CN101503660A - Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof - Google Patents
Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof Download PDFInfo
- Publication number
- CN101503660A CN101503660A CNA2009100776469A CN200910077646A CN101503660A CN 101503660 A CN101503660 A CN 101503660A CN A2009100776469 A CNA2009100776469 A CN A2009100776469A CN 200910077646 A CN200910077646 A CN 200910077646A CN 101503660 A CN101503660 A CN 101503660A
- Authority
- CN
- China
- Prior art keywords
- glucoamylase
- aspergillus niger
- enzyme
- gene
- engineering bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 52
- 108090000790 Enzymes Proteins 0.000 title claims description 118
- 102000004190 Enzymes Human genes 0.000 title description 114
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 83
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims abstract description 74
- 102100022624 Glucoamylase Human genes 0.000 claims abstract description 41
- 230000014509 gene expression Effects 0.000 claims abstract description 27
- 239000002773 nucleotide Substances 0.000 claims abstract description 23
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 8
- 230000004151 fermentation Effects 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 241000205091 Sulfolobus solfataricus Species 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 23
- 230000001580 bacterial effect Effects 0.000 claims description 18
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 claims description 14
- 238000003209 gene knockout Methods 0.000 claims description 7
- 230000009849 deactivation Effects 0.000 claims description 4
- 230000008676 import Effects 0.000 claims 1
- 239000012530 fluid Substances 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000000415 inactivating effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 113
- 239000000203 mixture Substances 0.000 description 79
- 239000000243 solution Substances 0.000 description 47
- 239000012634 fragment Substances 0.000 description 46
- 239000002609 medium Substances 0.000 description 33
- 230000000694 effects Effects 0.000 description 29
- 239000007788 liquid Substances 0.000 description 28
- 230000008859 change Effects 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 21
- 230000003321 amplification Effects 0.000 description 20
- 238000003199 nucleic acid amplification method Methods 0.000 description 20
- 101150108358 GLAA gene Proteins 0.000 description 17
- 238000012408 PCR amplification Methods 0.000 description 17
- 238000004321 preservation Methods 0.000 description 16
- 238000011160 research Methods 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 241000233866 Fungi Species 0.000 description 15
- 229920002472 Starch Polymers 0.000 description 15
- 108091008146 restriction endonucleases Proteins 0.000 description 15
- 239000008107 starch Substances 0.000 description 15
- 235000019698 starch Nutrition 0.000 description 15
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 14
- 230000000813 microbial effect Effects 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 11
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- 229930006000 Sucrose Natural products 0.000 description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 10
- 238000013461 design Methods 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 239000005720 sucrose Substances 0.000 description 10
- 238000004448 titration Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 235000015097 nutrients Nutrition 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 102000018120 Recombinases Human genes 0.000 description 8
- 108010091086 Recombinases Proteins 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 101150069003 amdS gene Proteins 0.000 description 7
- 238000000137 annealing Methods 0.000 description 7
- 230000004087 circulation Effects 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 108010048241 acetamidase Proteins 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 5
- 241000438227 Sulfolobus solfataricus P2 Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 229930182478 glucoside Natural products 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000003301 hydrolyzing effect Effects 0.000 description 5
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 5
- 229940097277 hygromycin b Drugs 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- PODWXQQNRWNDGD-UHFFFAOYSA-L sodium thiosulfate pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[O-]S([S-])(=O)=O PODWXQQNRWNDGD-UHFFFAOYSA-L 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 241000351920 Aspergillus nidulans Species 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 229920001353 Dextrin Polymers 0.000 description 4
- 239000004375 Dextrin Substances 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 235000019425 dextrin Nutrition 0.000 description 4
- 229940111205 diastase Drugs 0.000 description 4
- 230000009970 fire resistant effect Effects 0.000 description 4
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000008521 reorganization Effects 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 241000194108 Bacillus licheniformis Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 102000004139 alpha-Amylases Human genes 0.000 description 3
- 108090000637 alpha-Amylases Proteins 0.000 description 3
- 229940024171 alpha-amylase Drugs 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 3
- 229940059574 pentaerithrityl Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 108020005065 3' Flanking Region Proteins 0.000 description 2
- 108020005029 5' Flanking Region Proteins 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 241000228251 Aspergillus phoenicis Species 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 230000003416 augmentation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical group CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 101900018172 Aspergillus oryzae Acetamidase Proteins 0.000 description 1
- 101000914103 Bos taurus Chymosin Proteins 0.000 description 1
- 101100282617 Bovine herpesvirus 1.1 (strain Cooper) gC gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000959173 Rasamsonia emersonii Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101100370749 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) trpC1 gene Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 229930092411 Swietenocoumarin D Natural products 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241000193447 Thermoanaerobacter thermohydrosulfuricus Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 1
- HIVLDXAAFGCOFU-UHFFFAOYSA-N ammonium hydrosulfide Chemical compound [NH4+].[SH-] HIVLDXAAFGCOFU-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000003625 amylolytic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 241000617156 archaeon Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002478 diastatic effect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 101150073906 gpdA gene Proteins 0.000 description 1
- 101150095733 gpsA gene Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000050459 human LTF Human genes 0.000 description 1
- 229940116886 human interleukin-6 Drugs 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108010064037 prorennin Proteins 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 101150016309 trpC gene Proteins 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses engineering bacteria for expressing thermostable glucoamylase and use thereof. The engineering bacteria are recombinant engineering bacteria for expressing the thermostable glucoamylase which are obtained by inactivating a glucoamylase gene of aspergillus niger, inserting a glucoamylase gene of slfolobus solfataricus into a genome of the aspergillus niger and screening; the glucoamylase gene of the aspergillus niger has a nucleotide sequence which is at a 92183-94360 locus at a 5' end and has a genbank accession number of AM270061.1; and the glucoamylase gene of the slfolobus solfataricus has nucleotide sequence which is at a 6228-8096 locus at the 5' end and has a genbank accession number of AE006718. The engineering bacteria have the advantages that: the thermostable glucoamylase can be produced by the direct fermentation of the engineering bacteria; the expression is up to 3,000 U per ml of zymotic fluid; the production cost is low; and the produced glucoamylase can be separated and purified easily.
Description
Technical field
The present invention relates to express the engineering bacteria and the application thereof of thermophilic glucoamylase.
Background technology
Saccharifying enzyme, system is called α-1, and (α-1 4-glucan-glucohydrolase), has another name called glucoamylase (glucoamylase) to 4-dextran glucose hydrolysis enzyme, is a kind of circumscribed-type Glycosylase.It can discharge alpha-D-glucose from the non reducing end of starch, glycogen or oligomerization maltose.The Substratspezifitaet of saccharifying enzyme is lower, and except can be from non reducing end hydrolyzing alpha successively-1, the 4-glycosidic link can also hydrolyzing alpha-1, and therefore the 6-glycosidic link can all be hydrolyzed into glucose with amylopectin.In addition, saccharifying enzyme can also weakly hydrolyse α-1, the carbochain that 3-connects, it generally can both with starch very hydrolysis generate glucose.Therefore saccharifying enzyme is the main enzyme that amylolytic fermentation is produced alcohol, glucose syrup.
Now glucose production adopts enzyme process mostly both at home and abroad, and the enzyme process sugar refining technology generally adopts two-step approach.The first step is hydrolyzed into low-molecular-weight dextrin with α-Dian Fenmei with starch, is called liquefaction; Second step became glucose with saccharifying enzyme with dextrin hydrolysis, was called saccharification.During the higher starch fluid gelatinization of concentration, liquefaction rapidly, otherwise starch fluid viscosity rises equipment can't be moved, the starch of gelatinization can take place aging after temperature reduces and can't liquefy, sugar refining technology extensively adopts steam ejection liquefaction at present, and injection temperature is generally at 105～108 ℃, and the α-Dian Fenmei of using is mainly the high temperature resistant α-Dian Fenmei that derives from Bacillus licheniformis now, optimum temperature is more than 90 ℃, and optimal pH is 5.5～7.0.But the optimum temperature of saccharifying enzyme, pH etc. are all different with amylase, just can enter saccharifying after therefore needing to adjust temperature and pH.
