Luận án Tách dõng, biểu hiện và nghiên cứu tính chất của endochitinase từ bacillus thuringiensis phân lập ở Việt Nam

Trixton X100 with a concentration of 2% has increased the enzyme activity of rChiA by 14,6%. SDS with a concentration of 2%, enzyme activities of rChiA was only 66,8%. Organic solvents such as ethanol, acetone, methanol, n - butanol (10 - 30% concentrate) all reduced enzyme activity. Km and Vmax characteristic the specifity of each enzyme for each substrate. The Km smaller, the affinity greater of the enzyme with substrate and to the contrary. In our study, the Vmax, Km for purified recombinant ChiA, using the 4-MU-(GlcNAc)3 substrate, were 0,53 nM/min ± 0,087 and 0,41 mg/ml ± 0,06, respectively. When using colloidal chitin as the substrate, Vmax and Km were 0,63 µM/min ± 0,09 and 8,90 mg/ml ± 0,34. Thus, the 4-MU-(GlcNAc)3 substrate exhibits higher specificity than the chitin substrate. Because 4-MU-(GlcNAc)3 is the specific substrate for the endochitinase, the affinity of rChiA to the substrate is large. Using purified rChiA and colloidal chitin as the substrate source, chitin- derived oligosaccharides with degree of polymerization of 2 (GlcNAc2), (GlcNAc3) and (GlcNAc4) were detected. In addition, N-acetyl-D-glucosamine was detected, too. This may be due to the surface microstructure and the rate of chitin in different fungal cell walls, the enzyme rChiA inhibits the growth of two fungal pathogens, F. oxysporum and R. solani, but that does not affect to the Mucor sp. Fungal F. oxysporum, R. solani and Mucor sp. treated with rChiA, the amount of N-acetyl D- glucosamine released was 22,14 μg/ml, 18,36 μg/ml and 0 μg/ml, respectively. Recombinant chitinase not only as control agent against phytopathogenic fungi, but also for enhancing of insecticidal activity of protein Cry of B. 129 thuringiensis. 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Trình t so sánh trên Ngân hàng gen 10 20 30 40 50 60 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATGGCTATGA GGTCTCAAAA ATTCACACTG CTATTACTAT CCCTACTACT TTTCTTACCT EF581163.2 .......... .......... .......... .......... .......... .......... 70 80 90 100 110 120 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CTTTTTCTCA CAAATTTTAT TACTCCAAAT CTCGCATTAG CAGATTCACC AAAGCAAAGT EF581163.2 .......... .......... .......... .......... .......... .......... 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CAAAAAATTG TTGGGTACCT TCCTTCGTGG GGCGTTTACG GACGTAATTA TCAAGTTGCT EF581163.2 .......... ........T. .......... .......... .......... .......... 190 200 210 220 230 240 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GACATTGATG CATCAAAACT TACTCACCTT AACTATGCTT TCGCGGATAT TTGTTGGAAT EF581163.2 .......... .......... .......... .......... .......... .......... 250 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GGAAAACATG GAAACCCTTC TACTCATCCT GATAATCCAA ATAAACAAAC GTGGAACTGT EF581163.2 .......... .......... .......... .......... .......... .......... 310 320 330 340 350 360 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF AAAGAATCTG GTGTACCATT GCAAAATAAA GAGGTTCCTA ATGGTACTCT CGTACTCGGG EF581163.2 .......... .......... .......G.. .......... .......... .......... 