Header Page of 126 I HC THI NGUYấN TRNG I HC S PHM - NGUYN VN TUN TUYN CHN, NUễI CY CHNG ASPERGILLUS AWAMORI SINH TNG HP ENDO--1,4-GLUCANASE V NH GI TNH CHT Lí HểA CA ENDO--1,4-GLUCANASE LUN VN THC S KHOA HC SINH HC THI NGUYấN- 2009 Footer Page 1Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 I HC THI NGUYấN TRNG I HC S PHM - NGUYN VN TUN TUYN CHN, NUễI CY CHNG ASPERGILLUS AWAMORI SINH TNG HP ENDO--1,4-GLUCANASE V NH GI TNH CHT Lí HểA CA ENDO--1,4-GLUCANASE Chuyờn ngnh: Sinh hc thc nghim Mó s: 60 42 30 LUN VN THC S KHOA HC SINH HC NGI HNG DN KHOA HC: TS Quyn ỡnh Thi Thc hin ti: Vin Cụng ngh Sinh hcVin Khoa hc v Cụng ngh Vit Nam THI NGUYấN - 2009 Footer Page 2Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 LI CM N! Trc ht tụi xin gi li cm Sõu sc ti TS Quyn ỡnh Thi, Trng phũng Cụng ngh Sinh hc Enzyme, Phú vin trng Vin Cụng ngh Sinh hc, Vin Khoa hc v Cụng ngh Vit Nam, ó nh hng nghiờn cu, hng dn thớ nghim, sa lun v to mi iu kin v hoỏ cht cng nh trang thit b nghiờn cu tụi hon thnh lun Tụi xin chõn thnh cm n th cỏn b Phũng Cụng ngh Sinh hc Enzyme, Vin Cụng ngh Sinh hc ó giỳp tụi tn tỡnh sut quỏ trỡnh lm lun Tụi xin chõn thnh cm n Khoa Sinh hc, Khoa Sau i hc, trng i hc S phm, i hc Thỏi Nguyờn cựng cỏc thy cụ giỏo ó nhit tỡnh ging dy v to iu kin cho tụi hon thnh khoỏ hc Tụi cng xin chõn thnh cm n B mụn Hoỏ- Sinh hc, Ban ch nhim khoa Khoa hc C bn, Trng i hc Nụng Lõm, i hc Thỏi Nguyờn ó to iu kin cho tụi i hc Cui cựng, tụi xin chõn thnh cm n nhng ngi thõn gia ỡnh v bn bố ó giỳp v ng viờn tụi sut thi gian hc Thỏi Nguyờn thỏng nm 2009 Hc viờn Nguyn Vn Tuõn Footer Page 3Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 CC CH VIT TT APS Ammonium persulphate BSA Bovine serum albumin CMC Carboxyl methyl cellulose cs Cng s DNA Deoxyribonucleic acid DEAE Diethylaminoethyl C i chng EDTA Ethylene diamine tetraacetic acid IU International unit kb Kilo base kDa Kilo Dalton LB Luria and Bertani M Marker Nxb Nh xut bn OD Optical density PCR Polymerase chain reaction rRNA Ribosome ribonucleic acid SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TBE Tris base-Boric acid-EDTA TE Tris-EDTA v/v Volume/volume w/v Weight/volume Footer Page 4Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 MC LC M U CHNG TNG QUAN TI LIU 1.1 KHI NIM V CELLULASE V ENDO--1,4-GLUCANASE 1.2 NGUN GC V PHN LOI ENDO--1,4-GLUCANASE 1.2.1 Ngun gc 1.2.2 Phõn loi 1.3 CU TRC CA ENDO--1,4-GLUCANASE 1.3.1 Cu trỳc bc mt 1.3.2 Cu trỳc khụng gian 1.3.3 Cu trỳc ca trung tõm xỳc tỏc v vựng liờn kt c cht 1.4 C CH XC TC CA ENDO--1,4-GLUCANASE 11 1.5 NG DNG CA ENDO--1,4-GLUCANASE 12 1.5.1 Trong cụng nghip thc phm 12 1.5.2 Trong cụng nghip sn xut thc n gia sỳc 