The distribution of saccharifying enzyme in microorganism is very wide, belongs to glycoside hydrolysis 15 (GH15) protein families, and existing so far nearly 40 open reading frame that derive from filamentous fungus, yeast, eubacterium and ancient bacterium are divided into this family.What China was commonly used at present is the saccharifying enzyme of aspergillus niger, optimal pH 4.5, and 60 ℃ of optimum temperutures after therefore liquefaction is finished, need liquefying starch is cooled to 55～60 ℃, after pH transfers to 4.5～5.0, add an amount of saccharifying enzyme.Keep about saccharification 48h, dextrin just changes into glucose basically.Sometimes in order to shorten saccharification time, in saccharifying, usually adopt high temperature (88-100 ℃), this has just required resistant to elevated temperatures enzyme is to participate in saccharification.
At present, isolated polytype resistant to elevated temperatures saccharifying enzyme from many microorganisms, the thermostability report of relevant saccharifying enzyme is a lot.Zeikus etc. find that Clostridium thermohydro-sulfuricum saccharifying enzyme is in 50% starch solution, 70 ℃ of following enzyme complete stabilities, and still very stable in 10% alcohol liquid, still keep 50% even handle its enzymic activity of 1h down, and this kind of enzyme is not subjected to Ca at 85 ℃
2+, EDTA and α-, β-, the influence of cyclooctaamylose.The saccharifying enzyme GLUMI that reports such as Tomoko.TAKAHASHI come from A.saitoi is still keeping it all active more than 50 ℃, but the activity of this enzyme is subject to Hg
2+Inhibition.3 types of saccharifying enzyme GI that reports such as champion Chen obtain from aspergillus niger As3.4809 variant B-11 fermented liquid, GII, GIII, its optimum temperuture is 70 ℃.Cyclohexaamylose can make the thermostability of saccharifying enzyme improve down at 60 ℃.General saccharifying enzyme all has narrower pH value subject range, but optimal pH is generally 4.5～6.5.Its optimal pH scope of saccharifying enzyme GLUMI that reports such as Tomoko.TAKAHAS HI come from A.saitoi is 2.5～7.5, and optimal pH is 4.5.Hyun H H etc. reported once that the saccharifying enzyme pH value stabilization scope that A.niger produces was 2～11.The optimum temperuture that reports such as Mi-Sun Kim come from glucoamylase gene recombinant protein behind expression in escherichia coli of sulfolobus solfataricus is 90 ℃, optimal pH 5.5～6.0.The T1/2 of the reorganization Talaromyces emersonii glucoamylase that discoveries such as Bjarne Ronfeldt Nielsen are expressed in yeast is about 110 minutes under 70 ℃.
Sulfolobus solfataricus (sulfolobus solfataricus) belongs to the extreme thermophile bacteria in the ancient bacterium of spring, be located away from first in the gondola solfatara hot spring, form is spherical, lobate division is arranged, thermophilic acidophilic acid, optimum growth temp are 80 ℃, and optimum pH is 3.0, facultative autotrophy, can sulphur or other simple organics be nutrient.As the pattern bacterium of the ancient bacterium of research, the genome complete sequence determination of sulfolobus solfataricus P2 was all finished in calendar year 2001, and its genome is 2,992,245bp, 2977 albumen of encoding.These albumen have 1st/3rd, and sulfolobus solfataricus institute is peculiar, and the 40%th, ancient bacterium institute is peculiar, and 12% has homology with bacterium, and 2% has homology with fungi.
Filamentous fungus is as the host transformed bacterium, many other microorganisms such as incomparable advantage such as bacterium, yeast are arranged: 1) filamentous fungus has the ability of very strong secretion extracellular protein, and the recombinant protein of bacterial expression often exists with inclusion body form non-activity, indissoluble; 2) when bacterial expression external source eukaryotic gene, because of its translation with transcribe and be different from eukaryote,, might not express, but there is not similar situation in filamentous fungus even if therefore eukaryotic gene changes bacterium over to yet; 3) filamentous fungus can carry out correct translation post-treatment to expressing protein, comprises translation post-treatment such as the shearing of peptide chain and glycosylation; 4) filamentous fungus has quick fecundity and the short life cycle as bacterium, and is simpler than vegeto-animal cell cultures; 5) many filamentous funguss are used in foodstuffs industry for a long time as aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillu oryzae) etc., are generally acknowledged it is safe; 6) filamentous fungus has sophisticated zymotechnique and aftertreatment technology.This shows that filamentous fungus is an attractive allogeneic gene expression system.Especially this year, fast development along with the filamentous fungus transformation technology, many filamentous funguss are applied in industry, agricultural, the pharmaceutical industries, produced the recombinant protein of many fungies or non-originated from fungus, as glucoamylase (glucoamylase), ox renninogen (bovine chymosin), human lactoferrin (human lact2oferrin), hen's egg-white lysozyme (hen egg-white lysozyme) and plain 6 (humaninterleukin-6) of human leukocytes Jie etc.
Summary of the invention
The purpose of this invention is to provide engineering bacteria and the application thereof of expressing thermophilic glucoamylase.
The engineering bacteria of expression thermophilic glucoamylase provided by the present invention, it is glucoamylase gene deactivation with aspergillus niger (Aspergillus niger), and the expression cassette that will express the glucoamylase gene of sulfolobus solfataricus (sulfolobus solfataricus) is inserted in the genome of aspergillus niger (Aspergillus niger), and screening obtains expressing the recombinant bacterial strain of thermophilic glucoamylase; It is 5 of AY250996 ' end 1052-4252 position nucleotide sequence that the glucoamylase gene of described aspergillus niger (Aspergillus niger) has GENBANK Accession Number; It is 5 of AE006718.1 ' end 6228-8096 position nucleotide sequence that the glucoamylase gene of described sulfolobus solfataricus (sulfolobussolfataricus) has GENBANK Accession Number.
Also contain acetyl ammonia enzyme gene in the expression cassette of the glucoamylase gene of described expression sulfolobus solfataricus (sulfolobus solfataricus); Described acetyl ammonia enzyme gene have from GENBANK number for AM270228.1 5 ' end 31228-33207 position nucleotide sequence.
Above-mentioned glucoamylase gene deactivation with aspergillus niger (Aspergillus niger) can realize by technology such as transgenation, gene conversion (GeneConversion), gene disruption (gene desruption), homologous recombination.Specifically can utilize the method for gene knockout that the glucoamylase gene of described aspergillus niger (Aspergillus niger) is replaced with hygromycin gene; Hygromycin gene is expressed the thermophilic glucoamylase gene and the replacement of acetamidase expression of gene box of sulfolobus solfataricus again subsequently.
Described aspergillus niger (Aspergillus niger) can be aspergillus niger (Aspergillus niger) bacterial strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204).
The dedicated engineering bacteria of described expression thermophilic glucoamylase is preferably aspergillus niger (Aspergillus niger) WW1CGMCC No.2841.
Described aspergillus niger (Aspergillus niger) WW1 CGMCC No.2841, be preserved in Chinese microorganism strain preservation board of trustee reason person on December 25th, 2008 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC No.2841.
Described aspergillus niger (Aspergillus niger) WW1 CGMCC No.2841 is grown to bacterium colony white just on the Cha Shi solid medium, the back becomes accordion.30 ℃ of growth optimum temperutures.
Described aspergillus niger (Aspergillus niger) WW1 CGMCC No.2841 optimum medium is the Cha Shi substratum; Culture temperature is 30～33 ℃, and expressed saccharifying enzyme optimum temperuture is 90 ℃, optimum pH 6.0.
Above-mentioned engineering bacteria can be used for producing thermophilic glucoamylase, and concrete grammar is that the fermentation of the dedicated engineering bacteria of above-mentioned expression thermophilic glucoamylase is obtained thermophilic glucoamylase.