370 380 390 400 410 420 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GAACCATGGG CTGATGTTAC CAAATCGTAT CCTGGCTCAG GGACAACTTG GGAAGATTGC EF581163.2 .......... .......... .......... .......... .......... .......... 430 440 450 460 470 480 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GATAAATATG CCCGTTGCGG AAATTTCGGG GAACTAAAAC GATTAAAAGC TAAATATCCT EF581163.2 .......... .......... .......... .......... .......... .......... 490 500 510 520 530 540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CACTTAAAAA CAATTATTTC CGTTGGTGGC TGGACTTGGT CTAACCGCTT TTCTGATATG EF581163.2 .......... .......... .......... .......... .......... .......... 550 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GCCGCTGATG AAAAAACAAG AAAAGTATTT GCTGAATCTA CAGTAGCTTT TCTTCGCGCA EF581163.2 .......... .......... .......... .......... .......... .......... 610 620 630 640 650 660 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF TATGGGTTTG ATGGCGTAGA TTTAGACTGG GAATGTCCGG GCGTTGAAAC GATTCCTGGT EF581163.2 ........C. .......... .......... ....A..... .......... .......... 2 670 680 690 700 710 720 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GGTAGTTATC GTCCTGAAGA TAAACAAAAT TTCACTCTCC TTCTTCAAGA CGTCCGAAAT EF581163.2 .......... .......... .......... .......... .......... ........G. 730 740 750 760 770 780 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GCTTTGAATA AAGCAGGTGC TGAAGATGGC AAACAATATT TACTAACAAT CGCTTCAGGT EF581163.2 .......... .......... .......... .......... .......... .......... 790 800 810 820 830 840 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GCAAGCCAAC GCTACGCTGA TCATACAGAA CTAAAGAAAA TTTCTCAAAT ACTCGATTGG EF581163.2 .......... .......... .......... .......... .......... .......... 850 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATTAATATTA TGACATATGA TTTCCACGGC GGATGGGAAG CTACTTCTAA TCATAATGCA EF581163.2 .......... .......... .......... .......... .......... .......... 910 920 930 940 950 960 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GCTCTATATA AGGATCCAAA TGATCCAGCA GCAAATACGA ATTTTTACGT AGATGGTGCT EF581163.2 .......... ....C..... .......... .......... .......... .......... 970 980 990 1000 1010 1020 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATAAATGTTT ATACAAATGA AGGTGTTCCA GTCGATAAAC TAGTATTAGG CGTACCCTTT EF581163.2 .......... .......... .......... .......... .......... .......... 1030 1040 1050 1060 1070 1080 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF TACGGACGTG GCTGGAAAAG TTGTGGCAAA GAAAATAACG GACAATATCA ACCTTGCAAA EF581163.2 .......... .......... .......... .......... .......... .......... 1090 1100 1110 1120 1130 1140 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CCAGGTAGTG ATGGGAAACT TGCTTCTAAA GGTACTTGGG ATGATTACTC TACCGGTGAC EF581163.2 .......... .......... .......... .......... .......... .......... 1150 1160 1170 1180 1190 1200 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ACAGGTGTCT ATGATTACGG TGATTTAGCA GCCAATTACG TTAATAAAAA TGGTTTTGTA EF581163.2 .......... .......... .......... .......... .......... .......... 1210 1220 1230 1240 1250 1260 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CGCTACTGGA ATGACACAGC TAAAGTACCT TACTTATATA ATGCAACTAC AGGCACATTT EF581163.2 .......... .......... .......... .......... .......... .......... 