13 1.5.3 Trong cụng nghip sn xut dung mụi hu c 15 1.5.4 Trong cụng nghip sn xut giy v bt giy 15 1.5.5 Trong cụng nghip sn xut cht ty 16 1.5.6 Trong cụng ngh x lý rỏc thi sn xut phõn bún vi sinh 16 1.6 NH HNG CA IU KIN MễI TRNG N KH NNG SINH TNG HP ENDO--1,4-GLUCANASE 17 1.7 TNH CHT Lí HểA CA ENDO--1,4-GLUCANASE 20 1.8 MT S NGHIấN CU Cể LIấN QUAN VIT NAM 22 CHNG NGUYấN LIU V PHNG PHP 25 2.1 NGUYấN LIU V HểA CHT 25 2.1.1 Chng nm 25 2.1.2 Thit b thớ nghim 25 2.1.3 Húa cht 26 2.1.4 Mụi trng 26 Footer Page 5Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 2.1.5 Dung dch v m 27 2.2 PHNG PHP 28 2.2.1 Nuụi cy vi sinh vt 28 2.2.2 Xỏc nh hot tớnh endoglucanase 28 2.2.3 Kho sỏt nh hng ca mt s yu t lờn kh nng sinh tng hp endoglucanase 30 2.2.4 Tinh sch enzyme 32 2.2.5 in di SDS-PAGE 33 2.2.6 Xỏc nh tớnh cht lý húa ca endoglucanase 34 2.2.7 Xỏc nh hm lng protein tng s 35 2.2.8 Cỏc phng phỏp sinh hc phõn t 36 2.2.9 X lý s liu 41 CHNG KT QU V THO LUN 42 3.1 TUYN CHN V PHN LOI CHNG ASPERGILLUS AWAMORI SINH TNG HP ENDOGLUCANASE CAO 42 3.1.1 Tuyn chn 42 3.1.2 Phõn loi chng nm si da vo phõn on gene 28S rRNA 43 3.2 TI U CC IU KIN SINH TNG HP ENDOGLUCANASE 45 3.2.1 Kh nng sinh tng hp endoglucanase theo thi gian 45 3.2.2 Nhit nuụi cy 47 3.2.3 nh hng ca nng c cht cm ng 48 3.2.4 nh hng ca ngun carbon v nng ngun carbon 50 3.2.5 nh hng ca ngun nitrogen v nng ngun nitrogen 52 3.2.6 pH mụi trng nuụi cy ban u 54 3.3 TINH SCH ENDOGLUCANASE 55 3.3.1 Tinh sch qua ct sc ký lc gel sephadex G-100 55 3.3.2 Tinh sch qua ct sc ký trao i ion DEAE 56 3.4 NHNG TNH CHT Lí HểA CA ENDOGLUCANASE 57 3.4.1 nh hng ca nng c cht 57 Footer Page 6Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 3.4.2 Nhit phn ng ti u 58 3.4.3 pH phn ng ti u 60 3.4.4 bn nhit 61 3.4.5 bn pH 63 3.4.6 nh hng ca dung mụi hu c 64 3.4.7 nh hng ca ion kim loi 65 3.4.8 nh hng ca mt s cht ty 67 KT LUN V KIN NGH 69 TI LIU THAM KHO 71 PH LC 79 Footer Page 7Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 DANH MC CC BNG Bng 2.1 Cỏc thit b chớnh c s dng thớ nghim 25 Bng 2.2 Danh sỏch húa cht chớnh c s dng thớ nghim 26 Bng 2.3 Danh sỏch dung dch v m s dng thớ nghim 27 Bng 2.4 Thnh phn gel in di bin tớnh protein 33 Bng 3.1 Hot tớnh endoglucanase ca 26 chng A awamori 43 Bng 3.2 Trỡnh t nucleotide on gene 28S rRNA chng A awamori VTCC-F-099 79 Bng 3.3 Kh nng sinh tng hp endoglucanase theo thi gian 79 Bng 3.4 nh hng ca nhit nuụi cy 80 Bng 3.5 nh hng nng c cht cm ng 80 Bng 3.6 nh hng ca ngun carbon 50 Bng 3.7 nh hng ca nng lừi ngụ 80 Bng 3.8 nh hng ca ngun nitrogen 52 Bng 3.9 nh hng ca nng ammonium acetate 81 Bng 3.10 nh hng ca pH mụi trng nuụi cy ban u 81 Bng 3.11 Hot tớnh endoglucanase