CN1289665C - 重组米曲霉单宁酶及其表达和纯化方法 - Google Patents
重组米曲霉单宁酶及其表达和纯化方法 Download PDFInfo
- Publication number
- CN1289665C CN1289665C CN 200410027497 CN200410027497A CN1289665C CN 1289665 C CN1289665 C CN 1289665C CN 200410027497 CN200410027497 CN 200410027497 CN 200410027497 A CN200410027497 A CN 200410027497A CN 1289665 C CN1289665 C CN 1289665C
- Authority
- CN
- China
- Prior art keywords
- tannase
- gly
- aspergillus oryzae
- expression
- ser
- 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.)
- Expired - Fee Related
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 36
- 101900204346 Aspergillus oryzae Tannase Proteins 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title claims abstract description 10
- 108010038851 tannase Proteins 0.000 claims abstract description 72
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 51
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 239000013604 expression vector Substances 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 27
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 23
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 20
- 239000000047 product Substances 0.000 claims abstract description 9
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000008521 reorganization Effects 0.000 claims description 28
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 240000005708 Eugenia stipitata Species 0.000 claims description 7
- 235000006149 Eugenia stipitata Nutrition 0.000 claims description 7
- 239000012228 culture supernatant Substances 0.000 claims description 5
- 230000035800 maturation Effects 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 238000005571 anion exchange chromatography Methods 0.000 claims description 2
- 230000009977 dual effect Effects 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
- 230000006798 recombination Effects 0.000 claims 1
- 240000006439 Aspergillus oryzae Species 0.000 abstract description 11
- 235000002247 Aspergillus oryzae Nutrition 0.000 abstract description 11
- 241000235648 Pichia Species 0.000 abstract description 11
- 238000000855 fermentation Methods 0.000 abstract description 11
- 230000004151 fermentation Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 239000006228 supernatant Substances 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 3
- 239000002773 nucleotide Substances 0.000 abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 2
- 230000028327 secretion Effects 0.000 abstract description 2
- 238000005349 anion exchange Methods 0.000 abstract 1
- 238000004440 column chromatography Methods 0.000 abstract 1
- 229920001864 tannin Polymers 0.000 description 31
- 235000018553 tannin Nutrition 0.000 description 31
- 239000001648 tannin Substances 0.000 description 31
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 102220369447 c.1352G>A Human genes 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 11
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 239000013613 expression plasmid Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 241000235058 Komagataella pastoris Species 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 235000004515 gallic acid Nutrition 0.000 description 6
- 229940074391 gallic acid Drugs 0.000 description 6
