HOl NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A T A I NGUYEN SINH VAT L A N THCf 5
TOI l/U HOA DIEU KIEN NUOI CAY CHIJNG NAM SOfI Penicilium oxalicum
SENfH TONG HOP ENZYME CHITINASE DlTOC PHAN LAP TU" DAT
NGUYEN THI HA Trucmg Dgi hgc Cdn Tho Nhilu enzyme quan trgng dugc khai thae tu vi sinh vat va du-gc trng dung rgng rai trong doi song san xuat, do vi sinh val chiem khdi Iirgng Ion va co kha nang chuyen hoa vat chit trong tu nhien rat hiJu hieu. Khi so sanh vai cac nguon enzyme khac co nguon goc tir dgng vat va thyc va^ enzyme co nguon goc vi sinh co nhilu uu diem nhu hoat tinh cao, thai gian sinh tdng hgp ngan, nguyen lieu san xuat re tiln, co the san xu5t theo quy mo cong nghiep. Nhilu enzyme khai thae lir vi sinh val dugc t^p trung nghien cuu img dung irong thcri gian qua nhu protease, amylase, cellulase, peclinase... Ben canh do con co nhom enzyme chitinase c6 kha nang thuy phan lap yo chitin ciia loai giap xac cung dugc dac biet quan tam. Bai bao nay Irinh bay mgt s6 kel qua ve t6i uu hoa dieu kien nuoi ciy cac chiing N§m sgi dugc phan lap tir dSt d thanh pho Can Tha nham lao nguon chitinase vdi hoat tinh cao.
L VAT LIEU VA PHUONG PHAP NGHIEN CU'U I. Vat lieu
Cac chimg Nam sgi dugc phan lap tir dat d thanh phd Can Tha.
2.Phuxmgphap
Kiem tra hoat tinh chitinase ciia cac chimg Nam sgi phan lap dugc bang phuang phap do duong kinh vdng vd khuan. Khao sat anh hudng ciia cac yeu Id nhu thdi gian nudi cay, nhiet do, am dO, ham hrgng mudi, do pH, co chat den kha nang sinh tdng hop chitinase cua nam sgi.
Chgn 3 yeu to cd anh hudng dang ke len kha nang sinh tdng hgp chitinase cua chung Nam sgi nghien ciru de tdi uu hoa theo phuang phap quy hoach thuc nghiem.
II. KET QUA
1. Phan lap mot so chung Nam soi co kha nang sinh tong hop chitinase
Da phan lap dirge 70 chiing Nam sgi va tien hanh nudi cay cac chimg nay tren mdi trudng nudi cay nam sgi trong 4 ngay. Tien hanh khao sat kha nang sinh tdng hgp enzyme chitinase bang phucmg phap do dudng kinh vdng phan giai. Ket qua nghien ctiu cho thay co 10 chimg Nam sgi the hi?n hoat tinh chitinase du-gc trinh bay d bang 1.
Bang I Dudng kinh vdng phan giai chitin ciia 10 chiing Nam soi nghien ciru rr
1 2
Ten 4 4m 3 1 BU11 4 5
LX31 NB61
Duong kinh vitng phan giai (cm)
4,0 2,7 2,0 1,5 6,5
TT 6 7 8 9 10
Ten NB62 NB63 NB64 0U11 TN11
Du-o-ng kinh vong phan giai {cm]
2,5 5,5 2,2 3,5 1.7 Chung NB61 cd dudng kinh vong phan giai cao nhat (6,5cin) dugc chgn 6k nghien cuu tiep.
HQl NGHj KHOA HOC T O A N Q U O C V £ SINH T H A I V A TAI N G U Y £ N SINH VAT L A N THLf 5 2. Khao sat dac diem hinh thai va djnh danh chung NB61
* Dgc diem ve hinh thdi cua chung NB61:
Hinh 1. Hinh thai dgi the vd vi thi cua chiing ndm NB61
* Dinli danh bdng phuang phdp sinh hgc phan tir:
Chiing nam sgi NB6I nghien ciru dugc djnh danh den loai theo phuang phap sinh hgc phan lir. Trinh t\r gene nhan dugc dudi day dugc phan tich so sanh vdi cac gen da biet trong ca sd dir lieu tren trang web NCBI su dung chuong trinh BLAST.
CCCACCCOTGTTTATCGTACCTTGTTGCTTCGGCGGGCCCGCCTCAGGCCGCCGGGGGGC ATCCGCCCCCGGGCCCGCGCCCG CG A AG ACACA AA A ACG A ACTCTTGTCTG A AG ATTGC A GTCTGAGTACTTGACTAAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCAT CGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATC GAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGOGGCATGCCTGTCCGAGCGTC ATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCTCTCGCCCCCCGCTTCCGGGGGGCGG GCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCG CTCTGTAOGCCCGGCCGGCGCCCGCCGGCGAACACCATCAATCTTAACCAGGTTGACCTC GGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA.