The dedicated engineering bacteria of expression thermophilic glucoamylase of the present invention, remove the saccharifying enzyme α-1 of aspergillus niger itself, 4 and α-1,6 glucoside bond hydrolytic activities, replacement is with sulfolobus solfataricus (sulfolobus solfataricus) glucoamylase gene, make aspergillus niger express sulfolobus solfataricus (sulfolobus solfataricus) saccharifying enzyme, the saccharifying enzyme optimum temperuture of expressing is 90 ℃ a saccharifying enzyme, its optimal pH scope also is increased to 6.0, expression amount reaches the 3000U/ml fermented liquid, and (1h hydrolysis Zulkovsky starch produces the enzyme amount of 1mg glucose, be an enzyme activity unit), thereby realize the high expression of saccharifying enzyme in filamentous fungus of saccharification efficient that resistance toheat is good and optimum pH improves, reduce production cost, and increased the security of the saccharifying enzyme of expressing.
The present invention expresses the saccharifying enzyme of the dedicated engineering bacteria of thermophilic glucoamylase with Totomycin displacement aspergillus niger, and hygromycin gene is replaced by the thermophilic glucoamylase gene of sulfolobus solfataricus and acetamidase gene again subsequently; Can be to grow on the substratum supplied with of carbon source only containing ethanamide, make things convenient for the screening and the purifying of engineering bacteria positive transformant like this, and can not introduce too much microbiotic label, the security that has more improved engineering bacteria of the present invention.
Method of the present invention utilizes above-mentioned engineering bacterium fermentation to produce thermophilic glucoamylase, and production cost is low, characteristics such as the saccharifying enzyme separate easily of production and purifying.The thermophilic glucoamylase that this project bacterium produces, expression amount is up to the 3000U/ml fermented liquid, have higher optimum temperuture and optimal pH, the two is all close with the fire resistant alpha-diastase that derives from Bacillus licheniformis that present industry is used always, as use this recombinase to be used for alcohol industry and when Mashing process, can lower the temperature, saved the energy.
Description of drawings
Fig. 1 is aspergillus niger saccharification enzyme (glaA) gene knockout carrier pEasy-glaA
DirBuilding process
Fig. 2 is the structural representation of pWW
Fig. 3 is the optimal pH detected result of reorganization saccharifying enzyme
Fig. 4 is the optimum temperuture detected result of reorganization saccharifying enzyme
Fig. 5 is the heat stability test result of reorganization saccharifying enzyme
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
" glucase " definition among the present invention: (Glucoamylase EC.3.2.1.3), claims gamma amylase again to glucoamylase, is called for short saccharifying enzyme, and saccharifying enzyme has the extracellular enzyme of 5 prime excision enzyme activity by a series of microorganism secretions.The main effect of saccharifying enzyme is the α-D-1 on the carbochains such as hydrolyzed starch, dextrin, glycogen, 4 glucoside bonds and α-D-1,6 glucoside bonds.With glucose one by one hydrolysis get off, and obtain end product β-D-glucose.Saccharifying enzyme also can be faint hydrolyzing alpha-D-1,3 glucoside bonds, but hydrolyzing alpha-D-1,4 glucoside bonds fastest.
Embodiment 1, can express the structure of the engineering bacteria of thermophilic glucoamylase
One, the structure of the glaA gene knockout engineering bacteria of Aspergillus niger strain CICC2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204)
1, the clone of aspergillus niger saccharification enzyme (glaA) gene 5 ' flanking sequence and 3 ' flanking sequence
Go up glucoamylase gene (having GENBANK Accession Number is the 94354-92183 nucleotide sequence of 5 of AM270061.1 ' end) and the upstream and downstream sequences Design primer of announcing thereof according to NCBI:
The primer of the distolateral wing sequence of 5 of glaA ':
G1A5’FR1’F：5′-CAAGTATATGATGCGGTAGTGG-3′；
G1A5’FR1’R：5′-TGCTGAGGTGTAATGATGCTGG-3′。
The primer of the distolateral wing sequence of 3 of glaA ' adds XhoI and ApaI restriction enzyme site:
Hyg-G1A3’FR3’F：5’-CTCGAGACAATCAATCCATTTCGCTAT-3’
Hyg-G1A3’FR3’R：5’-GGGCCCTAACTCCGCGGAGGCTCATGG-3’
As template, is primer with G1A5 ' FR1 ' F and G1A5 ' FR1 ' R sequence with the genomic dna of Aspergillus niger strain CICC2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) respectively; Hyg-G1A3 ' FR3 ' F and Hyg-G1A3 ' FR3 ' R sequence are that primer carries out pcr amplification and obtains saccharifying enzyme 5 ' flanking sequence, 3 ' flanking sequence.
Above-mentioned pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes 56 ℃ of 30 seconds, annealing.
The primer of the distolateral wing sequence of 5 of above-mentioned glaA ' (G1A5 ' FR1 ' F and G1A5 ' FR1 ' R) pcr amplification obtains the amplified production of 1.5kb, show that through order-checking this product is the distolateral fin section of 5 of glaA ' (being 5 of AM270061.1 ' end 95854-94355 position Nucleotide from GENBANK number), with this fragment called after glaA5 ' FR.
Above-mentioned primer obtains about 1.5kb product to Hyg-G1A3 ' FR3 ' F and Hyg-G1A3 ' FR3 ' R pcr amplification, show that through order-checking this amplified fragments is the distolateral fin section of 3 of glaA ', with its called after glaA3 ' FR, this product has from GENBANK number holds 92182-90683 position Nucleotide for 5 of AM270061.1 '.
2, the structure of aspergillus niger saccharification enzyme (glaA) gene knockout carrier
Go up the following primer of Totomycin expression cassette sequences Design according to plasmid pSecTag2/Hygro A (available from invitrogen company):
Hyg aligning primer two ends add the NotI restriction enzyme site:
G1A5’FR-Hyg2’F：5’-GCGGCCGCGATGAAAAAGCCTGAACTCACCG-3’
G1A5’FR-Hyg2’R：5’-GCGGCCGCGCTATTCCTTTGCCCTCGGACGA-3’
With G1A5 ' FR-Hyg2 ' F and G1A5 ' FR-Hyg2 ' R is primer, is template with plasmid pSecTag2/Hygro A (available from invitrogen company), amplification hygromycin gene expression cassette.
Amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes 56 ℃ of 30 seconds, annealing.
Pcr amplification obtains about 1.0kb fragment, shows that through order-checking its nucleotides sequence classifies sequence 1 in the sequence table as, is hygromycin gene, with its called after HYG.
1.5kb glaA5 ' FR that above-mentioned amplification is obtained is cloned on the T carrier pEasy-T1 (available from full formula King Company), cuts through pcr amplification, enzyme and identifies and sequence verification, will checking correctly contains recombinant vectors called after pET-5 ' FR1 of 1.5kb glaA5 ' FR.The 1.0kb HYG that above-mentioned amplification is obtained is cloned on the T carrier pEasy-T1 (available from full formula King Company), cuts through pcr amplification, enzyme and identifies and sequence verification, will checking correctly contains the recombinant vectors called after pET-Hyg of HYG.1.5kb glaA3 ' FR that above-mentioned amplification is obtained is cloned on the T carrier pEasy-T1 (available from full formula King Company), cuts through pcr amplification, enzyme and identifies and sequence verification, will checking correctly contains recombinant vectors called after pET-3 ' FR1 of glaA3 ' FR.
The building process of aspergillus niger saccharification enzyme (glaA) gene knockout carrier as shown in Figure 1, after pET-Hyg cut with the NotI enzyme, the 1.0kb fragment that recovery obtains, the NotI restriction enzyme site that this fragment is inserted into pET-5 ' FR1 obtains recombinant vectors, carry out that enzyme is cut and sequence verification, empirical tests is shown contain Hyg and the segmental recombinant vectors called after of glaA5 ' FR pET-5 ' FR1-Hyg.PET-3 ' FR1 is cut with XhoI and ApaI enzyme, reclaim the 1.5kb fragment, be inserted between the XhoI and ApaI restriction enzyme site of pET-5 ' FR1-Hyg, obtain recombinant vectors, this recombinant vectors carried out enzyme is cut and sequence verification, empirical tests is shown contain glaA5 ' FR, Hyg and the segmental recombinant vectors called after of the glaA3 ' FR pEasy-glaA that connects successively
Dir(Fig. 1), this carrier is Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) glucoamylase gene knockout carrier.
3, Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) knocks out the bacterial strain 668-glaA of saccharifying enzyme (glaA) gene
DirStructure
Digest the plasmid pEasy-glaA of 10 micrograms with HindIII
Dir, on 0.8% sepharose, separate linear DNA, and isolate the linearizing pEasy-glaA of HindIII by agarose gel electrophoresis
DirPlasmid DNA fragment, glue reclaim this fragment.
The pEasy-glaA that above-mentioned separation is obtained
DirThe plasmid linear DNA carries out protoplast transformation and goes in Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204).Method for transformation see document (Yao Tingting, Wang Zhengxiang. the preparation of aspergillus niger protoplastis, regeneration and conversion condition. food and biotechnology journal, 2006 25 the volume 04 phase), concrete steps are as follows:
A, Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) is cultivated with the Cha Shi solid medium, picking includes the 4cm of substratum
2About the single bacterium colony and the substratum of size put into the triangular flask that includes 60ml Cha Shi liquid nutrient medium together, 30 ℃ of 200rpm shaking tables were cultivated 4 days, collected mycelium;
B, with the 1M Sorbitol Solution USP wash steps A obtain mycelium once, take by weighing weight in wet base, be the enzyme-added lysate of 1g:10ml according to mass volume ratio, 30 ℃, the slight shaking table enzymolysis of 80rpm, blood counting chamber detect the formation quantity of protoplastis and determine enzymolysis time, obtain protoplasma body fluid;
C, protoplasma body fluid is filtered, collect filtrate, 4 ℃ of centrifugal 10min of following 3200rpm, abandon supernatant liquor, with the 0.6M KCl solution and the S/C solution washing precipitation, centrifugal of precooling, the protoplastis precipitation is resuspended in an amount of S/C solution, makes protoplastis concentration reach 1-3 * 10 respectively
6Individual/ml, it is standby to put ice bath;
D, get the protoplastis suspension that 100ul step C obtains, add linearization plasmid (10ul) pEasy-glaA of about 1-10ug
Dir, use rifle head pressure-vaccum mixing gently, add 50ul PEG solution, put upside down mixing gently, ice bath 20 minutes;
Get the protoplastis that 100ul step C obtains simultaneously respectively, add ddH respectively
2O (10ul) with rifle head pressure-vaccum mixing and add 50ulPEG solution gently, puts upside down mixing, ice bath 20 minutes respectively gently;
E, change plasmid after taking out over to change ddH over to
2The difference of O slowly adds 1ml PEG solution, and room temperature is placed and to be added 2ml S/C solution after 5 minutes, puts upside down mixing gently, with change plasmid over to change ddH over to
2Being layered on respectively of O (minimum medium that contains the 200ug/ml hygromycin B) on the selectivity flat board changes ddH in addition over to
2Being layered on the control medium of O contrasts as protoplastis; 30 ℃ of cultivations.
The substratum that uses in the above-mentioned steps and the prescription of solution are as follows:
Cha Shi solid medium: contain the agar of 1.6% (quality percentage composition), sucrose 3.0% (quality percentage composition), NaNO
30.2% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), all the other are water; The solid medium of pH5.5-6.0
Cha Shi liquid nutrient medium: contain sucrose 3.0% (quality percentage composition), NaNO
30.2% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), all the other are water, the liquid nutrient medium of pH5.5-6.0.
Minimum medium: contain 1M sucrose, K
2HPO
40.1% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), KCl 0.05% (quality percentage composition), NaNO
30.2% (quality percentage composition), the agar of 1% (quality percentage composition), the 200ug/ml hygromycin B, all the other are the solid medium of water.
Control medium: 1M sucrose, NaNO
30.2% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), 1% agar.
Enzymatic lysis liquid: 1% (quality percentage composition) helicase, 1% (quality percentage composition) cellulase, 0.1% (quality percentage composition) N,O-Diacetylmuramidase, after the Sorbitol Solution USP configuration with 1M, with the membrane filtration sterilization in 0.45um aperture.
S/C solution: contain the 1M sorbyl alcohol, 50mM CaCl
2Solution.
PEG solution: contain 25% (quality percentage composition) PEG8000,50mM CaCl
2, 10mM Tris-HCl, the solution of pH7.5.
A transformant 30 ℃ of cultivations all grew ten after 9-10 days on the selectivity flat board surplus, protoplastis is in contrast grown up on control medium, illustrates that the digestion of protoplastis is no problem, and do not grow bacterium colony on selective medium.Transformant list bacterium colony on the picking selectivity flat board, be seeded in the Cha Shi liquid nutrient medium that contains hygromycin B (200ug/ml), cultivate after 3-4 days for 30 ℃, getting 200ul liquid is coated on the Cha Shi solid medium (containing hygromycin B 200ug/ml), separate continuously and go down to posterity 2 times, the transformant that contains the hygromycin B resistant gene survives.According to the distolateral fin section of 5 of glaA ' (is 5 of AM270061.1 ' end 95854-94355 position Nucleotide from GENBANK number), in conjunction with its position and upstream sequence, design authenticate forward primer (this primer is positioned at GENBANK number and is 5 of AM270061.1 ' end 96180-96161 position Nucleotide)
Up?glaA?5’FR?F：CTATCGTGTTTGTAGCAATG
According to the Totomycin sequence, design authenticate reverse primer:
In?Hyg?3‘FR?R：CTATTTACCCGCAGGACATA
The bacterial strain of getting survival extracts genomic gene, carries out augmentation detection with Up glaA 5 ' FR F and In Hyg 3 ' FR R, and it is that PCR detects the male bacterial strain that amplification obtains the segmental bacterial strain of 2.0kb.PCR is detected male recombinant bacterial strain called after aspergillus niger 668-glaA
Dir
Two, express the acquisition of the recombinant bacterial strain of sulfolobus solfataricus (sulfolobus solfataricus) SSG (SSG is the protein of ssg genetic expression)
1, the structure of the glucoamylase gene expression vector of sulfolobus solfataricus (sulfolobus solfataricus)
1) clone of aspergillus niger amdS gene
Go up aspergillus oryzae acetamidase gene order and the aspergillus niger genomic dna announced according to NCBI and compare the sequences Design primer of choosing the similarity maximum:
Acetamidase primer band Sca I and Not I restriction enzyme site
PgpdA-amdS?5F：5′-AGTACTATGGCCCTCACATCCTGGGAAC-3′；
amdS-TrpC?5R：5′-GCGGCCGCTCAAGCACTAGCCTTCTTGAATT-3′；
As template, is primer with PgpdA-amdS 5F and amdS-TrpC 5R with the genomic dna of Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204), carries out pcr amplification.
Amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes 56 ℃ of 30 seconds, annealing.
Amplification obtains the dna fragmentation of about 1.9kb, reclaim and check order after this fragment is connected to pEasy-T1 (purchase in full formula King Company) carrier, show that through order-checking this amplified fragments is acetamidase gene (being 5 of AM270228.1 ' end 31228-33207 position nucleotide sequence from GENBANK number), with its called after amdS, will contain the segmental recombinant vectors called after of this amdS pET-amdS.
The amdS fragment that plasmid pET-amdS is obtained 1.9kb after with Sca I and Not I double digestion is standby.
2) the segmental acquisition of PgpdA
According to the promotor PgpdA sequences Design primer of Aspergillus nidulans glyceraldehyde 3-phosphate dehydro-genase gene gpdA with Aspergillus nidulans (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) 2288) genomic dna obtains the PgpdA fragment of 2049bp for the template pcr amplification, its sequence is for 5 of Z32524.1 ' end 138-2186 position nucleotide sequence, as the promoter sequence of amdS from GENEBANK number.
Primer is with Bgl II and Sca I restriction enzyme site respectively, and primer sequence is as follows:
PgpdA-F：AGATCT?AGATCTTTCGACACTGAAATAC；
PgpdA-R：AGTACT?AGTACTTACTTAGGGGAAATAA。
Amplification obtains the fragment of about 2.0kb, reclaim be connected to after the PgpdA fragment that amplification obtains on the pEASY-T1 carrier through order-checking show correct after, use Bgl II and Sca I double digestion, obtain about 2.0kb fragment, with its called after PgpdA.