cỏc phõn on qua ct Sephadex G-100 81 Bng 3.12 Hot tớnh endoglucanase cỏc phõn on qua ct sc ký DEAE 82 Bng 3.13 Túm tt quỏ trỡnh tinh sch endoglucanase t chng A awamori VTCC-F-099 57 Bng 3.14 nh hng ca nng c cht n hot tớnh endoglucanase 82 Bng 3.15 nh hng ca nhit phn ng n hot tớnh endoglucanase 82 Bng 3.16 nh hng ca pH hn hp phn ng n hot tớnh endoglucanase 83 Bng 3.17 bn nhit ca endoglucanase 83 Bng 3.18 bn pH ca endoglucanase 84 Bng 3.19 bn pH theo thi gian ca endoglucanase 85 Bng 3.20 nh hng ca dung mụi hu c n hot tớnh endoglucanase 86 Bng 3.21 nh hng ca ion kim loi 66 Bng 3.22 nh hng ca mt s cht ty n hot tớnh endoglucanase 86 Footer Page 8Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page of 126 DANH MC CC HèNH Hỡnh 1.1 S thy phõn liờn kt -1,4-O-glucoside ca cellulase Hỡnh 1.2 Trỡnh t amino acid tng ng vi cu trỳc bc ca Cel12A t mt s chng vi sinh vt Hỡnh 1.3 Mụ hỡnh cu trỳc khụng gian (A); S trung tõm xỳc tỏc (B) ca Cel12A t H grisea Hỡnh 1.4 Cu trỳc vựng CBD ca Cel12A t Humicola grisea 10 Hỡnh 1.5 C ch thy phõn phõn t cellulose (A) v phc h cellulose (B) ca cỏc enzyme thuc phc h cellulase 11 Hỡnh 2.1 ng chun nng glucose 30 Hỡnh 2.2 Quy trỡnh tinh sch endoglucanase t chng A awamori VTCC-F-099 33 Hỡnh 2.3 ng chun Bradford dựng BSA lm chun 36 Hỡnh 3.1 Hot tớnh endoglucanase ca 26 chng A awamori nghiờn cu 42 Hỡnh 3.2 in di DNA tng s (A); Sn phm PCR (B): vi khuụn DNA tỏch chit t chng A awamori VTCC-F-099; Sn phm ct vector tỏi t hp bng XbaI v XhoI (C) 44 Hỡnh 3.3 Cõy phõn loi chng A awamori VTCC-F-099 45 Hỡnh 3.4 Kh nng sinh tng hp endoglucanase theo thi gian ca chng A awamori VTCC-F-099 46 Hỡnh 3.5 nh hng ca nhit nuụi cy n kh nng sinh tng hp endoglucanase ca chng A awamori VTCC-F-099 48 Hỡnh 3.6 nh hng ca nng CMC n kh nng sinh tng hp endoglucanase ca chng A awamori VTCC-F-099 49 Hỡnh 3.7 nh hng ca nng lừi ngụ n kh nng sinh tng hp endoglucanase ca chng A awamori VTCC-F-099 51 Hỡnh 3.8 nh hng ca nng ammonium acetate n kh nng sinh tng hp endoglucanase ca chng A awamori VTCC-F-099 53 Footer Page 9Sofhúa 126 bi Trung tõm Hc liu i hc Thỏi Nguyờn http://www.lrc-tnu.edu.vn Header Page 10 of 126 Hỡnh 3.9 nh hng ca pH mụi trng nuụi cy n kh nng sinh tng hp endoglucanase ca chng A awamori VTCC-F-099 54 Hỡnh 3.10 in di trờn gel polyacrylamide sn phm tinh sch endoglucanase t chng A awamori VTCC-F-099 qua ct Sephadex G-100 56 Hỡnh 3.11 in di sn phm tinh sch endoglucanase trờn gel polyacrylamide 57 Hỡnh 3.12 nh hng ca nng c cht lờn hot tớnh endoglucanase t chng A awamori VTCC-F-099 58 Hỡnh 3.13 nh hng ca nhit phn ng lờn hot tớnh endoglucanase t chng A awamori VTCC-F-099 60 Hỡnh 3.14 nh hng ca pH phn ng lờn hot tớnh endoglucanase