Ddi chilu trinh tir DNA vdi trinh t\r DNA cua cac chung NS tir ngan hang gen NCBI cho thdy ty le tuang ddng vdi loai Penicillium oxalicum la cao nhat (90%)
Kit hgp giii'a kel qua phan tich hinh thai va sinh hgc phan tu, chimg Nam sgi NB61 cd the thugc loai Penicillium oxalicum.
3. Anh hirdng cua mgt so yeu to moi tru'dng den qua trinh sinh tong hgp chitinase ciia Penicillium oxalicum
- Am dg moi trirang nuoi cay:
— 1 4 0 1 120 3 - 1 0 0 1 OBO B S 0 6 0
^ 0 4 0 g- 0 20
• ^ 0 00
' - -. , . ' ' S O ' j
0.81 , .
~ H H
n i i ^ H i ^ 4 ^ ^
50% 60% 70% B0%
__: 1
••' '• 1
= #
90%
Hmh 2. Biiu dd biiu thi dnh hirdng ciia dm do len khd ndng sinh chitinase ciia P. oxalicum 1002
HQI NGHI KHOA HOC T O A N Q U O C V E SINH T H A I V A T A I NGUYEN SINH VAT L A N THLf 5
Bilu do d hinh 2 cho thay, nam P. oxalicum phat trien tdt tren mdi trudng cd am dg 80%, hoat tinh en2yme chitinase dat cao nhit (l^OU/mg protein), khac biet cd y nghTa so vdi cac nghiem thurc khac, d im do thSp (50-70%) nam mdc khdng phat trien tdt, hoat tinh enzyme thSp.
Cl ^ dg cao (> 80%i), lugng nude du trong mdi tmdng nudi c§y se lam hd hoa vdi ca chSt va kit chat mdi tnrdng lai lam mdt dg xdp, thoang khi cua moi trudng ddng thdi cung gay kho Uian cho khuan ty phat trien. Dilu nay cho thay do am anh hudng kha Idn den qua trinh phat trien va sinh tdng hgp enzyme chitinase cua nam P. oxalicum
- Anh hirdng cda ndng dp NaCl
_ 1 20
•1 1 00 E
^ 0 60 f 0 40 S 0 20
^ 0 00
, „
0% 2%
BIN ^
4 % a a /
6% 8%
Hinh 3. Biiu do bieu thi dnh hir&ng ciia ndng do muoi NaCl den khd ndng sinh chilinase ciia P. oxalicum
Ket qua thi nghiem d hinh 3 cho thay, chiing Nam sgi P. oxalicum sinh tdng hgp chitinase tdt nhat d n6ng dg NaCl trong mdi trudng nudi cdy la 2%, hogt tinh enzyme thu nhan dugc la 1.12 U/mg protein, tang gap khoang 4 lan so vdi ddi chimg khdng bd sung NaCl. Tuy nhien khi ndng dp mudi tang han 2% kha nang sinh tdng hgp en^me cua P. oxalicum lai giam xudng. Dieu nay cho thay khoang Na' cdn thilt cho sir phat trien cua P. oxalicum sinh tdng hgp enzyme chitinase.
'Anh huang ciia ham lugng chitin:
160 1.49
1 100 —
£ 0 60 —
| 0 4 0 - o 0 20 —
0 00 0.80 ;•
0%
i n i ^ ^ °~^^
5% 10% 15%
Ham luvng ChiOn (%) 20%
Hinh -!. Biiu do bieu thj dnh hir&ng cua ham hrgng chitin den khd ndng sinh chitinase ciia P. oxalicum
Hojit tinh chitinase ciia P. oxalicum tang dan khi ham lugng chitin tang va dat circ dai 1,49 U/mg a ham lugng chitin bd sung la 10%), tuy nhien ham lugng chitin cao han lai irc che kha n5ng sinh enzyme cua nam.