- 102220023256 rs387907547 Human genes 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- RNKMOGIPOMVCHO-SJMVAQJGSA-N 1,3,6-trigalloyl glucose Chemical compound C([C@@H]1[C@H]([C@@H]([C@@H](O)[C@H](OC(=O)C=2C=C(O)C(O)=C(O)C=2)O1)OC(=O)C=1C=C(O)C(O)=C(O)C=1)O)OC(=O)C1=CC(O)=C(O)C(O)=C1 RNKMOGIPOMVCHO-SJMVAQJGSA-N 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 4
- 244000269722 Thea sinensis Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 235000013361 beverage Nutrition 0.000 description 4
- 102220369445 c.668T>C Human genes 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 235000013616 tea Nutrition 0.000 description 4
- 241000228245 Aspergillus niger Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 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 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000040739 Secretory proteins Human genes 0.000 description 2
- 108091058545 Secretory proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 235000013405 beer Nutrition 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 102220369446 c.1274G>A Human genes 0.000 description 2
- 229920002770 condensed tannin Polymers 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000015203 fruit juice Nutrition 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 235000015092 herbal tea Nutrition 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- ZKHSPLCIOVILMA-UHFFFAOYSA-N 2-(2-phenylphenyl)ethanol Chemical compound OCCC1=CC=CC=C1C1=CC=CC=C1 ZKHSPLCIOVILMA-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- QVZCXCJXTMIDME-UHFFFAOYSA-N Biopropazepan Trimethoxybenzoate Chemical compound COC1=C(OC)C(OC)=CC(C(=O)OCCCN2CCN(CCCOC(=O)C=3C=C(OC)C(OC)=C(OC)C=3)CCC2)=C1 QVZCXCJXTMIDME-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000252983 Caecum Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000146398 Gelatoporia subvermispora Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- MLBVKCMEYNDCIV-UHFFFAOYSA-N N1=CN=CC=C1.C(C1=CC=CC=C1)Cl Chemical compound N1=CN=CC=C1.C(C1=CC=CC=C1)Cl MLBVKCMEYNDCIV-UHFFFAOYSA-N 0.000 description 1
- 101150051118 PTM1 gene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- ZTHYODDOHIVTJV-UHFFFAOYSA-N Propyl gallate Chemical compound CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000007365 Sialoglycoproteins Human genes 0.000 description 1
- 108010032838 Sialoglycoproteins Proteins 0.000 description 1
- 241000194049 Streptococcus equinus Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical group C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000433 anti-nutritional effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 210000004534 cecum Anatomy 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
- 230000006706 cellular oxygen consumption Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 229920002705 flavono-ellagitannin Polymers 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- -1 gallic acid ester Chemical class 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 229920001461 hydrolysable tannin Polymers 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及一种重组米曲霉单宁酶的表达以及纯化方法。本发明通过PCR方法将编码米曲霉单宁酶成熟蛋白的核苷酸序列用从米曲霉基因中扩增出来,克隆到甲醇酵母表达载体pPIC9K,构建表达载体pPIC9K-TAN,采用电击法转化毕赤酵母KM71获得重组菌株,并用高密度发酵的方法进行重组单宁酶的诱导表达。表达产物分布于发酵上清液中,表达量达200mg/L。本发明建立了重组米曲霉单宁酶的纯化方法,采用DEAE阴离子交换柱层析一步纯化。本发明还包括构建了重组单宁酶在毕赤酵母中的组成型表达载体pGAPZαA-TAN和pGAPZαA-ETAN,并通过转化毕赤酵母菌株KM71H获得了具有单宁酶活性的工程菌株,能够以分泌形式表达重组单宁酶。本发明的重组米曲霉单宁酶具有生物活性。
