung cing nhanh v i hi$u qut cao. Chung nim k^ sinh con trung Penicillium sp. M4 dugc phSn lip ti^ xac r^p ch6t bdi nam gay b?nh trfin canh dong mia b ngoii thinh Hi Ngi c6 khi nang sinh ^ n ^ hop chitinase. Cic difiu I d ^ nuoi ciy thich hgp cho s^ sinh tong hgp chitinase (Ui du^c t& na trong mdi tniong I6n men r5n. Ngudn co chit l>6t ngo vdi dg diy 17 mm, do im co chit 60%, pH 6, b6 sung 1% co chit chitin cim img vi 0,9% ure lam ngufin nito vo co, thdi gian Ifin men li 5 ngiy d nhi§t dg 30°C la thich hgp nhat cho svr sinh long hgp chitinBse cua cbung nay. Hoft tinh chitinase cao nhit sau khi toi uu dieu k i ^ nuoi ciy ciia chi^g niy la (8,02 ± 0,02) U/ml. K6t qui tren c6 tinh khi dii cao khi ip dyng vio d i ^ kien thyc te vi nguyen Ii§u bo sung vio moi tnrang rat re tien, san c6 va cac difiu k i ^ nuoi cay pbit bgp vdl khi hfiu cua Vi^ Nam.
TO khoa: chitmase, difiu kifin t6i ini. nim ky sinh con bung, moi truong Ifin men ran, Penicillium sp. M4
utrBku
Hlu h^t cdc loSi can trung gay h^i deu c6 the b| nhiSm b^nh biri cic vi sinh v$t (Iwloazami, 2002). Cung nhif vi khuSn, nhi^u loSi n^m cb quan h§ cgng sinh ho§c hogi sinh v6l c6n ttOng, ttDng 66 c6 n h i ^ lodi n^m ky sinh. gSy hi$n tugng b$nh tj trin cdn tnJng. N^m gSy b^nh ttieo con dircmg chinh Id bao ti> nay milm phdt ttien thdnh h^ sgi an sdu vte khoang bgng, qua d u ^ g tiSu h6a. thfing qua cdc khf qudn vd chOng phfi kin cdc l5 khF con ttilng Idm chOng ch^L Nira cdn gdy b$nh cho cdn tmng bang cdch ti^t dgc t6 vd cdc enzyme thuy phdn nhir protease, lipase, chitinase (Madelin, 1963). ThuOc cdc enzyme thuy phdn nhif protease, lipase, chitinase... th) chitinase dang rSt du'g'c quan tdm vi nb c6 khd ndng thOy phdn chitin-nguydn li$u ca bdn cau thdnh nen ldp vi c6n trOng. Trong qud trlnh ky sinh cfin triing, hogt tlnh chitinase cdng mgnh thi t6c d$ digt c6n trfing cdng nhanh vd hi^u qud cao, ti4t ki^m thd'i gian. Di;a vdo d|ic tlnh ndy cOa nim, vigc nghien ci>u tao ra chitinase b6 sung vdo chS phSm sinh hpc Id r i t d n Uiiet Chitinase li enzyme thuy phdn chitin thdnh cdc don phdn N-acetylglucosamine, chitobiose hay chitotriose qua vigc thGy phdn lidn kit p-1,4-glucoside gii>a Ci vd C4cOa 2 phdn tt> N-acetyiglucosamine lidn tiep nhau trong chitin. Tuy nhidn, chitinase thifcmg phim hi$n nay cd gid ttidnh khd d i t VI v§y, vi0c nghidn cCru t6i iru dieu ki$n len men tang khd ndng sinh ting hpp chitinase cr nim ky sinh cdn trijng Id rat cd 'j' nghTa. Trong bdi bdo ndy, chOng tdi dd t6i ifu cdc diSu kiOn sinh t6ng hpp chitinase nhim tgo nguydn lidu cho vi^c Idm tdng higu qud didt r$p cOa c h l phim bdo ti> nim vira tlm ra ngudn nguydn lidu nudi c l y re ti^n, san cd bing phifong phdp Idn men ran.