1270 1280 1290 1300 1310 1320 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATTAGCTACG ATGACAATGA ATCTATGAAA TACAAAACAG ACTATATAAA GACGAAAGGT EF581163.2 .......... .......... .......... .......... .......... .......... 1330 1340 1350 1360 1370 1380 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF TTAAGTGGAG CAATGTTTTG GGAACTAAGC GGAGATTGCC GTACAAGTCC AAAATATAGT EF581163.2 .......... .......... .......... .......... .......... .......... 1390 1400 1410 1420 1430 1440 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF TGCAGTGGTC CAAAATTACT TGATACGCTA GTAAAAGAAT TACTTGGTGG ACCTATTAAT EF581163.2 .......... .......... .......... .......... .......... .......... 3 1450 1460 1470 1480 1490 1500 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CAAAAAGATA CTGAGCCACC AACGAATGTT AAAAACATTG TAGTTACGAA TAAAAATTCA EF581163.2 .......... .......... .......... .......... .......... .......... 1510 1520 1530 1540 1550 1560 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF AACTCAGTTC AATTAAACTG GACTGCATCT ACTGATAACG TAGGAGTTAC GGAATATGAA EF581163.2 .......... .......... .......... .......... .......... .......... 1570 1580 1590 1600 1610 1620 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATTACTGCTG GAGAAGAGAA ATGGAGTACA ACAACAAATA GCATTACAAT TAAAAACTTA kurstaki .......... .......... .......... .......... .......... .......... 1630 1640 1650 1660 1670 1680 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF AAACCTAATA CGGAATACAA ATTTTCAATA ATTGCCAAAG ATGCTGCTGG AAATAAATCG EF581163.2 .......... .......... .......... .......... .......... .........A 1690 1700 1710 1720 1730 1740 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF CAACCTACCG CTCTTACTGT CAAAACGGAT GAAGCTAATA CGACACCTCC TGATGGAAAT EF581163.2 .......... .......... .......... .......... .......... .......... 1750 1760 1770 1780 1790 1800 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF GGTACTGCTA CATTTTCAGT CACTTCGAAT TGGGGCAGCG GTTATAACTT CTCGATTATA EF581163.2 .......... .......... .......... .......... .......... .......... 1810 1820 1830 1840 1850 1860 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ATCAAAAATA ATGGAACGAA CCCCATTAAA AATTGGAAAT TAGAATTTGA TTATAGCGGC EF581163.2 .......... ....G..... ...T...... .......... .......... .......... 1870 1880 1890 1900 1910 1920 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF AATTTAACAC AAGTTTGGGA TTCTAAAATT AGTAGTAAAA CAAATAATCA TTATGTAATT EF581163.2 .......... .......... .......... .......... .......... .......... 1930 1940 1950 1960 1970 1980 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1_1_ChiF ACGAACGCAG GATGGGATGG TGAAATTCCT CCTGGTGGAT CTATTACAAT TGGCGGTGCA EF581163.2 .......... .....A.... .......... .......... .......... .......... 1990 2000 2010 2020 2030 ....|....| ....|....| ....|....| ....|....| ....|....| . MSS1_1_ChiF GGAACAGGTA ATCCTGCCGA ACTTTTAAAT GCCGTCATTA GCGAAAACTA G EF581163.2 .......... .......... .......... .......... .......... . Hình 1. So sánh trình tự nucleotide đoạn gen chiA của chủng MSS1.1 với trình tự EF581163.2 trên GenBank 4 10 20 30 40 50 60 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa MAMRSQKFTL LLLSLLLFLP LFLTNFITPN LALADSPKQS QKIVGYLPSW GVYGRNYQVA ABQ65137.2 .......... .......... .......... .......... ......F... .......... 