t chng A awamori VTCC-F-099 61 Hỡnh 3.15 nh hng ca nhit lờn bn endoglucanase t chng A awamori VTCC-F-099 62 Hỡnh 3.16 nh hng ca pH ti bn endoglucanase t chng A awamori VTCC-F-099 63 Hỡnh 3.17 nh hng ca pH ti bn endoglucanase t chng A awamori VTCC-F-099 theo thi gian 64 Hỡnh 3.18 nh hng ca dung mụi hu c lờn hot tớnh endoglucanase t chng A awamori VTCC-F-099 65 Hỡnh 3.19 nh hng ca cht ty lờn hot tớnh endoglucanase t chng A awamori VTCC-F-099 68 Footer Page 10 S of húa126 bi Trung tõm Hc liu i hc 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H, Tokuda G (2001), "Animal cellulases", Cell Mol Life Sci, 58: 1167-1178 63 Xu B, Janson JC, Sellos D (2001), "Cloning and sequencing of a molluscan endo--1,4-glucanase gene from the blue mussel, Mytilus edulis", Eur J Biochem, 268: 3718-3727 64 Yasuda K, Roneker KR, Miller DD, Welch RM, Lei XG (2006), "Supplemental dietary Inulin affects the bioavailability of Iron in corn and soybean meal to young pigs", J Nutr, 136: 3033-3038 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 78 Footer Page 88 of 126 http://www.lrc-tnu.edu.vn Header Page 89 of 126 PH LC Bng 3.2 Trỡnh t nucleotide on gene 28S rRNA chng A awamori VTCC-F-099 GCATATCAAT CCTCAGTAAC AAAGCTGGCT GATGCTTTGG GGCCGTCAGA TCCGTGCCCG TGGGAATGCA GCTAAATACT GATCGAAAGA GCACGTGAAA TCGCCCGCGG CCCCGTGGGC AGGCCCCTGG CAGGGGTGCA TCGGCACGGA TTGAAACACG Trỡnh t nucleotide AAGCGGAGGA AAAGAAACCA GGCGAGTGAA GCGGCAAGAG CCTTCGGAGT CCGCATTGTA GTGCGGCCCC CGTCTAAGTG GAGGGTGAGA ATCCCGTCTT TGTAAAGCTC CTTCGACGAG GCTCTAAATG GGTGGTAAAT GGCCGGAGAC CGATAGCGCA TGAAAAGCAC TTTGAAAGGA TTGTTGAAAG GGAAGCGCTT GGTTCAGCCG GCATTCGTGC GGGCCAGCGT CGGTTTGGGC AATGTAGTGC CCTCCGGGGC ATGCGGCCAG CCTGGACCGA CGCTGGCATA ATGGTCGTAA GACC V trớ ACCGGGATTG CTCAAATTTG ATTTGCAGAG CCCTGGAACG GGGCGGGGTG TCGAGTTGTT TTCATCTAAA CAAGTAGAGT GAGTTAAACA GCGACCAGAC CGGTGTACTT GGCCGGTCAA ACCTTATAGC GGAACGCGCT ACGACCCGTC 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 614 Bng 3.3 Kh nng sinh tng hp endoglucanase theo thi gian Thi gian (gi) OD1 OD2 OD3 24 48 72 96 0,30 0,39 0,39 0,45 0,31 0,38 0,39 0,43 120 144 168 192 216 240 0,28 0,27 0,21 0,23 0,22 0,22 0,30 0,26 0,30 0,23 0,29 0,24 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 79 Footer Page 89 of 126 Hot tớnh endoglucanase 0,31 0,38 0,39 0,44 IU/ml 0,41 0,002 0,49 0,006 0,49 0,002 0,55 0,012 % 74 89 91 100 0,29 0,26 0,25 0,23 0,25 0,23 0,39 0,36 0,35 0,33 0,35 0,33 71 66 64 60 64 60 0,008 0,002 0,043 0,001 0,036 0,011 http://www.lrc-tnu.edu.vn Header Page 90 of 126 Bng 3.4 nh hng ca nhit nuụi cy Nhit (C) 25 28 30 32 37 OD1 OD2 OD3 0,21 0,33 0,47 0,40 0,33 0,21 0,34 0,49 0,38 0,34 0,25 0,35 0,41 0,39 0,34 Hot