NGUYEN UEU VA PHl/aNG PHAP Nguyen ligu
Chdng nim Penidllium sp. M4 Oirac phdn Igp tCr xdc rdp chet b6i nam bgnh ttdn canh d6ng mia if phuang Ding Mai, qudn Hd Ddng, Hd Ndi.
Chitin huyin phO 1%: I g bdt chitin dugc them ti> tii' vdo 20 ml dung djch HGI ddm ddc, khuly d i u vd 0 trong 8 gl&., Thdm vdo h5n hgrp 200 ml ettianol Ignh (-20°C) vd de qua ddm. Ly tdm h6n hop d 4000 vdng/phiit, ttong 20 phiit, thu liy'' kit tua vd n>a tiia bing ddm phosphate 0,05 M cho d i n khi dgt pH trung tinli. Thdm vdo tua dung djch dgm phosphate din khi till tich 100 ml.
Phipomg phap
Cfipn Igc chiing nam c6 dgc lg>c diet r$p cao
Dung djch 0,05% Tween 80 de ttiu bao tt> tt> cdc dTa nudi nam sau 12 ngdy. Tdm ml dung djch nong dd 10* bdo tiWrt duvc phun trdn Id ngd (9x9 cm^) ddt sin 25 con rgp mudi hgi ngd. Sau khi phun bdo *> Idn r^p ngd, hdng ngdy, kiim tra s l lugng rdp cdn song sdt rgp miri sinh d 30°C va 70% dfi I m . d l Hnh ty le rgp chet ttieo cdng thi>c ciia Abbott (1925):
Rdp chit (%) = (1- A/B) x100; bx>ng do A: ty Ig (%) sd r§p song sdt tten Id ttii nghigm, B: ty Ig (%) so rgp song sdt trSnB dfii chiJng.
Chgn Igc chung nim kf sinh con triing co ho^t tinh chitinase cao: Uneo phuong phdp khuich tan trdn dTa ttigch sir dgng cc chat chitin cua Nguyen Ldn Dung vd ding tdc gid (1976).
Xic djnh ho^t tinh chitinase
Hogt tlnh chitinase dupe xdc d]nh dya vdo lu^ng dud-ng khi> N-acetyl-p-D-Glucosamine sinh ra trong phdn irng thiiy phdn. Khi cho chitinase tdc dgng vdi cc chat Id chitin huyen phO, N-acetyl-p-D-Glucosamine sinh ra ttong phdn i>iifl dupe djnh lupng qua phdn iing vdi thulc thu- DNS (3,5 - dinitrosalisylic acid), cho sdn phdm 3-amino-5 nitrosallcylic add hip thy dnh sdng kha kiln d budc sdng 540 nm.
Dich enzyme dupe chilt tach bdng nude cit (90 ml) tren may lie ttong v&ig 2 gid (200 vdng/phut), Ipc qua vdl man thu
; CONG NGHE SINH H p C T O A N Q U O C 2013
d|Ch lpc. Dem djch lpc ly tam 5000 vdng/phiit ttong 10 phut, ttiu dich nli Id djch enzyme ttid. 6ng ttit nghldm: 50 \ii dfch nK^ua**^^ + 50 pi chitin huyin phu 1%. H6n hpp dupe lac diu vd ii d 40°C trong vdng 120 phiit thi b l sung 100 |ji DNS. Hdn hpp dupe lac ddu rli dun cdch Uiuy d lOO^C ttong vdng 5 phOL Sau dd d l ngugi. pha loang d i n 1 ml vd dO OD d budc sdng 540 nm. Ong doi chimg: 50 ^1 djch enzyme thd + 100 p! DNS, lac diu, sau dd mdi ttidm 50 pi chitin huydn phu 1% vd tilp tyc Idm nhu frdn. MOt flon vj (unit) chitinase tuang dng vdi 1 jjmoi NAG (N-acetylglucosamine) t90 ra tu phdn dng thOy phdn eo chit chitin bxMig ihdi gian 1 phOL
Anh hwdng cua diiu kien m6l trvdng din kha ning sinh ^ng hpp r^itinase
Chiing nam Penicillium sp.M4 dupe nudi lac ttong mdi ttudng PDB d 30''C. Sau 4 ngdy, dich nudi cd mdt dd bdo ti>
5.10 /ml dupe cay vdo blnh ndn 250 ml chua ca chit ttieo fi Id 1 ml/10 g ca chit.