Description
技术领域
本发明涉及重组米曲霉单宁酶及其在甲醇酵母中的分泌表达、纯化。本发明还涉及重组米曲霉单宁酶在工业生产中的应用。
背景技术
单宁(tannin)又称鞣酸,在高等植物中广泛分布,包括单子叶植物、双子叶植物以及蕨类植物中。Howes列举了世界含单宁植物共87科600多种。单宁广泛存在于食品和饲料中,是食品尤其是饲料中的主要抗营养因子之一。单宁的抗营养作用主要有三个方面:(1)单宁和唾液蛋白、糖蛋白在口腔中相互作用,使组织产生收敛性,引起一系列的不适口反应。饲料中单宁可导致动物减少对饲料的采食量。当牧草中单宁含量超过20mg/g干物质时,家禽拒食。(2)单宁分子中存在大量酚羟基团和芳香环结构,可与蛋白质形成络合物,使蛋白质凝结沉淀,影响蛋白质的消化和吸收。单宁与蛋白质复合物(Tannin Protein Complex)在哺乳动物肠道中,难以被蛋白酶等分解。豆科牧草单宁含量过高则其饲料营养价值降低。(3)单宁能降低哺乳动物肠道消化酶的活性,并对胃肠黏膜产生不良影响。单宁与消化酶构成复合物,干扰酶生物活性。体外实验表明饲料中含有单宁浸提物(蚕豆壳等)降低了胰蛋白酶、糜蛋白酶、α-淀粉酶活性。单宁酸或氯化单宁酸导致鼠胃和十二指肠黏膜发生变化,引起胃黏液过度分泌,胃黏膜坏死,胆囊萎缩,小肠上皮细胞氧耗下降等不良反应。另外,单宁对反刍动物的毒性很大,牛羊采食含单宁的栎树和一些热带豆科树子实和茎叶时会发生急性中毒。在水溶液清凉茶饮料的生产中,茶中富含的单宁引起茶乳沉淀。单宁与啤酒和果汁中的蛋白质生成沉淀,会引起产品混浊。在食品、饮料、禽畜饲料的生产中必须去除不必要的单宁成分[9]。用单宁酶降解多余的单宁,是一种有广泛应用前景的工艺。
单宁是大多数微生物的拮抗物,对微生物有毒害作用,但一些微生物对单宁有抗性。这些微生物通过各种机制和方法降解单宁。1913年,Knudson分离到黑曲霉(Aspergillusniger)及青霉(Penicillum sp.)具有降解单宁酸的作用,首次对单宁发酵降解进行研究。近十年来,许多肠道细菌被发现具有降解单宁的能力。Osawa从树獭的粪便和盲肠中先后分离到降解单宁-蛋白质复合物的牛链球菌和肠杆菌。Nenuetr等人报告在马肠道分离出降解单宁的细菌。1994年Makkai发现毛孢属真菌(Sportrichum Preluerulentum)能有效降解栎树中的缩合单宁。白腐霉(Ceriporiopsis subvermispora)能对树叶中富含的缩合单宁进行化学修饰,从而提高树叶的消化性三倍。单宁酰基水解酶(EC3.1.1.20)通常称为单宁酶(Tannase).单宁酶水解单宁的酯键,产生没食子酸和葡萄糖。单宁酶是单宁生物降解的关键酶。单宁酶的进展性研究始于六十年代。先后从米曲霉和黑曲霉,念珠菌中分离纯化到单宁酶。也从某些细菌培养液中分离单宁酶。Adachi对米曲霉单宁酶的理化性质研究,证明单宁酶是典型的丝氨酸酯酶,与1994年Barthomeuf等的实验结果一致。
单宁酶在工业生产中用途广泛,日益受到人们的关注。在水溶性清凉茶饮料的生产中,单宁酶用于降解茶中富含的单宁,以避免单宁引起生成茶乳。单宁与啤酒和果汁中的蛋白质生成沉淀,引起混浊,单宁酶可作为澄清剂应用于此类饮料的生产中。饲料加工中,添加单宁酶可减少单宁的不良影响,提高饲料的利用率。在利用单宁沉淀蛋白质的特性纯化蛋白质时,单宁酶可用来除去沉淀物中的单宁。另外,单宁酶还用于生产没食子酸和没食子酸酯。没食子酸是一种用途广泛精细化工原料,医药上用于制甲氯苄嘧啶(为磺胺增效剂),联苯双酯(治肝病),克冠草(治冠心病)等药物。没食子酸丙酯、没食子酸甘油脂等用作食品的抗氧剂及防腐剂。没食子酸还用于合成染料、化学分析用试剂等用途。
单宁酶一直以来都从产单宁酶菌株发酵液中提取。1997年Lane等从米曲霉中制备了单宁酶制剂。但是产酶量相对少,又难以提纯,所以从产单宁酶菌株发酵液提取单宁酶成本高、效率低,难以推广应用。Hatameto于1996年克隆出米曲霉的单宁酶基因。将带有单宁酶基因的质粒PT1转化单宁酶产量低的菌株Aspergillus orygze Aol,提高宿主菌的单宁酶的产量。这为利用高效表达***生产重组单宁酶打下了基础。
作为新一代酵母表达***,甲醇酵母表达***与现有主要的重组蛋白生产***相比拥有多方面的优势。作为真核生物,甲醇酵母表达***拥有真核表达***如酿酒酵母表达***的优点,具有真核生物的蛋白质翻译后加工功能,有适于真核生物基因表达产物正确折叠的细胞内环境和糖链加工***,还能分泌外源蛋白质到培养基中利于纯化。这是大肠杆菌(E.coli)等原核表达***所没有的。与动物细胞表达***相比,甲醇酵母***具有繁殖快速、培养容易、成本低的特点,同时还容易进行分子遗传操作。甲醇酵母比酿酒酵母拥有优势表现在:重组株传代稳定、表达水平高及生产规模容易工业放大等。甲醇酵母***外源蛋白表达水平比酿酒酵母***高10-100倍。这是因为甲醇酵母拥有AOX启动子。AOX启动子是目前已研究过的启动子中最强的调控性真核启动子之一,因而由AOX启动子驱动的外源基因表达具有非常大的高效表达能力。自1987年Gzegy等首次在毕赤酵母(P.pastoris)中表达乙型肝炎表面抗原(HbsAg)以来,短短几年时间,至少有40种外源蛋白在P.pastoris中获得表达。1997年Waterham等分离克隆了毕赤酵母中3-磷酸甘油醛脱氢酶(GAP)启动子,它是一个组成型高效启动子。GAP启动子能够启动外源基因在细胞的生长过程中高效表达,不需进行诱导。由于GAP启动子的高效性以及操作上更为方便,因此它具有巨大的应用潜力,是AOX启动子的有效替代者。另外P.pastoris分泌表达有一个非常好的优点就是P.pastoris自身分泌蛋白质水平很低。这意味着在P.pastoris基本培养基上生长时,在培养中的大部分蛋白为目标蛋白质,这有利目标蛋白质的纯化。在分泌蛋白的糖基化修饰上,毕赤酵母不会象酿酒酵母的超糖基化,这是毕赤酵母的另一优点。
发明内容
本发明提供了一种新的米曲霉单宁酶及其编码基因;本发明还提供了上述新基因的表达以及表达产物的纯化方法和应用。
本发明通过设计了一对特异引物,将编码米曲霉单宁酶成熟蛋白(其编码的成熟肽序列如序列表中<400>2序列所示)的核苷酸序列(如序列表中<400>1序列所示)用PCR方法从米曲霉基因中扩增出来,克隆到毕赤酵母表达载体pPIC9K(购自Invitrogen公司),构建表达载体载体pPIC9K-TAN,采用电击法转化毕赤酵母KM71(购自Invitrogen公司)获得重组菌株。宿主菌类型,摇瓶培养条件,发酵培养条件等摸索和优化,重组蛋白的表达量达到200mg/L。
本发明还将单宁酶基因克隆到毕赤酵母表达载体pGAPZαA(购自Invitrogen公司)中,并通过PCR的方法对基因的表达框架进行了改造,构建了组成型表达载体pGAPZαA-TAN及pGAPZαA-ETAN。采用电击法转化毕赤酵母菌株KM71H(购自Invitrogen公司)获得重组菌株,并在摇瓶培养条件下以分泌形式表达。