tớnh endoglucanase IU/ml % 0,32 0,003 56 0,44 0,013 78 0,56 0,045 100 0,50 0,007 88 0,44 0,007 78 Bng 3.5 nh hng nng c cht cm ng Nng CMC (%) OD1 OD2 OD3 0,2 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 0,09 0,11 0,16 0,52 0,63 0,62 0,47 0,38 0,32 0,32 0,05 0,07 0,22 0,55 0,65 0,73 0,47 0,39 0,32 0,32 0,05 0,08 0,16 0,51 0,63 0,71 0,48 0,38 0,32 0,32 Hot tớnh endoglucanase IU/ml % 0,15 0,023 19 0,18 0,021 22 0,27 0,035 34 0,64 0,020 79 0,75 0,010 93 0,81 0,056 100 0,58 0,005 72 0,49 0,006 61 0,42 0,003 52 0,42 0,002 52 Bng 3.7 nh hng ca nng lừi ngụ Lừi ngụ (%) OD1 OD2 OD3 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 0,44 0,54 0,60 0,41 0,41 0,53 0,34 0,38 0,38 0,22 0,58 0,49 0,70 0,58 0,60 0,78 0,65 0,60 0,54 0,42 0,53 0,97 0,71 0,79 0,94 1,01 0,83 0,80 0,70 0,64 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 80 Footer Page 90 of 126 Hot tớnh endoglucanase IU/ml % 0,65 0,067 70 0,78 0,263 87 0,79 0,058 88 0,71 0,186 79 0,76 0,269 85 0,90 0,238 100 0,72 0,251 81 0,71 0,208 79 0,65 0,159 73 0,53 0,211 60 http://www.lrc-tnu.edu.vn Header Page 91 of 126 Bng 3.9 nh hng ca nng ammonium acetate Ammonium acetate (%) OD1 OD2 OD3 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 0,50 0,55 0,28 0,30 0,30 0,31 0,39 0,34 0,29 0,27 0,24 0,24 0,21 0,23 0,27 0,32 0,34 0,26 0,28 0,28 0,26 0,24 0,25 0,25 0,28 0,35 0,44 0,31 0,30 0,28 0,27 0,26 Hot tớnh endoglucanase IU/ml % 3,44 0,038 69 3,58 0,039 72 3,86 0,014 78 4,25 0,023 85 4,97 0,050 100 4,03 0,040 81 3,90 0,012 78 3,74 0,001 75 3,51 0,012 71 3,40 0,011 68 Bng 3.10 nh hng ca pH mụi trng nuụi cy ban u pH OD1 OD2 OD3 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 0,26 0,32 0,34 0,36 0,37 0,38 0,39 0,42 0,38 0,37 0,36 0,33 0,34 0,35 0,38 0,40 0,41 0,42 0,46 0,42 0,40 0,40 0,23 0,26 0,31 0,32 0,33 0,33 0,36 0,37 0,34 0,30 0,31 Hot tớnh endoglucanase IU/ml % 3,67 0,051 70 4,05 0,042 78 4,34 0,021 83 4,58 0,030 88 4,71 0,036 90 4,79 0,040 92 4,93 0,030 94 5,22 0,047 100 4,84 0,040 93 4,62 0,051 88 4,57 0,048 88 Bng 3.11 Hot tớnh endoglucanase cỏc phõn on qua ct Sephadex G-100 Phõn on 10 Hot tớnh endoglucanase IU/ml 1,79 1,83 2,01 4,02 4,89 4,52 4,58 7,21 IU/mg protein 213,71 169,95 183,17 330,37 288,36 149,94 78,66 109,57 Footer Page 91 S of húa126 bi Trung tõm Hc liu i hc Thỏi Nguyờn Phõn on 11 12 13 14 15 16 17 18 Hot tớnh endoglucanase IU/ml 6,66 6,81 6,57 6,46 4,03 3,70 2,70 2,14 IU/mg protein 80,07 82,83 87,70 106,64 72,49 93,57 103,29 224,31 http://www.lrc-tnu.edu.vn Header Page 92 of 126 Bng 3.12 Hot tớnh endoglucanase cỏc phõn on qua ct sc ký DEAE Phõn on 10 Hot tớnh endoglucanase IU/ml 0,76 0,73 0,82 1,53 1,30 1,13 1,07 1,78 IU/mg protein 