Ca chit: loi ngd. ggo, cam ggo. bOt ngd, hSn hpp: cdm gao/bdt ngd, b^t ngd/ldl ngd. ggo/bdt ngd, cdm ggo/ggo. cdm ggo/ldi ngo, ggo/loi ngo {1:1.w/w). Nude dupe bo sung bfong dng la 80%, 50%. 80%, 50%, 100%. 100%, 80%, 100%, 100%. 100% so vdi ea chiL cOng vdi 1% chitin lam ea chit cdm ung.
BQ diy CO chit, khd ndng sinh t6ng hpp chitinase dupe khdo sat d cdc dp ddy (mm) ca chat: 6, 9; 11; 13,5; 15; 17; 19;
21; 23 vd 25 (tuong duong vdi 6. 8. 10, 12, 14. 16. 18. 20. 22, 24 g co chlt/blnh tam gidc dung tich 250 ml). B l sung 50% dp am so vdi ca chdt bdt ngd vd 1% chitin Idm eo chit edm ung.
Dp im ccr chit ChOng nim dupe nudi tren 16 g co chit bdt ngd ( ttong binh tam gidc 250 ml. cho khd nang ting hpp chitinase cao) co bd sung 1% chitin vd nude dupe b i sung ttieo cac ti Id: 40. 50, 60, 70. 80, 90, 100% (nudc/ca ehit;
w/w).
Chit cim ung: Chiing nim dupe len men d cde ^eu kidn ^ i uu nhu ttdn va b l sung ca chit chitin d cdc ndng Ofy. 0%;
0,5%: 1%; 1,5%; 2%; 2.5% (so vdi eo chit).
Nhiit dp lin men: Chung nim dupe len men d edc dilu kidn t l i uu nhu tren r l i dem nudi d cde ddi nhi#t dO khde nhau:
20,26,28.30.37vd40°C.
pH mdi tnrdng: Chiing nam dupe Idn men d cdc dilu kidn toi uu nhu trdn nhung thay nude bang cdc hd dgm vdi ddi pH ti> 3-10 nhu: (i) 0,05M sodium citrate pH 3- 6; (ii) 0,05M sodium phosphate pH 7,8; (iii) 0,05M glycine-NaOH pH 9.10.
Nguin niter v6 ca. Chiing ndm duoc ldn men d eae dilu kign t l i uu nhu trdn vd b l sung 0,5% so vcri t h i tich cde nguon nita vd ca nhu: (NH4)2S04, NH4NO3, (NH4)2HP04, urd. KNO3, NaNOa.
Sau khi xdc djnh dupe nguln nita vd ca tot nhlt. d l t l i uu ning dO nita vd co chiing Penidllium sp. M4 dupe nudi trong mdi trudng ed ning dd nita v6 ca hJong ifng Id: 0.1%; 0,3%; 0,5%; 0,7%; 0,9% vd 1%.
Thdi gian lin men: Chiing nim Penidllium sp. M4 dupe len men d edc dilu kidn t l i uu (30°C; pH 6; 1% chitin; 16 g co chit bdt ngd; dd I m 60%; 0,9% ure so vdi t h i tich). Sau 4,5, 6, 7, 8, 9, 10 vd 12 ngdy idn men, enzyme thd dirpc ttiu de xdc dinh hogt tlnh.