本发明还摸索和优化了重组单宁酶蛋白的纯化条件,表达产物采用DEAE Sepharose(购自Amersham Biosciences公司)阴离子交换层析一步纯化,可得到纯度达95%以上的重组单宁酶蛋白。
本发明获得的重组单宁酶具有生物活性。
本发明构建了含有米曲霉单宁酶蛋白成熟肽编码序列的表达质粒pPIC9k-TAN(构建过程见图13)以及含有表达质粒pGAPZαA-TAN及pGAPZαA-ETAN(构建过程见图14)
本发明构建了米曲霉单宁酶成熟蛋白编码序列的表达质粒pPIC9K-TAN,由该表达质粒载体经EcoRI/NotI双酶切,可得到1.7kbp的片段,即为米曲霉单宁酶成熟肽编码序列。表达质粒pGAPZαA-TAN及pGAPZαA-ETAN中单宁酶基因通过XhoI和NotI克隆,由于单宁酶成熟蛋白编码序列中1405bp处含有一个XhoI酶切位点,表达质粒经XhoI/NotI双酶切可得到约300bp和1.4kb的两个片段。
本发明的表达质粒载体的复制方法:参照Sambrook(Sambrook,et al.1989,Molecularcloing.Cold Spring Harbor Labroratory Press.USA)方法,按CaCl2法在E.Coli.DH5α菌株中转化质粒,转化了pPIC9K-TAN的细菌用含100μg/ml氨苄青霉素的LB培养基进行培养,转化了pGAPZαA-TAN及pGAPZαA-ETAN的细菌用含有25μg/ml的低盐LB培养基进行培养,碱法提取质粒。
附图说明
图1为从米曲霉基因中PCR扩增的单宁酶基因电泳结果。1:PCR扩增的单宁酶基因;2:阴性对照;M:1kb DNAmarker。
图2为单宁酶甲醇酵母表达载体载体pPIC9k-TAN的酶切鉴定电泳结果。1:pPIC9k-TAN(EcoR I+Not I);2:pPIC9k(EcoR I+Not I);M1:1kb DNAmarker。
图3为整合了载体pPIC9k-TAN的重组甲醇酵母菌株PCR鉴定电泳结果。1:阳性克隆;2:阴性对照;M1:1kb DNA marker。
图4为重组单宁酶通过表达载体pPIC9k-TAN,用甲醇进行诱导表达的表达产物SDS-PAGE电泳结果。1:诱导前发酵上清液;2~5:经甲醇诱导后1~4天的发酵上清液;6:蛋白分子量标准。
图5为重组单宁酶的纯化结果SDS-PAGE电泳分析。1:发酵上清液;2:DEAE阴离子交换纯化的单宁酶(非还原);3:纯化的单宁酶去糖基化(非还原);4:蛋白分子量标准;5:DEAE阴离子交换纯化的单宁酶(还原);6:纯化的单宁酶去糖基化(还原)。
图6为重组单宁酶的最适反应温度曲线。
图7为重组单宁酶的热稳定性曲线。
图8为重组单宁酶的最适反应pH曲线。
图9为重组单宁酶的酸碱稳定性曲线。
图10为重组单宁酶通过表达载体pGAPZαA-TAN进行表达的细胞培养上清液SDS-PAGE电泳结果。1:蛋白分子量标准;2~9:培养2~9天的培养物上清液。
图11为转化了表达载体pGAPZαA-TAN及pGAPZαA-ETAN的毕赤酵母菌株KM71H的单宁平板筛选结果。A:转化了pGAPZαA-TAN的毕赤酵母菌株KM71H;B:转化了pGAPZαA-ETAN的毕赤酵母菌株KM71H。
图12为表达载体pGAPZαA-TAN及pGAPZαA-ETAN中单宁酶的表达框架示意图。A:表达载体pGAPZαA-TAN中单宁酶基因的表达框架;B:表达载体pGAPZαA-ETAN中单宁酶基因的表达框架。
图13为单宁酶甲醇酵母表达载体载体pPIC9k-TAN的构建示意图。
图14为表达载体pGAPZαA-TAN及pGAPZαA-ETAN构建示意图。
具体实施方式
下面结合附图及实施例对本发明作进一步说明,将有助于本领域的普通技术人员理解本发明,但不以任何形式限制本发明。
实施例一、米曲霉单宁酶基因的PCR扩增
米曲霉基因组的提取参考《现代分子生物学实验技术》的实验方案,根据单宁酶基因编码的成熟蛋白两端序列和毕赤酵母表达载体pPIC9K(购自Invitrogen公司)的多酶切位点,合成一对引物,序列如下:
上游引物,5’G
GAATTCGCTTCTTTTACCGATGTGTGCAC3’(单下划线部分为EcoRI酶切序列);
下游引物,
(单下划线部分为NotI酶切序列,双下划线为终止密码子)。
PCR扩增、基因克隆皆按常规方法进行。PCR产物约1700bp,如图1,编码570个氨基酸残基。
实施例二、单宁酶甲醇酵母表达载体pPIC9k-TAN的构建
PCR扩增的米曲霉单宁酶基因用EcoRI和NotI双酶切克隆到甲醇酵母表达载体pPIC9K相应的酶切窗口,获得表达载体pPIC9K-TAN,构建过程见图13,经酶切鉴定(图2)和测序分析表明克隆的基因为目的基因。
实施例三、酵母菌株的转化及阳性克隆的筛选
表达质粒pPIC9K-TAN用PmeI单切线性化,采用电击法转化毕赤酵母KM71(购自Invitrogen公司),同时转化同样线性化的空载体pPIC9K作为对照。在MD固体培养平板筛选阳性克隆,并用含G418抗性的YPD平板进一步筛选阳性克隆。转化子通过PCR的方法进一步进行了鉴定,PCR引物为α-factor sequencing primer(5’-TACTATTGCCAGCATTGCTGC-3’)和3’AOX1 sequencing primer(5’-GCAAATGGCATTCTGACATCC-3’),采用Taq DNA聚合酶进行PCR反应,以提取的阳性克隆基因组为模板,反应循环条件为,94℃5分钟,94℃45秒,52℃30秒,72℃2分钟,32个循环,72℃10分钟,4℃保存。琼脂糖凝胶电泳检测显示图3,PCR扩增得到一特异性约1.8kb DNA条带,与预期的大小相符。
实施例四、重组米曲霉单宁酶的高密度发酵诱导表达
从YPD平板上选取单克隆,接种于20ml BMGY培养基中,30℃、240rpm培养20h。以1∶50的比例接种到300ml BMGY培养基中,30℃、240rpm培养至OD600=2~6,用以接种发酵罐。
7L发酵罐,加入3L发酵基础培养基,121℃灭菌20min,调节温度至30℃,用氨水调节pH至5.5,加入PTM1(4.35ml L-1),接入种子菌(1∶10)。发酵过程中,温度控制在30℃,通气量维持在2vvm,转速控制在300~800rpm之间以维持溶氧20%以上。
发酵分为三个阶段:生长期,从加入种子菌,培养约16~24h,直到将发酵罐中甘油耗尽,表现为溶氧突然上升;之后进入甘油促生长期,补加50%甘油(含有PTM1,12ml L-1),补料速度为18ml L-1h-1,持续4~6h;最后进入诱导期,用氨水调节pH至5.0,流加100%甲醇(含有PTM1,12ml L-1),流速从1ml L-1h-1经10h线性升至3ml L-1h-1,持续120h。发酵上清用SDS-PAGE检测蛋白表达,如图4。
实施例五、重组米曲霉单宁酶的纯化
发酵液于4℃8000rpm离心30min,上清经Sephendex G-25脱盐,更换为10mmol/L柠檬酸pH5.5缓冲液,再经DEAE Sepharose Fast Flow离子交换层析柱纯化,洗脱体系为10mmol/L柠檬酸pH5.5缓冲液,NaCl线性梯度(0~0.5mol/L),pH5.5。洗脱样品用SDS-PAGE进行分析鉴定(图5)。合并目的蛋白组分,双蒸水透析,透析后样品冷冻干燥,-20℃保存。
实施例六、重组米曲霉单宁酶的活性鉴定