3) the segmental acquisition of TtrpC
According to the terminator TtrpC sequences Design primer of Aspergillus nidulans tryptophane synthetic gene trpC with Aspergillus nidulans (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) 2288) genomic dna obtains the TtrpC fragment of 763bp for the template pcr amplification, the sequence of NCBI from GENEBANK number for X02390.1 5 ' hold 3406-4168 position nucleotide sequence, this fragment is as the terminator of amdS.
Primer sequence is as follows:
amdS-TtrpC?6F：5′-GCGGCCGC?GGATCCACTTAACGTTACTGA-3′；
TtrpC-G1A3’FR?6R：5′-AAGCTT?TCGAGTGGAGATGTGGAGTGG-3′；
With NotI and this fragment of HindIII double digestion, obtain the 0.76kb fragment, after the TtrpC fragment that the recovery amplification obtains through the correct fragment called after TtrpC of sequence verification.
4) with the amplification of 3 ' FR sequence of different restriction enzyme sites
With carrier pET-3 ' FR1 template, design following two pairs of primers respectively with different restriction enzyme sites, carry out pcr amplification.Wherein the primer sequence with ApaI and BglII restriction enzyme site is:
Ssg-G1A3’FR?3F：5′-GGGCCCACAATCAATCCATTTCGCTATA-3′；
G1A3’FR-PgpdA?3R：5′-AGATCTTAACTCCGCGGAGGCTCATGG-3′。
Band HindIII and AatII restriction enzyme site primer sequence are:
TrpC-G1A3’FR?7F：5′-AAGCTTACAATCAATCCATTTCGCTA-3′；
G1A3’FR?7R：5′-GACGTCTAACTCCGCGGAGGCTCATGG-3′。
Amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes 56 ℃ of 30 seconds, annealing.
Amplification obtains having 3 ' FR fragment of the 1.5kb of different restriction enzyme sites.This amplification is obtained having 3 ' FR fragment called after, 3 ' flank P1 fragment of ApaI and BglII restriction enzyme site; 3 ' FR fragment called after, the 3 ' flank P2 fragment that has HindIII and AatII restriction enzyme site that above amplification is obtained.
5) clone of the glucoamylase gene of sulfolobus solfataricus (sulfolobus solfataricus)
The ssg gene order of the sulfolobus solfataricus P2 that announces according to NCBI, analyze Aspergillus niger strain CICC2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) middle glucoamylase gene promoter sequence, utilization fusion round pcr is synthetic to contain Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) in the fragment of ssg gene order (having GENBANKAccession Number is that 5 of AE006718 ' holds the 6228-8096 nucleotide sequence) of saccharifying enzyme 5 ' terminal sequence (being 5 of AM270061.1 ' end 95854-94355 position Nucleotide) and sulfolobus solfataricus P2 from GENBANK number, with its called after 5 ' FR-SSG.Its concrete preparation method is as described below:
Design Overlap PCR primer sequence is as follows, wherein, G1A 5 ' FR1F band AatII restriction enzyme site, Ssg2R band ApaI restriction enzyme site:
G1A?5’FR1F：5′-GACGTCCAAGTATATGATGCGGTAGTG-3′；
G1A5’FR-ssg1R：
5′-TTCCTATGGAGGAAACTCTCATTGCTGAGGTGTAATGATGCTGG-3′；
G1A5’FR-ssg2F：
5′-CCAGCATCATTACACCTCAGCAATGAGAGTTTCCTCCATAGGAA-3′；
Ssg2R：5′-GGGCCCTTATATATGGTTTAAGAGCT-3′。
With pET-5 ' FR1 is template, with above-mentioned sequence (G1A5 ' FR1F and G1A5 ' FR-ssg1R) is that primer carries out pcr amplification, amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 56 ℃ of 30 seconds, annealing, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes.Obtain the G1A5 ' FR fragment of 1.5kb.
With hyperthermophilic archaeon strain sulfolobus solfataricus P2 (available from Chinese common micro-organisms culture presevation administrative center, catalog number (Cat.No.) 1.2371
T) genomic dna is template, above-mentioned G1A5 ' FR-ssg2F and Ssg2R are primer, carry out pcr amplification, amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 56 ℃ of 30 seconds, annealing, 1 minute, extend 72 ℃, 2 minutes, 72 ℃, extended 10 minutes.PCR obtains the ssg fragment fragment of 1.8kb.
G1A5 ' the FR and the ssg fragment that obtain with above-mentioned PCR are template, use primer G1A5 ' FR1F and Ssg2R to carry out pcr amplification, amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 56 ℃ of 30 seconds, annealing, 1 minute, extend 72 ℃, 3 minutes, 72 ℃, extended 10 minutes.
3.3kb fragment with pcr amplification obtains is 5 ' FR-ssg, 5 ' FR-ssg is connected to pEasy-T1 upward cuts evaluation correctly, called after pET-5 ' FR-ssg through order-checking and enzyme.Show the nucleotide sequence that about 3.3kb fragment 5 ' FR-ssg that this pcr amplification obtains has sequence 2 in the sequence table through order-checking, the 1-1500 at 5 of sequence 2 ' end is that above-mentioned Aspergillus niger strain CICC2204 is (available from Research for Industrial Microbial Germ preservation administrative center in sequence table, catalog number (Cat.No.) is 2204) middle saccharifying enzyme 5 ' terminal sequence is the SSG gene order of above-mentioned sulfolobus solfataricus P2 from 5 of sequence 2 ' the 1501-3369 at end.
6) structure of the recombinant vectors of the glucoamylase gene of expression sulfolobus solfataricus (sulfolobus solfataricus)
With the above-mentioned PgpdA fragment that obtains with is connected through the amdS fragment of double digestion after obtain the 4034bp fragment, and obtain the 4797bp fragment after TtrpC 763p fragment through NotI and the acquisition of HindIII double digestion is connected.This fragment with obtain the 6297bp fragment after 3 ' flank P1 fragment with the 1.5kb behind ApaI and the BglII double digestion is connected, with through HindIII with obtain the 7797bp fragment after 3 ' flank P2 fragment (1510bp) after the AatII enzyme is cut is connected.
After pET-5 ' FR-ssg cut with AatII and ApaI enzyme, be connected (selecting the two to hold ways of connecting), obtain the 11.1kb fragment at ApaI with the fragment of the above-mentioned 7.8kb that obtains.
Cut with this fragment and through the AatII enzyme and to obtain new recombinant expression vector after pGEM-T Vector (available from the promega) carrier segments that reclaims the back is connected, this carrier is carried out PCR and sequence verification, with the correct segmental recombinant vectors called after of the above-mentioned 11.1kb pWW (its structural representation as shown in Figure 2) that contains of checking.
2, the screening of the conversion of recombinant aspergillus niger expression vector pWW and transformant
1) conversion of recombinant aspergillus niger expression vector pWW
Recombinant aspergillus niger expression vector pWW is after the linearizing of AatII complete degestion, with the bacterial strain aspergillus niger 668-glaA of the step 3) of step 1 preparation
DirFor starting strain prepares protoplastis, carry out the PEG-protoplast transformation, method for transformation see document (Yao Tingting, Wang Zhengxiang. the preparation of aspergillus niger protoplastis, regeneration and conversion condition. food and biotechnology journal, 2006 25 04 phases of volume), concrete steps are as follows:
A, with aspergillus niger 668-glaA
DirCultivate with the Cha Shi solid medium, picking includes the 4cm of substratum
2About the single bacterium colony and the substratum of size put into the triangular flask that includes 60ml Cha Shi liquid nutrient medium together, 30 ℃ of 200rpm shaking tables were cultivated 4 days, collected mycelium;
B, with the 1M Sorbitol Solution USP wash steps A obtain mycelium once, take by weighing weight in wet base, be the enzyme-added lysate of 1g:10ml according to mass volume ratio, 30 ℃, the slight shaking table enzymolysis of 80rpm, blood counting chamber detect the formation quantity of protoplastis and determine enzymolysis time, obtain protoplasma body fluid;
C, protoplasma body fluid is filtered, collect filtrate, 4 ℃ of centrifugal 10min of following 3200rpm, abandon supernatant liquor, with the 0.6M KCl solution and the S/C solution washing precipitation, centrifugal of precooling, the protoplastis precipitation is resuspended in an amount of S/C solution, makes protoplastis concentration reach 1-3 * 10 respectively
6Individual/ml, it is standby to put ice bath;
D, get the protoplastis suspension that 100ul step C obtains, add linearization plasmid (10ul) pWW of about 1-10ug, with rifle head pressure-vaccum mixing gently, adding 50ulPEG solution is put upside down mixing, ice bath 20 minutes gently;
Get the protoplastis that 100ul step C obtains simultaneously respectively, add ddH respectively
2O (10ul) with rifle head pressure-vaccum mixing and add 50ulPEG solution gently, puts upside down mixing, ice bath 20 minutes respectively gently;
E, change plasmid after taking out over to change ddH over to
2The difference of O slowly adds 1ml PEG solution, and room temperature is placed and to be added 2ml S/C solution after 5 minutes, puts upside down mixing gently, with change plasmid over to change ddH over to
2Dull and stereotyped go up (minimum medium that contains 10mM ethanamide and 20mM CsCl) selected in being layered on respectively of O, changes ddH in addition over to
2Being layered on the control medium of O contrasts as protoplastis; Cultivated 5-6 days for 30 ℃.