KfiT QUA vA T H A O L U ^
Chpn Ipc ch^ng n i m ky sinh cdn triing tong htyp chitinase boat ttnh cao
Tir mlu d i t vd xde edn tning chit to^n cdnh ding mfa ngogi ttidnh Hd Ngi, chiing tdi da phdn ldp dupe 6 chiing nim (khdng ttinh bdy d bdi ndy), trong dd ed 2 chung chpn Ige Fusarium sp. M3 vd Penicillium sp. M4 cd hieu Ipc digt r$p ngd cao nhlt lln lupt Id 88.4% vd 92,3% sau 7 ngdy phun, vdi so lupng 10^ bdo h>/ml, d 30°C, dg Im 70%. Chitinase eiia 2 chung dupe xdc dinh bing khuich tdn frdn dTa thgch (Hinh 1), djch ngogi bdo cOa ehung Fusarium sp. M3 vd Penicillium sp.M4 tgo viing phdn hOy co chat chitin trdn dTa ttiach lln lupt Id 14 mm vd 30 mm.
Hlnh 1. Ho^t tlnh cUa chitinase ngo^l bio tir chfing Fusarium sp. M3 (giing M3), Penicillium sp. M4 (gilng ^M].
Tif Hinh 1 cho thiy cd hai chiing ddu cd hogt tfnh diitinase, trang dd chung Penicillium sp.M4 cd dudng kfnh vdng phdn gidi Idn gip 2,1 lln chiing Fusarium sp.M3. So sdnh vdi cdng bd cua Nguyen Thi Ha (2012), ehiing Penicillium citrinum vd Aspergillus protuberus ed tiudng kfnh phdn gidi lan lupt Id 26,5 ± 0,18 vd 25,5 ± 0,05 mm ttii chung Penidllium sp.^M cd hogt tfnh cao han. Tir k i t qua trdn, chiing tdi da c h ^ chung Penicillium sp. M4 de toi uu hda edc dieu kidn nudi cly.
Anh hudng ciia cdc d l l u kign moi truwng d i n kha nang sinh t i n g hpp chitinase Ccr chit
Nam spi cd khd ndng sd dyng nhilu nguon carbon khdc nhau, cd lodi chf thieh hop vdi mdt hogc m$t so it nguon carbon, cd lodi Igi thich hpp vdi nhilu nguon cartjon. Nguln carbon cd ttil Id cde logi eartjohydrate don gidn nhu dudng don, dudng ddi hodc phuc tgp nhu tinh bdt, cellulose. Nguon cartxjn phirc tgp nay thudng tdn tgi d hlu hit cde san phim, p h i phim nhu triu cdm, Idi ngd, ggo, bdt ngd. . NhO'ng ehit ndy thudng duac sd dgng Idm nguln carbon cho qud frlnh sinh ting hpp chitinase.
Trong 10 mdi ttudng da khao sat, mdi tivdng b$t ngd eho hogt tinh chitinase cao nhlt vdi {6,05 ± 0,06) U / m l « tnfdng cdm gao + bdt ngd cho hoat tlnh chitinase cao thu 2 vdi (5,6 ± 0,03) U/ml (P< 0.01) (Hlnh 2a). K i t q u i n d v ^ khde bidt vdi nghidn cihi oia Parameswaran vd ding tac gia (2005) da chgn cdm mi Idm ca chit de c h O l l ^ j Penidllium aculeatum NRRL 2129 sinh ting hpp chitinase (Parameswaran e^ al., 2005). Tu nhung kit qua dgt dupt^^
ngd dupe chpn Idm mdi tnrdmg trong qud ttinh ldn men thu djch enzyme the.
Dg diy car chit
Dd ddy CO d i l t dnh hudng rat ldn din khd ndng smh tong hpp chitinase oia nam kf sinh edn trCing. Khi dd ddy co chii qua nhd, nude tir co chit se bj bay hoi nhanh khdng ddm bdo d | I m cho qud trtnh ldn men. Ngupc Igi, khi dd ddyco chat qua Idn, t* Id trong dli giira b l mdt tilp xiic vd khli lupng ca chit nhd, dg lim thOng- thodng khf gidm, khd nSrq thodt nhidt kdm dnh hudng bit Icri tdi qud frinh sinh ting hpp chitinase. Trong nghidn cdu ndy, Idn men dupe thvchi^n trong blnh ndn 250 ml. dudng kinh ddy 8 em. Dd ddy ea chit 17 mm thfch hgrp chit cho chiing Penicillium sp. M4 sinh chitinase. Tu kit qud tten, dd ddy co chit 17 mm (tijong duong 16 g eo chat/ binh dung tfch 250 ml) dupe chgn diflin hdnh cdc nghidn cdu tilp ttieo (Hinh 2b).