重组米曲霉单宁酶的活性鉴定参考Beverini(1990)的方法。1IU的酶活定义1分钟水解1μmol单宁酸所需的酶量。酶活检测我们制备的重组米曲霉单宁酶酶活达到50,000IU/g。
实施例七、重组米曲霉单宁酶最适反应温度及热稳定性测定
在不同温度(10℃~70℃),测定酶活性。测定热稳定性时,则将酶液于不同温度下保温10分钟后,测定其残余的酶活性。测得重组单宁酶的最适反应温度为40℃,热稳定性范围为10~40℃(图6,图7)。
实施例八、重组米曲霉单宁酶最适反应pH及酸碱稳定性测定
配制不同pH值的Na2HPO4-柠檬酸缓冲液(pH2.2~8.0),0.5个pH单位为梯度。测定最适反应pH值时,将单宁酶溶解于去离子水中,用不同pH值的Na2HPO4-柠檬酸缓冲液稀释5倍;然后测定酶活。测定酸碱稳定性时,则将单宁酶溶解于不同pH值的缓冲液中,在30℃保温一小时后加入20倍体积的pH5.5的50mM柠檬酸缓冲液,然后测定酶活。测定结果,重组单宁酶的最适反应pH值为pH6.0,酸碱稳定性范围为pH4~6(图8,图9)。
实施例九、重组单宁酶毕赤酵母组成型表达载体pGAPZαA-TAN及pGAPZαA-ETAN的构建
根据表达载体pGAPZαA(购自Invitrogen公司)的部分信号肽序列及单宁酶编码序列设计合成引物:
上游引物:
(1):5’CCG
CTCGAGAAAAGA GCTTCTTTTACCGATGTGTGCAC3’(单下划线部分为载体信号肽序列中的XhoI酶切位点),
下游引物:5’TCGCTGGACTGGCCATAAAAGC3’(下游引物为单宁酶编码序列中约480bp处的一段序列)。
以pPIC9K-TAN为模板,分别用两组引物进行PCR扩增,扩增得到的片段大小约为500bp,用XhoI和SphI双酶切后切成两个片段,回收其中约100bp的片段;将质粒pPIC9K-TAN用SphI和NotI双酶切,回收大小为1.4kb的单宁酶基因片段;将这两个片段连接到表达载体pGAPZαA相应的XhoI/NotI酶切窗口中,构建成表达载体pGAPZαA-TAN及pGAPZαA-ETAN,构建过程见图14;表达载体中单宁酶基因的表达框架如图12,在pGAPZαA-TAN中单宁酶直接连接于载体信号肽末端,在pGAPZαA-ETAN中单宁酶基因连接于毕赤酵母蛋白Ste13蛋白酶切割位点末端,即在信号肽序列及单宁酶之间含有4个氨基酸残基EAEA;经酶切和DNA测序鉴定载体构建成功。
实施例十、毕赤酵母菌株的转化和阳性克隆的筛选
表达载体pGAPZαA-TAN及pGAPZαA-ETAN分别用AvrII进行线性化,采用电击法转化毕赤酵母KM71H,同时转化同样线性化的空载体pGAPZαA作为对照。转化后的细胞用zeocine抗性平板筛选出抗性克隆,再用含0.2%单宁酸的YNB+0.5%葡萄糖平板筛选出单宁酶活性菌株。其中具有单宁酶活性的重组菌株能够在菌落周围形成一个透明的单宁降解圈(图11)。
实施例十一、重组单宁酶通过表达载体pGAPZαA-TAN进行组成型表达
从YPD平板上挑取单菌落,接种至50mlYPD培养基,30℃、240rpm培养,每隔24小时添加终浓度为0.5%的葡萄糖溶液,并取培养上清液进行SDS-PAGE分析,在分子量约100kDa处有一特征蛋白条带,即为重组单宁酶产物(图10)。
序列表
<110>中山大学
<120>重组米曲霉单宁酶及其表达和纯化方法
<160>2
<210>1
<211>1710
<212>DNA
<213>米曲霉(Aspergillus oryzae)
<220>
<221>mat peptide
<222>(1)…(570)
<400>1
gct tct ttt acc gat gtg tgc acc gtg tct aac gtg aag gct gca ttg 48
Ala Ser Phe Thr Asp Val Cys Thr Val Ser Asn Val Lys Ala Ala Leu
1 5 10 15
cct gcc aac gga act ctg ctc gga atc agc atg ctt ccg tcc gcc gtc 96
Pro Ala Asn Gly Thr Leu Leu Gly Ile Ser Met Leu Pro Ser Ala Val
20 25 30
acg gcc aac cct ctc tac aac cag tcg gct ggc atg ggt agc acc act 114
Thr Ala Asn Pro Leu Tyr Asn Gln Ser Ala Gly Met Gly Ser Thr Thr
35 40 45
acc tat gac tac tgc aat gtg act gtc gcc tac acg cat acc ggc aag 192
Thr Tyr Asp Tyr Cys Asn Val Thr Val Ala Tyr Thr His Thr Gly Lys
50 55 60
ggt gat aaa gtg gtc atc aag tac gca ttc ccc aag ccc tcc gac tac 240
Gly Asp Lys Val Val Ile Lys Tyr Ala Phe Pro Lys Pro Ser Asp Tyr
65 70 75 80
gag aac cgt ttc tac gtt gct ggt ggt ggt ggc ttt tcc ctc tct agc 288
Glu Asn Arg Phe Tyr Val Ala Gly Gly Gly Gly Phe Ser Leu Ser Ser
85 90 95
gat gct acc gga ggt ctc gcc tat ggc gct gtg gga ggt gcc acc gat 336
Asp Ala Thr Gly Gly Leu Ala Tyr Gly Ala Val Gly Gly Ala Thr Asp
100 105 110
gct gga tac gac gca ttc gat aac agc tac gac gag gta gtc ctc tac 384
Ala Gly Tyr Asp Ala Phe Asp Asn Ser Tyr Asp Glu Val Val Leu Tyr
115 120 125
gga aac gga acc att aac tgg gac gcc aca tac atg ttc gca tac cag 432
Gly Asn Gly Thr Ile Asn Trp Asp Ala Thr Tyr Met Phe Ala Tyr Gln
130 135 140
gca ctg gga gag atg acc cgg atc gga aag tac atc acc aag ggc ttt 480
Ala Leu Gly Glu Met Thr Arg Ile Gly Lys Tyr Ile Thr Lys Gly Phe
145 150 155 160
tat ggc cag tcc agc gac agc aag gtc tac acc tac tac gag ggt tgc 528
Tyr Gly Gln Ser Ser Asp Ser Lys Val Tyr Thr Tyr Tyr Glu Gly Cys
165 170 175
tcc gat gga gga cgt gag ggt atg agt caa gtc cag cgc tgg ggt gag 576
Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Val Gln Arg Trp Gly Glu