The substratum that uses in the above-mentioned steps and the prescription of solution are as follows:
Cha Shi solid medium: contain the agar of 1.6% (quality percentage composition), sucrose 3.0% (quality percentage composition), NaNO
30.2% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), all the other are water; The solid medium of pH5.5-6.0.
Cha Shi liquid nutrient medium: contain sucrose 3.0% (quality percentage composition), NaNO
30.2% (quality percentage composition), K
2IIPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), all the other are water, the liquid nutrient medium of pH5.5-6.0.
Minimum medium: contain 1M sucrose, K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), 10mM ethanamide, the solid medium of 20mM CsCl and 1% (quality percentage composition) agar.
Control medium: 1M sucrose, NaNO
30.2% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), 1% agar.
Enzymatic lysis liquid: 1% (quality percentage composition) helicase, 1% (quality percentage composition) cellulase, 0.1% (quality percentage composition) N,O-Diacetylmuramidase, after the Sorbitol Solution USP configuration with 1M, with the membrane filtration sterilization in 045um aperture.
S/C solution: contain the 1M sorbyl alcohol, 50mM CaCl
2Solution.
PEG solution: contain 25% (quality percentage composition) PEG8000,50mM CaCl
2, 10mM Tris-HCl, the solution of pH7.5.
2) screening of transformant
AmdS genes encoding acetamidase gene, this endonuclease capable acts on ethanamide, and decomposing ethanamide is acetate and ammonium, as the carbon source of cell growth.The faint expression in Aspergillus niger strain CICC 2204 (available from Research for Industrial Microbial Germ preservation administrative center, catalog number (Cat.No.) is 2204) of this gene, the faint expression of endogenic acetamidase simultaneously can be inhibited by adding 20mM CsCl.By being that thereby growth filters out the positive colony that homologous recombination takes place on the substratum supplied with of carbon source only containing ethanamide, stable transformant shows as dominant growth and bacterium colony increases gradually.Aspergillus niger strain CICC 2204 protoplastiss are grown in contrast on minimum medium, contrast can grow up the explanation protoplastis digestion no problem, and Aspergillus niger strain CICC 2204 protoplastiss (minimum medium that contains 10mM ethanamide and 20mM CsCl) on selective medium do not grow or grow less bacterium colony but can not continued growth, are positive transformant at the above-mentioned transformant of selecting can grow on the substratum that obtains.The transformant that the picking step 1) obtains (contains 1M sucrose, K at the minimum medium that contains 10mM ethanamide and 20mM CsCl
2HPO
40.1% (quality percentage composition), 7H
2OFeSO
40.001% (quality percentage composition), 7H
2OMgSO
40.05% (quality percentage composition), KCl 0.05% (quality percentage composition), 10mM ethanamide, the solid medium of 20mM CsCl and 1% (quality percentage composition) agar), cultivate screening 2 times, obtain positive transformant for 30 ℃.
For positive transformant is identified, according to the distolateral fin section of 5 of glaA ' (is 5 of AM270061.1 ' end 95854-94355 position Nucleotide from GENBANK number), in conjunction with its position and upstream sequence, design authenticate forward primer (this primer is positioned at GENBANK number and is 5 of AM270061.1 ' end 96180-96161 position Nucleotide)
Up?glaA?5’FR?F：5′-CTATCGTGTTTGTAGCAATG-3′
According to the synthetic authenticate reverse primer of glucoamylase gene (having GENBANK Accession Number is the 6228-8096 nucleotide sequence of 5 of AE006718 ' end) of sulfolobus solfataricus,
In?Ssg?R：5′-CTATATATAATACAGTAGTTTC-3′
Extract bacterial strain aspergillus niger 668-glaA
DirAnd the genomic dna of transformant, carry out the PCR Molecular Identification, carry out augmentation detection with primer Up glaA 5 ' FR F and In Ssg R, it is that PCR detects the male bacterial strain that amplification obtains the segmental bacterial strain of 2047bp, and result's screening obtains PCR and detects the correct commentaries on classics pWW bacterial strain of male.And the contrast bacterium purpose band that do not increase.
3) transformant thermophilic glucoamylase enzyme activity determination
The mono-clonal of the above-mentioned transformant of picking includes the 4cm of substratum respectively
2About the size single bacterium colony, put into together to be switched to substratum 100ml shake-flask culture base (soybean cake powder 2% (quality percentage composition) is housed, corn steep liquor 2% (quality percentage composition), W-Gum 12% (quality percentage composition), all the other are water) the 500ml triangular flask in, get original bacteria strain aspergillus niger 668-glaA simultaneously
DirIn contrast, shake training 4 days under 30 ℃, get fermented liquid supernatant liquid and test diastatic activity respectively.
Thermophilic glucoamylase enzyme activity determination method is as described below:
Draw 1ml fermented liquid supernatant liquid respectively, move in the volumetric flask, be diluted to the 10ml scale with damping fluid respectively, the 100ml scale, the 1000ml scale (decide according to enzyme height alive, and concentration is high more by dilute strength, extension rate should be big more) fully shake up, to be measured as sample.When preparing enzyme liquid to be measured, the difference that sample liquid concentration consumes 0.05mol/L Sulfothiorine standard titration solution in the time of should being controlled at the blank and sample of titration is at 4.5ml～5.5ml, and scope is interior, and (enzyme activity is about 120U/ml～150U/ml).
Get two 50ml colorimetric cylinders of A, B, add 25ml20g/L Zulkovsky starch solution and 5ml0.05mol/L acetate-sodium acetate buffer solution (pH6.0) respectively, shake up.Preheating 5min～10min in 80 ± 0.2 ℃ water bath with thermostatic control.In the B pipe, add the above-mentioned liquid to be measured of 2.0ml, shake up, clock immediately.Behind accurate response 30min under this temperature, in A, B two pipes, respectively add the 0.2ml200g/L sodium hydroxide solution immediately, shake up, simultaneously two pipes are taken out, use water cooling rapidly, and in the A pipe, add the above-mentioned liquid to be measured of 2.0ml (as blank).
Draw each 5.0ml of reaction solution in above-mentioned A, B two pipes, in two iodine flasks, accurately add 0.1mol/L iodine reference liquid (press the GB/T601 preparation and demarcate) 10.0ml, add the 15ml0.1mol/L sodium hydroxide solution again, the limit edged shakes up, and places 15min in the dark place, takes out.Use the water wash bottle cap, add 2mol/L sulphuric acid soln 2ml, with 0.05mol/L Sulfothiorine standard titration solution (press GB/T601 preparation with demarcate) titration blue-violet solution, until just colourless be its terminal point, the volume (V of the blank and sample consumption Sulfothiorine standard titration solution of record respectively
A, V
B).