I I III
7 6
f 9 i '
5 2 0
1 1 ll
e 9 1 1 13.5 IS 17 19III 1 1 1
E)$ cUy co> c h i t
A
211
1 1
23
J
251
Hlnh 2. Anh hudng cda cdc ngudn coi chit (a), dg ddy cc chit (b) d4n kha ndng sinh ting hpp chlHnase ciia chung ninl Penicillium sp. M4.
LN: Lfii ngd, G: gao, CG. cdm ggo, BN: b^t ngd, CB: cdm ggo + b$t ngd, BL: bdt ngd + idi ng5, GB: 990 + b$t ngd, CGG: cdm g^
gao, CL: cdm 990 + idi ngd, GL: ggo + loi ngd.
Dp im ccr chit
Dd I m ban diu dnh hudng r i t ldn din si/ sdn sinh cde logi enzyme ttiuy phdn khi Idn men rln vl nd dnh hudng din]
tdng tnfdng cua sinh vgt. Dp I m co ehit thay dli lidn tge ta^sng qud trlnh Idn men, do vdy vide t l i inj dd I m cho oa\
Id rat quan trpng. Dd I m ca chit thip se khdng dii lupng nude cho nim sinh ttirdng, ngupc lgi dd I m qud cao cOng hudng tidu cpe din sy sinh ttudng do dd thodng khf gidm (Moo etai. 1993).
e 1 5
1"
Chitin
_ _.
1 1 1 ,
0 0-S 1 1.9 2 N&ns <i« chitin (%)
1
25 Hinh 3 hph hiiimg cua d$ am co- chit (a), nong dd chitin trong mdi trufimg (b) din khd nang sinh ting hpp chitinase dt._
Chung nim Penicillium sp.M4.
DO Im ban diu bo sung bdng 60% ca chit (tuong duang vdi dd I m mdi tnfdng eo chat 37,5%) chCing Penicillium 39.
M4 sinh tdng hpp enzyme chitinase cao nhlt vdi (6,36 ± 0,03) U/ml {P< 0,01) (Hlnh 3a). Kit qud ndy ed s\f khdc bilta vdi nghidn edu cua Patidar vd dong tdc gia (2005) khi chiing Penicillium Chrysogenum PPCS1 vd PPCS 2 dgt hogtM cliitinase rao nhlt khi nufii d dd I m ban dau Id 80% vd 120% tuong dng (Patidar e( al., 2005). Tip kit qud trdn, dOfl be sung bang 60% co ehit dupe sd dyng d l tiln hdnh cdc budc nghidn cdu tiep theo.
Ndng d^ cvchat cam dng
Ca chit edm dng ddnq vai trd Id chit cdm dng eho qud frinh tdng hpp mpt logi enzyme tifOng dng. Khi b i sungi vdo mdi trudng nufii cly se tdc dfing din sinh ttudng vd khd nang smh tong hpp chitinase cua chung nim. Tronf het cdc trudng hpp, khi nfing dp chitin khodng 1 -1,5% ti^ng mfii tnfdng, vi sinh vgt cd khd nang sinh chitinase tt, Felse. Panda, 1999). Tu hinh 3b cho tiily. khi khfing sd dung chitin ttxing mdi trudng nudi cly, chiing Penicillium i vdn smh tfing hop chitinase nhung thap (0,15 ± 0,02) U/ml) {P< 0,01). Chung nam Penidllium sp. M4 cho h "
chitinase cao nhdt khi nfing dfi chitin Id 1 % vd cd xu hudng gidm khi nong dfi chitin vupt qud muc 1 %.