180 185 190
gag tat gac ggt gcg att act ggt gcc ccg gct ttc cgt ttc gct cag 624
Glu Tyr Asp Gly Ala Ile Thr Gly Ala Pro Ala Phe Arg Phe Ala Gln
195 200 205
caa cag gtt cac cat gtg ttc tcg tcc gaa gtg gag caa act ctg gac 672
Gln Gln Val His His Val Phe Ser Ser Glu Val Glu Gln Thr Leu Asp
210 215 220
tac tac ccg cct cca tgt gag tcg aag aag atc gtg aac gcc acc att 720
Tyr Tyr Pro Pro Pro Cys Glu Ser Lys Lys Ile Val Asn Ala Thr Ile
225 230 235 240
gct gct tgc gac ccg ctt gat gga aga acc gac ggt gtt gtg tcc cgg 768
Ala Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val Val Ser Arg
245 250 255
acg gat ctt tgc aag ctt aac ttc aat ttg acc tct atc atc ggt gag 816
Thr Asp Leu Cys Lys Leu Asn Phe Asn Leu Thr Ser Ile Ile Gly Glu
260 265 270
cct tac tac tgt gct gcg gga act agc act tcg ctt ggt ttc ggc ttc 864
Pro Tyr Tyr Cys Ala Ala Gly Thr Ser Thr Ser Leu Gly Phe Gly Phe
275 280 285
agc aat ggc aag cgc agc aat gtc aag cgt cag gcc gag ggc agc acc 912
Ser Asn Gly Lys Arg Ser Asn Val Lys Arg Gln Ala Glu Gly Ser Thr
290 295 300
acc agc tac cag ccc gcc cag aac ggc acg gtc acc gca cgt ggt gta 960
Thr Ser Tyr Gln Pro Ala Gln Asn Gly Thr Val Thr Ala Arg Gly Val
305 310 315 320
gct gtc gcc cag gcc atc tac gat ggt ctc cac aac agc aag ggc gag 1008
Ala Val Ala Gln Ala Ile Tyr Asp Gly Leu His Asn Ser Arg Gly Glu
325 330 335
cgc gcg tac ctc tcc tgg cag att gcc tct gag ctg agc gat gct gag 1056
Arg Ala Tyr Leu Ser Trp Gln Ile Ala Ser Glu Leu Ser Asp Ala Glu
340 345 350
acc gag tac aac tct gac act ggc aag tgg gag ctc aac atc ccg tcg 1104
Thr Glu Tyr Asn Ser Asp Thr Gly Lys Trp Glu Leu Asn Ile Pro Ser
355 360 365
acc ggt ggt gag tac gtc acc aag ttc att cag ctc ctg aac ctc gac 1152
Thr Gly Gly Glu Tyr Val Thr Lys Phe Ile Gln Leu Leu Asn Leu Asp
370 375 380
aac ctt tcg gat ctg aac aac gtg acc tac gac acc ctg gtc gac tgg 1200
Asn Leu Ser Asp Leu Asn Asn Val Thr Tyr Asp Thr Leu Val Asp Trp
385 390 395 400
atg aac act ggt atg gtg cgc tac atg gac agc ctt cag acc acc ctt 1248
Met Asn Thr Gly Met Val Arg Tyr Met Asp Ser Leu Gln Thr Thr Leu
405 410 415
ccc gat ctg act ccc ttc caa tcg tcc ggc gga aag ctg ctg cac tac 1296
Pro Asp Leu Thr Pro Phe Gln Ser Ser Gly Gly Lys Leu Leu His Tyr
420 425 430
cac ggt gaa tct gac ccc agt atc ccc gct gcc tcc tcg gtc cac tac 1344
His Gly Glu Ser Asp Pro Ser Ile Pro Ala Ala Ser Ser Val His Tyr
435 440 445
tgg cag gcg gtt cgt tcc gtc atg tac ggc gac aag acg gaa gag gag 1392
Trp Gln Ala Val Arg Ser Val Met Tyr Gly Asp Lys Thr Glu Glu Glu
450 455 460
gcc ctg gag gct ctc gag gac tgg tac cag ttc tac cta atc ccc ggt 1440
Ala Leu Glu Ala Leu Glu Asp Trp Tyr Gln Phe Tyr Leu Ile Pro Gly
465 470 475 480
gcc gcc cac tgc gga acc aac tct ctc cag ccc gga cct tac cct gag 1488
Ala Ala His Cys Gly Thr Asn Ser Leu Gln Pro Gly Pro Tyr Pro Glu
485 490 495
aac aac atg gag att atg atc gac tgg gtc gag aac ggc aac aag ccg 1536
Asn Asn Met Glu Ile Met Ile Asp Trp Val Glu Asn Gly Asn Lys Pro
500 505 510
tcc cgt ctc aat gcc act gtt tct tcg ggt acc tac gcc ggc gag acc 1584
Ser Arg Leu Asn Ala Thr Val Ser Ser Gly Thr Tyr Ala Gly Glu Thr
515 520 525
cag atg ctt tgc cag tgg ccc aag cgt cct ctc tgg cgc ggc aac tcc 1632
Gln Met Leu Cys Gln Trp Pro Lys Arg Pro Leu Trp Arg Gly Asn Ser