The thermophilic glucoamylase unit of activity: 1ml enzyme liquid or 1g enzyme powder are under 80 ℃, the condition of pH6.0, and 1h hydrolysis Zulkovsky starch produces 1mg glucose, is an enzyme activity unit, and symbol is: U/ml (or U/g).
Enzyme activity unit calculates by formula (1):
X＝(V
A-V
B)×c×90.05×32.2/5×1/2×n×2＝579.9×(V
A-V
B)c×n...........(1)
In the formula:
The enzyme activity unit of X---sample, unit are every milliliter of enzyme activity unit (U/ml) or the every gram of enzyme activity unit (U/.g);
V
A---when titration is blank, consume the volume of Sulfothiorine standard titration solution, unit is a milliliter (mL);
V
B---during the titration sample, consume the volume of Sulfothiorine standard titration solution, unit is a milliliter (mL);
The accurate concentration of c---Sulfothiorine standard titration solution, unit is every liter (mol/L) of mole;
90.05---the molar mass of glucose, unit is every mole (g/mol) of gram;
32.2---the cumulative volume of reaction solution, unit are milliliter (mL);
5---draw the volume of reaction solution, unit is a milliliter (mL);
1/2---be converted to the amount of 1mL enzyme liquid;
N---extension rate;
2---react 30min, be converted into the enzyme activity coefficient of 1h.
With sample determination result's arithmetical mean value representation, the revision of the convention to three position effective digital.
Result's tolerance: the twice independent test result's who under repeated condition, obtains absolute difference be not more than these two measured values arithmetical av 10%, be prerequisite to be no more than 5% under the situation greater than the arithmetical av 10% of these two measured values.
The result shows, original bacteria strain aspergillus niger 668-glaA
DirThe work of thermophilic glucoamylase enzyme be zero because there is not the thermophilic glucoamylase gene of sulfolobus solfataricus in original strain, and the saccharifying enzyme of himself is also knocked out.This with we the expection consistent.10 positive transformant vigor being measured are had nothing in common with each other, and the enzyme that one of them bacterial strain produces is lived the highest, and enzyme is lived and is 3000U/ml.
With this bacterial strain called after aspergillus niger (Aspergillus niger) WW1.This aspergillus niger (Aspergillus niger) WW1, be preserved in Chinese microorganism strain preservation board of trustee reason person on December 25th, 2008 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC No.2841.
The compound method of the reagent that above-mentioned enzyme activity determination uses is as described below:
0.05mol/L acetate-sodium acetate buffer solution (pH6.0): take by weighing sodium acetate (CH
3COONa3H
2O) 6.7g is with water dissolution and be settled to 1000ml.The pH value of above-mentioned damping fluid should be drawn glacial acetic acid (about 0.5ml-1ml) and use acidometer to be proofreaied and correct.
20g/L Zulkovsky starch solution: take by weighing Zulkovsky starch (2 ± 0.001) g, mix well with less water then, slowly impouring in the ebullient water, is boiled, is stirred until transparent, cooling, and water is settled to 100ml.This solution needs preparation on the same day.
Embodiment 2, prepare thermophilic glucoamylase in a large number
1, substratum preparation
Strain activation and culture base: sucrose 3.0% (quality percentage composition), K
2HPO
40.1% (quality percentage composition), 7H2,OFe,SO4 0.001% (quality percentage composition), 7H2,OMg,SO4 0.05% (quality percentage composition), KCl 0.05% (quality percentage composition), 10mM ethanamide and 20mM CsCl, all the other are water, regulate pH to 5.5-6.0,121 ℃ of sterilization 30min.
First order seed substratum: soybean cake powder 3% (quality percentage composition), corn steep liquor 2.5% (quality percentage composition), W-Gum 15% (quality percentage composition), alpha-amylase (Novi's letter
Profit can be come fire resistant alpha-diastase Supra
Lot number: NBSG4213) 12U/ gram W-Gum is handled 30min for 80 ℃, polyoxyethylene polyoxypropylene tetramethylolmethane ether (Polyoxyethylene Polyoxypropylene PentaerythritolEther-PPE) 0.05% (quality percentage composition), all the other are water, 121 ℃ of sterilization 30min.
Fermention medium: W-Gum 20% (quality percentage composition), soybean cake powder 5% (quality percentage composition), corn steep liquor 3% (quality percentage composition), sulphur ammonium 0.8% (quality percentage composition), calcium chloride 0.05% (quality percentage composition), alpha-amylase (Novi's letter
Profit can be come fire resistant alpha-diastase Supra
Lot number: NBSG4213) 12U/ gram W-Gum is handled 30min for 80 ℃, polyoxyethylene polyoxypropylene tetramethylolmethane ether (Polyoxyethylene Polyoxypropylene Pentaerythritol Ether-PPE) 0.05% (quality percentage composition), all the other are water, 121 ℃ of sterilization 30min.
2, fermentative preparation thermophilic glucoamylase
Seed culture: aspergillus niger (Aspergillus niger) WW1 is inoculated into strain activation and culture base (Cha Shi substratum) activation, choose single bacterium colony from flat board, be linked in the first order seed substratum (100ml/500ml triangular flask), 30 ℃, 150r/min cultivates 2d, and whether microscopy also is coated with dull and stereotyped the detection and pollutes.
High density fermentation: seed is after the first order seed culture medium culturing, be that 10% inoculum size is received in the 5L fermentor tank that the 2.5L fermention medium is housed by volume, 30 ℃, 200 rev/mins (fermentor tank is a Shanghai Baoxing Biology Equipment Engineering Co., Ltd, model: stirring velocity BIOTECH-5BG) is carried out fermentation culture, after about 16 hours, beginning with mass percentage concentration is that 25% ammoniacal liquor is regulated pH, makes the pH of fermented liquid be controlled at 4.2-4.5.By regulating rotating speed, tank pressure and air flow quantity, speed makes dissolved oxygen greater than 20%, cultivates about 120 hours.Timing sampling observation of cell growing state and enzyme are lived in the culturing process.The enzyme activity determination method is carried out according to the described thermophilic glucoamylase activity determination method of the step 3) of step 2 in the step 2 of embodiment 1.The result shows that aspergillus niger (Aspergillus niger) WW1CGMCC No.2841 thermophilic glucoamylase enzyme is lived and is 3000U/ml.
The zymologic property research of embodiment 3, saccharifying enzyme
1, the detection of optimal pH
With the further preliminary study recombinase active of fermented supernatant fluid of engineering bacteria aspergillus niger (Aspergillus niger) WW1CGMCC No.2841 and the relation of pH, get the nutrient solution supernatant liquor of positive transformant fermentation 140h, the pH value of change damping fluid (pH2.3～pH7.0), the mensuration enzyme is lived, all the other conditions of enzyme activity determination are carried out according to the described thermophilic glucoamylase activity determination method of the step 3) of step 2 in the step 2 of embodiment 1, enzyme soprano's alive relative enzyme work is decided to be 100%, and remaining enzyme is lived and the highest enzyme is lived, and ratio is that its relative enzyme is lived.
The result as shown in Figure 3, the optimal pH of recombinase is 6.0 as seen from Figure 3.At present the optimum pH of the alpha-amylase that uses during starch liquefacation in the alcohol industry production also be pH6.0, as using this enzyme can save accent one step of pH when the saccharification, provides cost savings.
2, the detection of enzyme reaction temperature
Culture supernatant with the positive transformant 140h of shake-flask culture, measuring enzyme under different temperature of reaction lives, all the other conditions of enzyme activity determination are carried out according to the described thermophilic glucoamylase activity determination method of the step 3) of step 2 in the step 2 of embodiment 1, enzyme soprano's alive relative enzyme work is decided to be 100%, and remaining enzyme is lived and the highest enzyme is lived, and ratio is that its relative enzyme is lived.
The result as shown in Figure 4, the detected result of recombinase optimum temperuture shows (Fig. 4): the optimum temperuture of recombinase is 90 ℃, still has activity in the time of 100 ℃.Its optimum temperuture is close with the fire resistant alpha-diastase that derives from Bacillus licheniformis that industry is at present used always, as uses this recombinase to be used for alcohol industry and can lower the temperature when Mashing process, has saved the energy.