Nhiit d^
Nhidt dd nudi cljr ylu t l quan tnpng, dnh hudng ldn din sinh tnfdng vd sinh ting hpp enzyme (Ramesh, Lorwai*
1987). Nhigt dd toi uu eho sy sinh trudng ciia da s l nim spi Id tu 28 - 32*'C, toi da dudi 50°C. Chung Penidllium sft *
; CONG NGHE SINH HOC TOAN Q U 6 C 2013
c6 khd nang smh cMnase eao trong dai nhigt dp khd rfing ti> 26 - 37°C vd eho hogt tinh eao nhat d 30°C (Hinh 4a). Kit qua ndy khd phii hpp vdi nghidn curu aia Li Thi Hud (2010),Trrc/K)derTna harzianum cho hogt tlnh chitinase cao nhat d 30 C. vd cd sy Wide bigt vdi nghien ciru eiia Patidar vd Oong tac gid (2005) khi nim PenicBlium chrysogenum eho hogt tlnh chitinase cao nhlt d 24''C (Patidar ef al., 2005).
fM m6i frvdng lin men
Gid tri pH mfii truflng ban d i u dnh hudng quan frong din khd ndng sinh long hpp chitinase cOa cdc chiing nim spi. TOy thugc tdng loai. tung ehung md pH mdi ttvdng ban S u thieh hpp Id awt, ttung tfnh hay kiem. Tuy nhidn, hlu hit cdc lodi nim ky sinh ddu ua pH ttilp. Chiing Penicillium sp. M4 sinh chitinase rao nhdt tgi pH 6 vdi (6,59 ± 0,02) U/ml (P< 0,01) (Hinh 4b). Kit qua trdn phil hpp vdi cdng bo cua Ingold (1967) cho ^ n g pH (4-6) ttiich hpp cho nam sinh ting hpTJ enzyme cao.
ILIIIILL.
3 4 5 6 7 0 9 IDPH
Hlnh 4. Anh htrang cua nhi$t d$ (a), pH (b) din khd nang sinh dng hpp chiflnase cda chung Penicillium sp. M4.
Ngu6n nitarvd ctr
Niti7 vd ca dupe cung cap tu nhilu nguln khdc nhau nhu muli amonium, muli nitrate. Nguln nito vd co phii hpp cA\o khd ndng sinh ting hpp chitinase php thufic tdng chdng nim. Ning dp nita t l i uu cho khd ndng sinh ting hpp chitinase d^ nim cdn phg thufic vdo fi Id cOa nito so vdi carbon. Cf ddy, tifit ngd dupe su dgng lam nguln rarfoon nhung ddng thdi nd cQng cd t h i cung c i p nito cho nim. Tuy nhidn, hdm lupng nita ed t h i chua phO hpp cho vige sinh enzyme eiia nim. Nguln nita vd ca cd dnh hudng ro rdt din khd nang sinh tong hpp chitinase cGa chOng nim. Trong s l sau nguln nito vd CO dupe khao sat thi hogt tinh chitinase cua chiing Penidllium sp. M4 diu tdng khi bd sung thdm edc nguln nito vd CO vdo mdi ttudng nudi ely (hlnh 5a). Tuy nhien, tiogt b'nh chitinase cda chiJng dgt mdc cao nhlt khi mdi trudng b l sung thdm urd (7,77 ± 0,06) U/ml tang ldn 1,44 lan so vfii vide nudi trong mdi trudng khdng b l sung ure (5,41 ± 0,07) U/ml {P< 0,01). Kit qud ndy ed sy khde vdi nghidn edu cua Aghaeizadeh Fatemah vd dfing tde gid (2008) khi nim Penidllium aculeatum PTCC5167 eho hogt tinh cao nhlt khi b l sung KNO3 (Aghaeizadeh Fatemah ef ai, 2008).
Hinh 5. Anh hudng cua m^t s l nguln nito- Penlcimum sp. M4.
CC (a), nong dp ur£ (b) den kha n3ng sinh ting hpp chitinase cua chiinB nim UrI dupe chting minh Idm tang kha ndng ting hpp chitinase, chiing tfii tiln hdnh t l i uu nong dg urd d cdc ning dfi khdc nhau. ChOng Penidllium sp. M4 ed khd ndng sinh chitinase rao nhdt (8,00 ± 0,02) U/ml (P< 0,01) d nong dfi ure 0,9% vd cd 301 hudng gidm khi ning dd ure bo sung vupt qud 0.9% (hinh 5b). Vi vdy, nong dp ure 0,9% dupe chpn eho nhung nghiin cuu tiep theo.