530 535 540
agc ttc gac tgt gtc aac gac gag aag tcg att gac agc tgg acc tac 1680
Ser Phe Asp Cys Val Asn Asp Glu Lys Ser Ile Asp Ser Trp Thr Tyr
545 550 555 560
gag ttc cca gcc ttc aag gtc cct gta tac 1710
Glu Phe Pro Ala Phe Lys Val Pro Val Tyr
565 570
<210>2
<211>570
<212>PRT
<213>米曲霉(Aspergillus oryzae)
<220>
<221>mat peptide
<222>(1)…(570)
<400>2
Ala Ser Phe Thr Asp Val Cys Thr Val Ser Asn Val Lys Ala Ala Leu
1 5 10 15
Pro Ala Asn Gly Thr Leu Leu Gly Ile Ser Met Leu Pro Ser Ala Val
20 25 30
Thr Ala Asn Pro Leu Tyr Asn Gln Ser Ala Gly Met Gly Ser Thr Thr
35 40 45
Thr Tyr Asp Tyr Cys Asn Val Thr Val Ala Tyr Thr His Thr Gly Lys
50 55 60
Gly Asp Lys Val Val Ile Lys Tyr Ala Phe Pro Lys Pro Ser Asp Tyr
65 70 75 80
Glu Asn Arg Phe Tyr Val Ala Gly Gly Gly Gly Phe Ser Leu Ser Ser
85 90 95
Asp Ala Thr Gly Gly Leu Ala Tyr Gly Ala Val Gly Gly Ala Thr Asp
100 105 110
Ala Gly Tyr Asp Ala Phe Asp Asn Ser Tyr Asp Glu Val Val Leu Tyr
115 120 125
Gly Asn Gly Thr Ile Asn Trp Asp Ala Thr Tyr Met Phe Ala Tyr Gln
130 135 140
Ala Leu Gly Glu Met Thr Arg Ile Gly Lys Tyr Ile Thr Lys Gly Phe
145 150 155 160
Tyr Gly Gln Ser Ser Asp Ser Lys Val Tyr Thr Tyr Tyr Glu Gly Cys
165 170 175
Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Val Gln Arg Trp Gly Glu
180 185 190
Glu Tyr Asp Gly Ala Ile Thr Gly Ala Pro Ala Phe Arg Phe Ala Gln
195 200 205
Gln Gln Val His His Val Phe Ser Ser Glu Val Glu Gln Thr Leu Asp
210 215 220
Tyr Tyr Pro Pro Pro Cys Glu Ser Lys Lys Ile Val Asn Ala Thr Ile
225 230 235 240
Ala Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val Val Ser Arg
245 250 255
Thr Asp Leu Cys Lys Leu Asn Phe Asn Leu Thr Ser Ile Ile Gly Glu
260 265 270
Pro Tyr Tyr Cys Ala Ala Gly Thr Ser Thr Ser Leu Gly Phe Gly Phe
275 280 285
Ser Asn Gly Lys Arg Ser Asn Val Lys Arg Gln Ala Glu Gly Ser Thr
290 295 300
Thr Ser Tyr Gln Pro Ala Gln Asn Gly Thr Val Thr Ala Arg Gly Val
305 310 315 320
Ala Val Ala Gln Ala Ile Tyr Asp Gly Leu His Asn Ser Arg Gly Glu
325 330 335
Arg Ala Tyr Leu Ser Trp Gln Ile Ala Ser Glu Leu Ser Asp Ala Glu
340 345 350
Thr Glu Tyr Asn Ser Asp Thr Gly Lys Trp Glu Leu Asn Ile Pro Ser
355 360 365
Thr Gly Gly Glu Tyr Val Thr Lys Phe Ile Gln Leu Leu Asn Leu Asp
370 375 380
Asn Leu Ser Asp Leu Asn Asn Val Thr Tyr Asp Thr Leu Val Asp Trp
385 390 395 400
Met Asn Thr Gly Met Val Arg Tyr Met Asp Ser Leu Gln Thr Thr Leu
405 410 415
Pro Asp Leu Thr Pro Phe Gln Ser Ser Gly Gly Lys Leu Leu His Tyr
420 425 430
His Gly Glu Ser Asp Pro Ser Ile Pro Ala Ala Ser Ser Val His Tyr
435 440 445
Trp Gln Ala Val Arg Ser Val Met Tyr Gly Asp Lys Thr Glu Glu Glu
450 455 460
Ala Leu Glu Ala Leu Glu Asp Trp Tyr Gln Phe Tyr Leu Ile Pro Gly
465 470 475 480
Ala Ala His Cys Gly Thr Asn Ser Leu Gln Pro Gly Pro Tyr Pro Glu
485 490 495
Asn Asn Met Glu Ile Met Ile Asp Trp Val Glu Asn Gly Asn Lys Pro
500 505 510
Ser Arg Leu Asn Ala Thr Val Ser Ser Gly Thr Tyr Ala Gly Glu Thr
515 520 525
Gln Met Leu Cys Gln Trp Pro Lys Arg Pro Leu Trp Arg Gly Asn Ser
530 535 540
Ser Phe Asp Cys Val Asn Asp Glu Lys Ser Ile Asp Ser Trp Thr Tyr
545 550 555 560
Glu Phe Pro Ala Phe Lys Val Pro Val Tyr
565 570
Claims (3)
1.一种米曲霉单宁酶基因的重组方法,根据单宁酶基因编码的成熟蛋白两端序列和毕赤酵母表达载体pPIC9K的多酶切位点,合成一对引物,序列如下:
上游引物,5’G
GAATTCGCTTCTTTTACCGATGTGTGCAC3’;
下游引物,5’GCG