79,74 56,86 55,75 71,65 62,10 57,80 56,16 57,59 Phõn on 11 12 13 14 15 16 17 18 Hot tớnh endoglucanase IU/ml 2,01 1,91 1,91 1,62 1,01 0,98 0,60 0,49 IU/mg protein 48,75 43,89 40,60 34,68 23,12 30,99 24,09 24,49 Bng 3.14 nh hng ca nng c cht n hot tớnh endoglucanase CMC (%) 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 Hot tớnh endoglucanase (IU/mg protein) 84,15 0,005 119,71 0,007 179,43 0,008 260,87 0,010 295,95 0,013 377,49 0,060 283,28 0,021 232,33 0,009 173,19 0,006 163,45 0,006 % 22 32 48 69 78 100 75 62 46 43 Bng 3.15 nh hng ca nhit phn ng n hot tớnh endoglucanase Nhit 30 37 40 45 50 55 60 65 70 Hot tớnh endoglucanase (IU/mg protein) 45,37 0,005 60,67 0,025 93,11 0,004 95,35 0,001 109,09 0,004 64,95 0,006 50,85 0,001 45,76 0,003 34,65 0,004 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 82 Footer Page 92 of 126 % 42 56 85 87 100 60 47 42 32 http://www.lrc-tnu.edu.vn Header Page 93 of 126 Bng 3.16 nh hng ca pH hn hp phn ng n hot tớnh endoglucanase Hot tớnh endoglucanase (IU/mg protein) 37,87 0,009 144,65 0,002 226,68 0,002 184,88 0,007 118,54 0,005 95,25 0,004 90,58 0,003 82,20 0,072 84,54 0,010 Nhit 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 % 17 64 100 82 52 42 40 36 37 Bng 3.17 bn nhit ca endoglucanase Nhit Hot tớnh endoglucanase Thi gian (gi) (IU/mg protein) C 30 40 12 24 36 48 60 72 12 24 410,23 399,02 388,99 385,77 382,75 285,43 258,34 236,23 368,43 355,86 346,61 0,005 0,001 0,008 0,004 0,002 0,004 0,001 0,006 0,007 0,001 0,005 36 48 60 72 12 24 314,85 277,92 254,15 228,53 361,13 324,98 302,38 0,006 0,006 0,007 0,005 0,001 0,004 0,025 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 83 Footer Page 93 of 126 % 100 97 95 94 93 70 63 58 90 87 84 77 68 62 56 88 79 74 http://www.lrc-tnu.edu.vn Header Page 94 of 126 50 60 70 36 210,12 0,001 48 60 72 12 24 36 48 60 150,88 130,33 124,09 255,61 179,33 131,79 91,36 65,83 59,60 0,024 0,005 0,006 0,014 0,009 0,006 0,001 0,008 0,001 72 12 24 36 48 60 72 38,94 202,22 143,77 112,50 54,92 26,57 23,94 23,35 0,001 0,002 0,010 0,007 0,002 0,003 0,001 0,000 51 37 32 30 62 44 32 22 16 15 49 35 27 13 6 Bng 3.18 bn pH ca endoglucanase Hot tớnh endoglucanase m pH i chng 6,5 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 Acetate Phosphate (IU/mg protein) 234,57 56,48 266,53 322,93 347,29 285,43 241,78 229,21 245,38 223,27 210,21 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 84 Footer Page 94 of 126 0,008 0,028 0,008 0,020 0,004 0,004 0,011 0,008 0,020 0,004 0,004 % 100 24 114 138 148 122 103 98 105 95 90 http://www.lrc-tnu.edu.vn Header Page 95 of 126 Bng 3.19 bn pH theo thi gian ca endoglucanase pH 4,0 4,5 5,0 5,5 6,0 Thi gian (gi) 