Thdri gian ISn men
r. V'
0
ll
2 31 III.
Th^'iglanlngty)
Hlnh 6. Anh hudng cda thdi gian din kha ning sinh tong hpp chitinase cua chung Penicillium sp. M4.
Mil mdt lodi ed mfit thdi gian ldng ttifdng t l i uu khde nhau, thudng thi hogt tfnh enzyme manh nhlt d thdi diem bdo h>
mAi b i t d I u h l n h ttidnh. T h d i g i a n Idn m e n d e dat hogt tfnh chitinase c a o nhat edng n g a n ttii ehi phf s d n x u i t edng tiiip.
Chiina Pentoillium s p . M4 e d k h d n d n g sinh l o n g h p p chitinase c a o n h l t (8,02 ± 0,02) U/ml s a u 5 n g d y Idn m e n {P<J),Oij fHlnh 61 O i l u ndy phii h<7p v d i nghien e d u eua Lee v d d i n g tdc gid (2009) v l chiing Penicillium sp. L Y G 0 7 0 4 cd%^
tlnh c a o nhat s a u 3 ngay Idn m e n ( L e e ef al., 2009). Khi Idn m e n cOa ehiing n i m PeniclBlum s p . M 4 v d i c o c h i t bfit ngi, hogt tlnh chitinase flat d u p e s a u k h i tfii u u ede dieu kifin nufii c l y d a t a n g 1,33 l l n : t i r (6,05 ± 0,06) U/ml ldn (8,021 0 02) U/ml Hoat tfnh chitinase d a t d u p e t u o n g d o i c a o s o v d i m d t s o nghien e d u v l hogt tfnh chitinase cua nim Pento/ffum'khde tten ttil g i d i , Aghaeizadeh vd dong tac g i d (2008) b d o c d o h o g t tfnh chitinase d g t d u p e 1,66 U/ml d mai frudng t l i u u V d i cde n g u l n n g u y d n lidu d u p e c h g n frdn. k i t q u d ndy eo finh Wia ttii c a o khi d p d g n g v d o t h y c t l vl cdc n g u y l n ligu ndy r e tien v d s i n c d . B e n cgnh d d , p h u o n g p h d p ldn m e n r a n d d v d n h d n h , ehi phf d d u t u v d v d n hdnh ttilp lgi 0$t d u p e nang suat eao.
K ^ L U A N
C h i i n g n i m k^ sinh cfin friing Penicillium sp. M4 d u p e p h d n l$p t d cdnh d d n g m f a ngogi ttidnh H d Ndi c d khd ndng sinh t i n g h p p chitinase rao tt^n mfii frudng ran vdi n g u l n c o t ^ l t bfit n g d e d d f i d d y 17 m m . d f i I m c a chat 6 0 % , 1 % ra c h i t chitin cdm u n g . pH 6 . ndng d f i urd 0.9%, nhidt dfi 30''C ttong t h d i g i a n nuoi c d y 5 ngdy. Hogt tfnh chitinase dgt ttong i J l u kidn Idn m e n toi u u Id (8,02 ± 0.02) U/mi tang 1,33 l l n so v d i ngufin e a c h i t bfit n g d c h u a d u p e tdi u u .
TAI LI^U THAM KHAO
NguySn LSn Dung. NguySn Ddng D u e Ddng Hdng Mien, NguySn VTnh Phudrc, Nguyin Dinh Q u y i n , Nguyln Phiing Tien. Ph?m Via Ty (1976). Mdt s i phuwng phdp ngtuSn ci>u Vi sinh vat hpc. NXB Khoa hpc ky thu$t. Hd N^i.
NguySn Thj Hk (2012). T l i uu d i l u ki$n nudi cay chiing Aspergillus pmtubems smh long hpp enzym chitinase dugx; phan ldp tir rOng ngdp mdn C i n Gidr, Tap chl Khoa hgc- D?i hoc Cin Tha. 22b: 2 & ^ 5 .