HQI NGHI KHOA HQC T O A N Q U O C V E SINH THAI VA TAI N G U Y £ N SINH VAT LAN THO" 5 - Anh hirdng cdapH moi trudng nuoi cay:
Hmh 5. Biiu do bieu thj dnh huang ciia pH mdi trirdng din khd ndng sinh chitinase ciia P. oxalicum
Ve anh hudng cOa pH, qua bieu dd d hinh 5 cho thay, qua trinh sinh long hgp enzymes khdng dat hieu qua cao khi lang dg pH mdi trudng nudi cay. O cac gia Iri pH ldn nhu 5,5 va 6,0 ho^it tinh (U/mg protein) cua enzyme do nam sgi sinh tdng hgp khdng cao. Tuy nhien qua trinh sinh tdng hgp diln ra manh d pH 4,0 va dat cao nhdt d pH 4.5 vdi boat tinh rieng enzyme ghi nh^n dat dugc cao nhdt la 1.18 U/mg. Ci gia trj pH tir 5-6, ho?t tinh enzyme giam ma,nh, dieu nay chimg td yeu td pH mdi Irudng cd anh hudng vd cimg quan trgng den kha nang sinh tong hgp enzyme cua nam sgi P. oxalicum.
-Anh hu&ng cda thdi ginn nuoi cay:
Hinh 6. Bieu do bieu thi dnh hircmg ciia yiii to ihai gian nudi cdy den hogt tinh enzyme chilinase cua ndm sgi P. oxalicum
Bieu do xac djnh hoat tinh rieng enzyme thu nhan dugc theo thdi gian (hinh 6) cho thay P. oxalicum cd kha nang sinh long hgp chitinase rdt manh ngay tir liic bat dau nudi cay va ho?t linh rieng enzyme thu dugc dai cao nhai 0.96 U/mg vao ngay thir 2 chirng td nam mdc rat dl thich nghi vdi mdi trudng va bdt ddu diln ra cac phan irng sinh hoa sir dung chdt dinh duang ciia moi tnrdng de sinh trudng va phat triln. Sang ngay thu: 3 hoat tinh rieng enzyme bat dau giam manh do qua trinh sinh ldng hgp enzyme sdp kit ihiic va ngudn dinh duang tu mdi trudng cung c^in.
HQl NGHI KHOA HQC T O A N Q U O C V E SINH THAl V A T A I NGUYEN SINH VAT LAN THLf 5 -Anh hirdng cUa nhiet do moi tnr&ng nuoi cdy:
Hinh 7. Biiu do bieu thj dnh hirdng ciia nhiit do moi tnrdng nuoi cdy den khd ndng sinh chitinase ciia P. oxalicum
Bieu do the hien anh hudng ciia yen td nhiet do mdi trudng nudi cay den kha nang sinh tong hgp enzyme chitinase cua chiing Nam sgi P. oxalicum (liinh 7) clio thay sinh ldng hgp enq'me dat hi?u qua cao nhat (hoat tinh rieng enzyme cao nhat 1,65 U/mg) khi nudi cay d 40''C.
3. Quy hoach thuc nghiem toi uu hda dilu kien nuoi cay chiing Nam soi P. oxalicum sinh tong hgp chitinase cao
Tir ket qua tren cho thdy 3 ylu td dm dg, pH va nhiet do cd anh hudng manh din kha nang sinh tdng hgp enzyme cua nam sgi. Do do, ba yeu td mdi trudng la: Am do, nhiet dd va pH dirge chpn de tiep tuc nghien cuu Irong thi nghiem khao sat tdi uu.
3dng 2 Stf do va ket qua bo tri thi nghiem thyc nghiem
TN
1 2 3 4 5 6 7
e
9 10 11
Cac y l u t6 tlieo ty if thirc Z,
45 35 45 35 45 35 45 35 40 40 40
Z i 85 75 75 85 85 75 76 85 80 80 80
ZJ 5 5 5 5 4 4 4 4 4.5 4.5 4.6
Cac y l u t6 theo ty i f ma hoa
X, X,
1 -1 1 -1 1 -1 1 -1
X i X j
HTR enzyme
Y 1.568 1.484 1.373 1.705 1 589 1.624 1.776 1.654 1 092 1.108 1.138
HQl NGHj KHOA HQC T O A N Q U O C V £ SINH THAI V A TAI N G U Y £ N SINH VAT LAN THO' 5 Tir sd lieu thi nghiem, la xac djnh dugc cac he sd hdi quy. Sau khi Ioai bd cac h? sd khong cd y nghTa thi phuong trinh hoi quy cd dang: Y = l.597-0.064Xj-0.042X|X., + 0.071X,X, Tir phuong trinh hdi quy nhan thay mudn tang gia trj cua hoat tinh enzyme chitinase ciia nam sgi P. oxalicum can phai tang gia tri am do, giam nhiet do va pH cua mdi trudng nuoi cay. Do do, tilp tuc nghien cuu tdi uu hda dieu kien nudi cay mdi trudng bang phirang phap dudng ddc nh^t bit ddu tir miic co sd la 0. Chgn budc nhay la 2 ddi vdi am dg, 2 ddi vdi nhiet dg va 0.2 ddi vcri pH mdi tnrdng. Ket qua dugc trinh bay d bang 3.