3, enzyme stability detects
Get the culture supernatant of the positive transformant 140h of shake-flask culture, under 60 ℃, 80 ℃ and 100 ℃, be incubated 0,10,20,30 respectively, 60min, rapid cool to room temperature, measuring the activity of residual enzyme according to the described thermophilic glucoamylase activity determination method of the step 3) of step 2 in the step 2 of embodiment 1, is 100% with the enzyme work of uninsulated enzyme liquid.It is that relative enzyme is lived that remaining enzyme is lived with the ratio of living with the enzyme of uninsulated enzyme liquid.
The detected result of recombinase optimum temperuture shows as shown in Figure 5: recombinase is in the time of 80 ℃, and the enzyme activity transformation period is more than 60min; 100 ℃ of enzymes are lived and are reduced comparatively fast, and enzyme activity still is higher than 60% behind the insulation 10min, and residual enzyme activity is 28% behind the insulation 60min.From this glucoamylase gene, this enzyme has glycosylation site, and has glycosylase in this Archimycetes, so may there be glycosylation in this enzyme.Optimize so be more suitable in fungal expression system, expressing.
Sequence table
<160>2
<210>1
<211>1026
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
<210>2
<211>3369
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
Claims (6)
1, expresses the engineering bacteria of thermophilic glucoamylase, it is glucoamylase gene deactivation with aspergillus niger (Aspergillus niger), and the expression cassette that will express the glucoamylase gene of sulfolobus solfataricus (sulfolobus solfataricus) imports in the genome of aspergillus niger (Aspergillus niger), and screening obtains expressing the recombinant bacterial strain of thermophilic glucoamylase;
It is the 94354-92183 nucleotide sequence of 5 of AM270061.1 ' end that the glucoamylase gene of described aspergillus niger (Aspergillus niger) has GENBANK Accession Number; It is the 6228-8096 nucleotide sequence of 5 of AE006718 ' end that the glucoamylase gene of described sulfolobus solfataricus (sulfolobussolfataricus) has GENBANK Accession Number.
2, engineering bacteria according to claim 1 is characterized in that: also contain acetyl ammonia enzyme gene in the expression cassette of the glucoamylase gene of described expression sulfolobus solfataricus (sulfolobus solfataricus); Described acetyl ammonia enzyme gene have from GENBANK number for AM270228.1 5 ' end 31228-33207 position nucleotide sequence.
3, engineering bacteria according to claim 1 is characterized in that: the method for described glucoamylase gene deactivation with aspergillus niger (Aspergillus niger) is the method for gene knockout.
4, engineering bacteria according to claim 1 is characterized in that: described engineering bacteria of expressing thermophilic glucoamylase is aspergillus niger (Aspergillus niger) WW1 CGMCC No.2841.
5, the described arbitrarily application of engineering bacteria in producing thermophilic glucoamylase of expressing thermophilic glucoamylase among the claim 1-4.
6, application according to claim 5 is characterized in that: the method for described production thermophilic glucoamylase is that any described engineering bacterium fermentation among the claim 1-4 is obtained thermophilic glucoamylase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100776469A CN101503660B (en) | 2009-02-10 | 2009-02-10 | Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100776469A CN101503660B (en) | 2009-02-10 | 2009-02-10 | Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101503660A true CN101503660A (en) | 2009-08-12 |
| CN101503660B CN101503660B (en) | 2011-07-27 |
Family
ID=40976037
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100776469A Expired - Fee Related CN101503660B (en) | 2009-02-10 | 2009-02-10 | Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101503660B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849636A (en) * | 2012-11-28 | 2014-06-11 | 丰益(上海)生物技术研发中心有限公司 | Optimized gene encoding rhizomucor miehei lipase, aspergillus niger strain transformed from optimized gene and use thereof |
| CN105462864A (en) * | 2015-12-25 | 2016-04-06 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain capable of producing alpha-glucosaccharase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369016A (en) * | 1999-07-30 | 2002-09-11 | 荷兰应用科学研究会(Tno) | Novel means of transformation of fungi and their use for heterologous protein production |
-
2009
- 2009-02-10 CN CN2009100776469A patent/CN101503660B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849636A (en) * | 2012-11-28 | 2014-06-11 | 丰益(上海)生物技术研发中心有限公司 | Optimized gene encoding rhizomucor miehei lipase, aspergillus niger strain transformed from optimized gene and use thereof |
| CN103849636B (en) * | 2012-11-28 | 2019-03-12 | 丰益(上海)生物技术研发中心有限公司 | Encode the optimization gene of rhizomucor miehei lipase, by Aspergillus niger strain of the genetic transformation and application thereof |
| CN105462864A (en) * | 2015-12-25 | 2016-04-06 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain capable of producing alpha-glucosaccharase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101503660B (en) | 2011-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102787130B (en) | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method | |
| CN102676480B (en) | Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy | |
| CN102286443B (en) | Method for producing mixture of recombinant isoamylase, alpha-amylase and glucamylase | |
| CN109385413B (en) | Glucoamylase TlGA1931 and gene and application thereof | |
| CN103215300B (en) | Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose | |
| CN104962594B (en) | A method of improving Glucoamylase of Aspergillus niger inversion rate of glucose | |
| CN103571812A (en) | Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant | |
| CN103224949B (en) | Bacillus subtilis for high-efficiency expression of recombination pullulanase and obtaining method thereof | |
| CN106755015A (en) | The preparation method and producing enzyme technique of a kind of new Pullulanase gene and superior strain | |
| CN104630189B (en) | A kind of carbohydrase Pullulanase joint product and its production method | |
| CN103834629A (en) | Recombinant high-temperature pullulanase and preparation method thereof | |
| CN102260694B (en) | Acidproof medium-temperature alpha-amylase and preparation method thereof | |
| CN103614303B (en) | A kind of Li's Trichoderma strains of expressing saccharifying enzyme | |
| CN102220244B (en) | Hypocrea lixii strain and method for preparing dextran enzyme with the Hypocrea lixii strain | |
| CN103421794A (en) | Expression element composed of aspergillus niger and expression vector composed of expression element, and recombined aspergillus niger and construction method and application thereof | |
| CN101878308B (en) | A process for the preparation of ethanol from starch | |
| CN103146726B (en) | Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof | |
| CN101503660B (en) | Engineering bacteria expressing thermophilic saccharifying enzyme and use thereof | |
| CN102994476B (en) | Saccharifying enzyme | |
| CN107201373B (en) | A kind of maltogenic amylase and its gene, the engineering bacteria containing the gene and its application | |
| CN104911106B (en) | A kind of method that thermophilic loose penicillium bacterial strain and its bacterial strain prepare dextranase | |
| CN102061295A (en) | Vitreoscilla hemoglobin gene expression box and method for improving yield of saccharifying enzyme produced by aspergillus niger | |
| CN105925594A (en) | Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch | |
| CN105505896A (en) | Preparation method of transglucosidase | |
| CN102392003B (en) | Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: WANG BING Free format text: FORMER OWNER: BEIJING ZHONGTIAN-NOAH SPORTS SCIENCE CO., LTD. Effective date: 20100910 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100049 ROOM 511, YUEFU COMMERCIAL BUILDING, NO.B-1, YONGDING ROAD, HAIDIAN DISTRICT, BEIJING TO: 100036 2101, BUILDING 11, NO.4, CUIWEI ROAD, HAIDIAN DISTRICT, BEIJING |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20100910 Address after: 100036 Beijing city Haidian District Cuiwei Road No. 4 Building No. 11 2101 Applicant after: Wang Bing Address before: 100049, room 1, Yuefu business building, No. 511, Yongding Road, Beijing, Haidian District Applicant before: Beijing ZhongTian-Noah Sports Science Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20151109 Address after: 102629, Beijing, Zhongguancun science and Technology Park, Daxing pharmaceutical industry base, Yongxing Road, No. 25, block B, Room 102, Daxing District Patentee after: Beijing Zhongke Xingguan biotechnology Limited by Share Ltd Address before: 100036 Beijing city Haidian District Cuiwei Road No. 4 Building No. 11 2101 Patentee before: Wang Bing |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110727 Termination date: 20190210 |