Ld Thj Hud (2010). Khdo sdt khd ndng sinh t i n g hpp enzyme chitinase cCia mdt so chung n i m spi thudc gilng Aspergillus, Tnchodenna vd i>ng dvng. Luin vBn Tftgc sT, DHSP TP. H6 Chl Minh.
Agheizadeh Fatemeh, Faezi Ghasemi M. Ghasemi M, GhaemI N. Dastan K (2006). Optimization of chitinase production by Penicillium aculeatum PTCC 5167 in submerged culture fermentation. J Bio Sci. 2(5):13-24.
Mhur Felse P, Panda T (1999). Self-directing opBmization of parameters for extracellular chitinase production by Tnchoderma harzianum in batch mode. Process ^ochem. 6-7(34): 563-566.
Madelin MF (1963). Diseases caused by Hyphomycetous fungi, 233-271. In E. A Steinhaus (ed.). insect Pathology: An Advanced
Treatise. Vol. 2. Acaderrtic Press, NY. a Moazami N (2002). Biopestddes production. Encyclopedia of Bkilo^al physiological and Health Sciences, Encyclopedia Of Ufa
Support Systems | Moo Y, Moreira MAR, Tengerdy RP (1993). Principles of solid substrate fennentation. Fungal Technology, Edward Arnold, London, DO.
117-144.
Parameswaran Binod, Tunde Pusztahelyi. Viviana Nagy, Chandran Sandhya. George Szakacs, Istvan Pocsi, /tehok Pandey (2005).
Production and purification of extracellular chitinases from Peniaiium aculeatum NRRL 21 under solid-state fennentation. Enzyme Micrt^lal Technon{36): 880-887,
Patidar P, /\giaw^ D. Baneijee T, Patil S (2005). Optimisation of process parameters for i^itinase production by soil isolates <il Penicillium chrysogenum under solW substrate fermentation. Process Biochem 40(9): 2962-2967.
Ramesh MV, LcHisane BK (1987) Solid state fennentation for production of alpha amylase by Bacillus megaterium 16 M. Biotechnd Lett. 5(9): 323-328.
Lee YG , Chung KG ,Wi SG. Lee JC . Bae HJ (2009) Purification and properties of a chrtinase fram Penicillium sp LYG 0704. Profwi Express and Punf. 2(65)' 244-250.
OPTIMIZATION OF SOLID FERMENTATION CONDITIONS FOR PRODUCING CHITINASE BY PENICILLIUM SP. M4 ISOLATED FROM SUGARCANE FIELDS
V u Thf T h a n h , V u V a n H a n h , N g h i e m N g o c HAinh, Q u y e n D i n h T h i Institute of Biotechnology. Viet Nam Academy of Science and Technology
SUMMARY
Enlomopathogenic fiingi often affect certain types of tissue, such as the 1^1 tissue and other tissues which are dissolved by enzymes (chitinases, proteases, lipases) from ftingi. In particular, the stronger chitinase activity, the faster the insect is killed Enlomopathogenic fiingus Penicillium sp. M4 isolated from cadaver's bugs in sugarcane fields outside Hanoi was capable of bio- synthesizing chitinase. The proper culture conditions for the bio-synthesis of chitinase were optimized by using solid fermentation.
Carbon source of com starch with 17 mm in thickness, moisture content of 60%, pH 6, an addition of chilin (1%, w/w) as inducer and 0.9% (w/w) urea as inorganic nitrogen source, 5 days of fermention time and at 30"C of incubation temperature were optimized for the bio-synthesis of chitinase The highest chitinase activity produced from optimized culture was (8.02 ± 0.02) U/ml. The result K applicable to apply in real conditions, since materials for the culture medium are very cheap and available, the culture conditions Kf suitable to the climate in Vietnam.
Keywords chitinase, parasitic insects, Penicillium sp.M4, optimum conditions, solid fermentation.
Aulhor for coTespondence:Tel. 84-4-379 17948; Email: [email protected]