70 80 90 100 110 120 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa DIDASKLTHL NYAFADICWN GKHGNPSTHP DNPNKQTWNC KESGVPLQNK EVPNGTLVLG ABQ65137.2 .......... .......... .......... .......... .........E .......... 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa EPWADVTKSY PGSGTTWEDC DKYARCGNFG ELKRLKAKYP HLKTIISVGG WTWSNRFSDM ABQ65137.2 .......... .......... .......... .......... .......... .......... 190 200 210 220 230 240 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa AADEKTRKVF AESTVAFLRA YGFDGVDLDW ECPGVETIPG GSYRPEDKQN FTLLLQDVRN ABQ65137.2 .......... .......... .......... .Y........ .......... .........S 250 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa ALNKAGAEDG KQYLLTIASG ASQRYADHTE LKKISQILDW INIMTYDFHG GWEATSNHNA ABQ65137.2 .......... .......... .......... .......... .......... .......... 310 320 330 340 350 360 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa ALYKDPNDPA ANTNFYVDGA INVYTNEGVP VDKLVLGVPF YGRGWKSCGK ENNGQYQPCK ABQ65137.2 .......... .......... .......... .......... .......... .......... 370 380 390 400 410 420 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa PGSDGKLASK GTWDDYSTGD TGVYDYGDLA ANYVNKNGFV RYWNDTAKVP YLYNATTGTF ABQ65137.2 .......... .......... .......... .......... .......... .......... 430 440 450 460 470 480 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa ISYDDNESMK YKTDYIKTKG LSGAMFWELS GDCRTSPKYS CSGPKLLDTL VKELLGGPIN ABQ65137.2 .......... .......... .......... .......... .......... .......... 490 500 510 520 530 540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa QKDTEPPTNV KNIVVTNKNS NSVQLNWTAS TDNVGVTEYE ITAGEEKWST TTNSITIKNL ABQ65137.2 .......... .......... .......... .......... .......... .......... 550 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa KPNTEYKFSI IAKDAAGNKS QPTALTVKTD EANTTPPDGN GTATFSVTSN WGSGYNFSII ABQ65137.2 .......... .......... .......... .......... .......... .......... 610 620 630 640 650 660 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| MSS1.1_aa IKNNGTNPIK NWKLEFDYSG NLTQVWDSKI SSKTNNHYVI TNAGWDGEIP PGGSITIGGA ABQ65137.2 .......... .......... .......... .......... .....N.... .......... 670 ....|....| ....|. MSS1.1_aa GTGNPAELLN AVISEN ABQ65137.2 .......... ...... Hình 2. So sánh trình tự amino acid của protein ChiA t chủng MSS1.1 với trình tự của chủng HD73 có mã số ABQ65137.2 trên ngân hàng dữ liệu 5 Ph l c 2. Trình tự nucleotide của gen chiA và amino acid của Chitinase có nguồn gốc t chủng B. thuringiensis var. kurstaki MSS1.1 được đăng ký trên Ngân hàng cơ sở dữ liệu GenBank 6 Ph l c3. Bản ồ vector biểu hiện pET28b(+) 7 Ph l c 3. Bản đồ vector biểu hiện pET28b(+) 8 Ph l c 4. Kết quả chạy kiểm tra mô hình của thuật toán tối ưu Bảng 1. Kết quả theo mô hình Số TT Lactose (mM) Thời gian (giờ) Mật ộ TB (OD 600 nm) Hoạt ộ (U/ml) 1 6,00 9,00 0,80 7,36 2 7,00 6,00 1,00 5,14 3 5,00 6,00 0,60 1,99 4 7,00 12,00 1,00 4,88 5 7,00 6,00 0,60 5,59 6 5,00 12,00 1,00 3,17 7 5,00 12,00 0,60 1,14 8 5,00 6,00 1,00 4,02 9 7,00 12,00 0,60 5,34 10 5,81 6,41 0,75 6,39 11 6,44 12,00 0,62 5,51 12 6,86 6,07 0,80 6,71 13 5,15 6,08 0,85 4,87 14 6,16 9,61 0,65 6,61 15 6,86 11,82 0,94 5,82 16 6,66 6,57 0,69 6,83 17 