12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72 Hot tớnh endoglucanase (IU/mg protein) 259,02 0,002 217,52 0,031 166,28 0,005 105,38 0,001 73,33 0,001 67,88 0,004 54,24 0,004 36,70 0,002 322,35 0,016 310,56 0,014 269,06 0,005 267,21 0,006 264,19 0,004 185,66 0,000 122,34 0,009 104,90 0,004 310,76 0,008 301,21 0,002 281,14 0,004 277,53 0,001 275,00 0,004 202,42 0,006 149,52 0,008 116,68 0,003 253,47 0,004 244,02 0,001 217,62 0,008 210,02 0,005 203,59 0,004 174,26 0,006 175,82 0,002 173,58 0,007 209,43 0,001 198,72 0,001 192,58 0,000 143,09 0,008 137,73 0,006 136,56 0,005 125,45 0,005 115,42 0,010 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 85 Footer Page 95 of 126 % 100 84 64 41 28 26 21 14 100 96 83 83 82 58 38 33 100 97 90 89 88 65 48 38 100 96 86 83 80 69 69 68 100 95 92 68 66 65 60 55 http://www.lrc-tnu.edu.vn Header Page 96 of 126 Bng 3.20 nh hng ca dung mụi hu c n hot tớnh endoglucanase Dung Mụi i chng Acetone n- Butanol Ethanol isopropanol Methanol Nng (%) 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 Hot tớnh endoglucanase (IU/mg protein) 372,91 0,016 349,04 0,031 333,75 0,004 325,95 0,004 287,28 0,003 225,12 0,025 213,53 0,003 229,60 0,001 198,52 0,001 195,79 0,002 365,02 0,050 276,27 0,044 258,24 0,004 307,64 0,001 271,30 0,011 266,82 0,004 % 100 94 89 87 77 60 57 62 53 53 98 74 69 82 73 72 Bng 3.22 nh hng ca mt s cht ty n hot tớnh endoglucanase Cht ty Nng (%) i chng Tween 20 Tween 80 SDS Triton X-100 0,0 0,5 1,0 1,5 2,0 0,5 1,0 1,5 2,0 0,5 1,0 1,5 2,0 0,5 1,0 1,5 2,0 Hot tớnh endoglucanase (IU/mg protein) 213,53 0,004 191,22 0,008 167,83 0,004 160,43 0,006 131,30 0,008 177,48 0,001 160,43 0,004 144,55 0,003 136,56 0,001 97,01 0,005 83,17 0,001 51,70 0,001 33,48 0,004 175,73 0,004 147,67 0,002 134,32 0,003 125,56 0,010 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 86 Footer Page 96 of 126 % 100 90 79 75 61 83 75 68 64 45 39 24 16 82 69 63 59 http://www.lrc-tnu.edu.vn Header Page 97 of 126 S húa bi Trung tõm Hc liu i hc Thỏi Nguyờn 87 Footer Page 97 of 126 http://www.lrc-tnu.edu.vn ... Tuyển chọn, nuôi cấy chủng Aspergillus awamori sinh tổng hợp endo-β-1,4-glucanase đánh giá tính chất lý hóa endo-β-1,4glucanase” với mục tiêu: (1) Tuyển chọn chủng Aspergillus awamori sinh tổng. .. - NGUYỄN VĂN TUÂN TUYỂN CHỌN, NUÔI CẤY CHỦNG ASPERGILLUS AWAMORI SINH TỔNG HỢP ENDO-β-1,4-GLUCANASE VÀ ĐÁNH GIÁ TÍNH CHẤT LÝ HÓA CỦA ENDO-β-1,4-GLUCANASE Chuyên ngành: Sinh học thực nghiệm... sinh trƣởng khả sinh tổng hợp loại enzyme vi sinh vật chịu tác động nhiều yếu tố khác nhƣ: thời gian nuôi cấy, tốc độ lắc sục khí, lƣợng giống đƣợc tiếp vào ban đầu 2.7 TÍNH CHẤT LÝ HÓA CỦA ENDO-β-1,4-GLUCANASE