Being i Ket qua nuoi cay P. oxalicum theo phuong phap leo doc
TT 1 2 3 4 5
Nhiet df 40 38 36 34 32
Am do 60 82 84 86 88
pH moi truing 4 5 4.3 4 1 3 9 3.7
HTR enzyme chitinase 1.520 1.603 1 824 1.548 1.515
So lieu d bang 3 cho thay, dieu kien nudi cay tren mdi trudng ban ran tdi uu cho nam s^i P. oxalicum sinh tdng hgp enzyme phan giai ca chat chitin la: Mdi trudng cd am dp 84%, dp pH mdi trirdng 4.1, ca chat cam irng chitin 10%t, nhiet dp mdi trudng 36°C, ham lugng muoi 2%, thdi gian nudi cay48h.
III. KET LUAN
Trong 70 chiing Nam sgi phan lap tir dat d thanh phd Can Tha cd 10 chiing the hi?n hoat tinh chitinase, trong do Penicillium oxalicum cd kha nang sinh ldng hgp enzyme chitinase cao nhat. Cac yeu td mdi trudng nhu: Am do, dd mudi, nhiet dg, ham lugng ca chat cam irng chitin, thdi gian nudi cay, pH mdi trudng diu cd anh hudng din kha nSng sinh tdng hgp enzyme chitinase ciia nam sgi P. oxalicum, trong dd ba nhan td la am dg, pH va nhiet dg cd anh hudng nhieu nhat. Mdi trudng ban ran tdi uu cho nam sgi P. oxalicum sinh tdng hgp enzyme phan giai ca chat chitin la mdi trudng cd am do 84%, dp pH 4.1, ca chat cam ung chitin 10%i, nhiet dp 36''C, ham lugng mudi 2%, thdi gian nudi cdy 48h.
TAI LIEU THAM KHAO
1. Vo Bich Giang, 2005. Thirc tap enzyme hgc, Trudng Dai hoc Su ph?im Tp. Ho Chi Minh.
2. Nguyen Due Lirong, 2004. Cdng nghe enzyme, NXB. Dai hoc Qudc gia Tp. H6 Chi Minh, tr.27-108.
3. Nguyen Due Luong, 2003. Thi nghiem cdng nghe sinh hoc tap I, NXB. Dai hgc Quoc gia Tp. Ho Chi Minh, tr. 200-250.
4. Ngujen Due Lup-ng, Phan Thj HuyIn, NguySn Anh Tuyet, 2006. Thi nghi|m cong ngh? sinh hpc tap 2, NXB. Dai hoc Qu6c gia Tp. Ho Chi Minh.
5. Nguyin Minh Tuyln, 2004. Quy hoach thuc nghiem, NXB. Khoa hpc vi Ky thuat Ha Npi, tr.84-100.
6. Pankaj Patidar, Deepti Agrawal, Tushar Banerjee, Shridhar Patil, 2005. Optimisation of process parameters for chitinase production by soil isolates of Penicillium chrysogenum under solid substrate fermentation. Process Biochemistry, 40, 2962-2967.
7. Yoon Gyo Lee, Ki-Chul Chung, Seung Gon Wic, Jae Chang Lee, Hyeun-Jong Bae, 2009.
Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expression and Purification, 65,244-250.
1006
HQl NGHI KHOA HQC T O A N Q U 6 C V £ SINH T H A I V A TAI NGUYEN SINH VAT L A N THLf 5 OPTIMISATION OF CULTURE CONDITIONS FOR CHITINASE PRODUCTION
BY Penicillium oxalicum ISOLATED FROM SOIL
NGUYEN THl HA
SUMMARY
This paper present results of the research aimed at isolating fungi strains which exposed high chitinase activity and at studying the medium conditions affecting chilinase production of a chosen fungi stain. By determination of the halo's diameters of colonies on culture medium with chitin as a specific substrate, ten fungi strains with high chiUnase activity were Isolated. Among isolated, one strain with highest diitinase production was selected. This strain was morphologically and biomolecularly determined as Penicillium oxalicum. The suitable conditions for high chitinase production of Penicillium oxalicum were as follows: Humidity of 80%. chitin of 10%, sodium chloride of 2%, 48 h incubation, pH of 4,5 and temperature of 40°C. Also, the interaction of three factors such as pH, temperature, and incubation time were studied. The optimum conditions were defined as follows: Humidity of 84%, pH of 4,1 and temperature of 36°G.