5,48 8,14 1,00 5,95 18 5,01 9,34 0,67 3,54 19 5,91 9,20 0,88 7,22 20 5,18 9,90 0,65 3,88 21 5,23 9,04 0,74 5,18 22 6,83 11,37 0,88 6,55 23 6,45 7,30 0,75 7,39 24 5,37 10,94 0,71 4,76 25 5,36 6,67 0,88 5,78 26 6,67 7,71 0,70 7,26 27 5,44 11,29 0,91 5,41 28 5,62 11,44 0,66 4,70 9 29 6,30 6,47 0,87 7,09 30 5,16 8,71 0,83 5,43 31 5,66 7,47 0,95 6,50 32 5,11 9,05 0,93 5,23 33 6,63 6,72 0,92 6,85 34 6,93 10,69 0,63 6,40 35 6,19 9,78 0,93 7,17 36 5,24 9,08 0,92 5,66 37 6,69 6,37 0,94 6,43 38 5,55 6,97 0,88 6,37 39 6,71 9,91 0,66 6,97 40 5,23 10,18 0,82 5,30 41 5,29 7,33 0,91 5,77 42 6,71 10,08 1,00 6,31 43 5,63 11,97 0,99 5,00 44 5,32 6,35 0,79 5,38 45 5,99 9,16 0,95 6,98 46 5,11 6,47 0,74 4,35 47 5,05 11,99 0,63 1,88 48 5,66 10,33 0,60 4,76 49 5,35 11,78 0,84 4,85 50 6,76 9,12 0,91 7,18 51 5,45 11,45 0,69 4,56 52 6,85 6,53 0,72 6,85 53 5,24 11,01 0,73 4,41 54 6,44 6,41 0,89 6,98 55 5,76 11,97 0,94 5,52 56 5,68 8,76 0,93 6,74 57 5,42 9,14 0,98 5,92 58 6,72 9,84 0,78 7,48 59 5,96 7,18 0,92 6,98 10 60 5,57 6,75 0,98 5,96 61 6,58 9,43 0,65 6,94 62 5,40 8,45 0,65 4,95 63 5,16 9,09 0,73 4,78 64 6,85 11,59 0,89 6,35 65 6,93 7,01 0,98 6,07 66 5,35 7,86 0,76 5,73 67 5,09 11,01 0,95 4,40 68 5,57 7,12 0,89 6,46 69 6,35 9,81 0,92 7,24 70 5,91 11,89 0,96 5,66 71 5,84 9,95 0,86 6,97 72 6,09 10,02 0,80 7,27 73 6,71 7,07 0,64 6,69 74 6,90 8,52 0,64 6,91 75 6,83 7,98 0,70 7,26 76 6,63 11,03 0,70 6,79 77 6,42 10,72 0,70 6,90 78 6,04 8,02 0,99 6,72 79 5,85 8,14 0,89 7,13 80 5,60 6,81 0,91 6,34 81 6,75 6,60 0,79 7,10 82 5,95 7,94 0,76 7,16 83 5,20 8,77 0,80 5,47 84 6,88 6,13 0,97 5,83 85 6,15 8,12 0,87 7,49 86 6,53 11,38 0,99 6,01 87 5,60 6,52 0,96 6,03 88 6,19 9,37 0,91 7,35 89 5,82 6,64 0,93 6,59 90 5,74 7,12 0,65 5,70 11 91 6,61 11,14 0,97 6,20 92 5,86 7,31 0,80 7,00 93 5,18 10,59 0,77 4,73 94 5,62 6,77 0,93 6,29 95 6,06 8,56 0,66 6,68 96 6,35 11,00 0,83 7,08 97 5,81 10,06 0,70 6,28 98 5,15 11,34 0,74 3,96 99 5,78 10,02 0,89 6,80 100 6,46 6,71 0,66 6,68 Bảng 2. Các phương án lựa chọn nằm trong vùng tối ưu Số TT Lactose (mM) Thời gian (giờ) Mật ộ TB (OD600 nm) Hoạt ộ (U/ml) 1 6,00 9,00 0,80 7,36 19 5,91 9,20 0,88 7,22 23 6,45 7,30 0,75 7,39 26 6,67 7,71 0,70 7,26 29 6,30 6,47 0,87 7,09 35 6,19 9,78 0,93 7,17 50 6,76 9,12 0,91 7,18 58 6,72 9,84 0,78 7,48 69 6,35 9,81 0,92 7,24 72 6,09 10,02 0,80 7,27 75 6,83 7,98 0,70 7,26 79 5,85 8,14 0,89 7,13 81 6,75 6,60 0,79 7,10 82 5,95 7,94 0,76 7,16 85 6,15 8,12 0,87 7,49 88 6,19 9,37 0,91 7,35 92 5,86 7,31 0,80 7,00 96 6,35 11,00 0,83 7,08 12 Ph l c 5: Kết quả thử nghiệm của enzyme tái tổ hợp Bảng 1. T lệ sâu tơ chết (%) sau các khoảng thời gian thử nghiệm khác nhau Mẫu thí nghiệm Thời gian th nghiệm (giờ) 0 24 36 48 60 72 SP10.6 0 26,67 63,33 82,22 93,33 100 rChiA 0 0 3,33 6,67 7,78 8,89 SP10.6 + rChiA 0 51,11 81,10 100 - - Bảng 2. T lệ sâu khoang chết (%) sau các khoảng thời gian thử nghiệm khác nhau Mẫu thí nghiệm Thời gian th nghiệm (giờ) 0 24 36 48 60 72 SP10.6 0 27,78 53,33 69,99 78,89 83,33 rChiA 0 0 3,33 4,44 5,56 6,67 SP10.6 + rChiA 0 33,33 67,78 84,44 85,56 87,78 Chú thích: SP10.6: Protein tinh thể t chủng B. thuringiensis SP10.6 rChiA: Enzyme chitinase tái tổ hợp SP10.6 + rChiA: Hỗn hợp giữa protein tinh thể t chủng SP10.6 và chitinase tái tổ hợp. 13