GCGGCCGC GTATACAGGGACCTTGAAGGC3’;经PCR扩增得到如SEQ ID NO:1所示的米曲霉单宁酶基因。
2.权利要求1所得的米曲霉单宁酶基因的表达方法,通过表达载体pPIC9K-TAN在毕赤酵母菌株KM71中采用甲醇进行诱导表达,或者通过表达载体pGAPZαA-TAN和pGAPZαA-ETAN在毕赤酵母菌株KM71H中进行组成型表达,不需诱导;两种方式的表达产物均分泌于胞外,通过获取培养物上清液进行纯化得到如SEQ ID NO:2所示的重组米曲霉单宁酶。
3.如权利要求2所述的米曲霉单宁酶基因的表达方法,其特征是所述的培养物上清液的纯化是用DEAE阴离子交换柱层析一步纯化。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410027497 CN1289665C (zh) | 2004-06-09 | 2004-06-09 | 重组米曲霉单宁酶及其表达和纯化方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410027497 CN1289665C (zh) | 2004-06-09 | 2004-06-09 | 重组米曲霉单宁酶及其表达和纯化方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1584023A CN1584023A (zh) | 2005-02-23 |
CN1289665C true CN1289665C (zh) | 2006-12-13 |
Family
ID=34601369
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200410027497 Expired - Fee Related CN1289665C (zh) | 2004-06-09 | 2004-06-09 | 重组米曲霉单宁酶及其表达和纯化方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1289665C (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103436505A (zh) * | 2013-08-13 | 2013-12-11 | 广西大学 | 一种制备单宁酶的方法 |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102260656B (zh) * | 2011-05-31 | 2012-12-12 | 天津市畜牧兽医研究所 | 重组黑曲霉单宁酶及其表达和纯化方法 |
CN106490450B (zh) * | 2016-10-25 | 2019-06-28 | 中南林业科技大学 | 一种栓皮栎种仁脱苦脱涩方法 |
CN110564746A (zh) * | 2019-08-05 | 2019-12-13 | 集美大学 | 一种耐酸型单宁酶及其基因和应用 |
CN110527690A (zh) * | 2019-08-05 | 2019-12-03 | 集美大学 | 一种耐热型单宁酶及其应用 |
CN110527633B (zh) * | 2019-08-05 | 2022-03-22 | 集美大学 | 一种米曲霉及其在制备单宁酶中的应用 |
CN110551702B (zh) * | 2019-08-05 | 2022-07-19 | 集美大学 | 重组塔宾曲霉单宁酶及其表达和应用 |
CN113801863B (zh) * | 2021-09-18 | 2023-06-23 | 中国农业科学院麻类研究所 | 一种罗布麻单宁酶全细胞催化剂工程菌株及其制备方法和应用 |
-
2004
- 2004-06-09 CN CN 200410027497 patent/CN1289665C/zh not_active Expired - Fee Related
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103436505A (zh) * | 2013-08-13 | 2013-12-11 | 广西大学 | 一种制备单宁酶的方法 |
CN103436505B (zh) * | 2013-08-13 | 2014-09-17 | 广西大学 | 一种制备单宁酶的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN1584023A (zh) | 2005-02-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1249239C (zh) | 合成有机产物的方法和材料 | |
CN1053013C (zh) | 微生物植酸酶的克隆及表达 | |
CN1029977C (zh) | 胰岛素类似物的制备方法 | |
CN1230537C (zh) | 来自曲霉属融合产物中经加工的重组乳铁蛋白和乳铁蛋白多肽片断的表达 | |
CN1628168A (zh) | 用于在假丝酵母细胞中生产有机产物的方法和物质 | |
CN1289665C (zh) | 重组米曲霉单宁酶及其表达和纯化方法 | |
CN1990871A (zh) | 重组人纤溶酶原Kringle 5(hk5)生产方法 | |
CN1793375A (zh) | 重组人神经生长因子的酵母表达***及制备重组人神经生长因子的方法 | |
CN1301307A (zh) | 在丝状真菌突变细胞中生产多肽的方法 | |
CN1192103C (zh) | 生产植酸酶的方法 | |
CN1788087A (zh) | 醋酸菌的乙醇脱氢酶基因 | |
CN1237208A (zh) | 用于产朊假丝酵母转化的方法和材料 | |
CN101050467A (zh) | 一种高活性鸡溶菌酶的基因克隆、表达与应用 | |
CN1766098A (zh) | 一种甘露聚糖酶及其编码基因与应用 | |
CN1834237A (zh) | β-甘露聚糖酶及其表达方法与专用工程菌 | |
CN1594542A (zh) | 黑曲霉菊粉内切酶基因和表达该基因的重组毕赤酵母菌株 | |
CN1924001A (zh) | 用甲基营养酵母生产人热休克蛋白70的方法 | |
CN1258317A (zh) | 登革病毒基因表达的方法 | |
CN1238501C (zh) | 外切-β-1,4-葡聚糖酶/内切-β-1,4-木聚糖酶及其应用 | |
CN1295248C (zh) | 一种小盐芥钠氢泵蛋白基因tnhx1及其抗盐应用 | |
CN1280417C (zh) | 一种人酪氨酸酶表达载体及其应用 | |
CN1724672A (zh) | 一种组成型表达载体及其应用 | |
CN1268751C (zh) | 表达人粒细胞巨噬细胞集落刺激因子的酵母重组株及rhGM-CSF的纯化方法 | |
CN101062942A (zh) | 来源于烟曲霉的与活性氧杀伤相关蛋白及其编码基因 | |
CN1769456A (zh) | 重组人那希力肽的生产方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
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: 20061213 Termination date: 20160609 |