CN111500553A - 高活性的聚半乳糖醛酸酶的制备和应用 - Google Patents
高活性的聚半乳糖醛酸酶的制备和应用 Download PDFInfo
- Publication number
- CN111500553A CN111500553A CN202010333827.XA CN202010333827A CN111500553A CN 111500553 A CN111500553 A CN 111500553A CN 202010333827 A CN202010333827 A CN 202010333827A CN 111500553 A CN111500553 A CN 111500553A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- polygalacturonase
- 15min
- pcr
- plasmids
- 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.)
- Pending
Links
- 108010059820 Polygalacturonase Proteins 0.000 title claims abstract description 32
- 230000000694 effects Effects 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 37
- 239000013256 coordination polymer Substances 0.000 claims abstract description 3
- 239000013612 plasmid Substances 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000001976 enzyme digestion Methods 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000012163 sequencing technique Methods 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 7
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 108010006785 Taq Polymerase Proteins 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 238000010367 cloning Methods 0.000 claims description 6
- 239000012154 double-distilled water Substances 0.000 claims description 6
- 239000003623 enhancer Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 6
- 230000009466 transformation Effects 0.000 claims description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 241001506991 Komagataella phaffii GS115 Species 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 239000003957 anion exchange resin Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000004520 electroporation Methods 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 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 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 239000000600 sorbitol Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 230000035772 mutation Effects 0.000 abstract 2
- 238000000855 fermentation Methods 0.000 description 20
- 230000004151 fermentation Effects 0.000 description 20
- 239000000243 solution Substances 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 239000000047 product Substances 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 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 7
- 108010093305 exopolygalacturonase Proteins 0.000 description 7
- 229960001031 glucose Drugs 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 6
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 239000007640 basal medium Substances 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000001814 pectin Substances 0.000 description 6
- 229920001277 pectin Polymers 0.000 description 6
- 235000010987 pectin Nutrition 0.000 description 6
- 238000011218 seed culture Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 101150051118 PTM1 gene Proteins 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000012526 feed medium Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000002572 peristaltic effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000770536 Bacillus thermophilus Species 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 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 1
- 241000588698 Erwinia Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000233732 Fusarium verticillioides Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 241000218483 Streptomyces lydicus Species 0.000 description 1
- 241000204666 Thermotoga maritima Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 229910052925 anhydrite Inorganic materials 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229930189065 blasticidin Natural products 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及高活性的聚半乳糖醛酸酶的制备和应用,(polygalacturonase,XP_011073814.1)在第308位、第309位上发生突变,由原来的CP突变为WR其有意效果在于,获得的突变体聚半乳糖醛酸酶比野生型酶活性高8.82%。
Description
技术领域
本发明属于基因工程与酶工程领域,具体涉及高活性的聚半乳糖醛酸酶的制备和应用。
背景技术
果胶酶是指能够分解果胶物质的多种酶的总称,广泛地应用于食品、纺织、造纸等工业,具有重要的经济价值,聚半乳糖醛酸酶是研究较多的果胶酶,它能够通过在氧桥中引入水而催化聚半乳糖醛酸链的水解断裂,从而迅速的降低果胶溶液的黏度,且不会生成甲醇,在食品工业特别是果汁澄清中有重要意义。动物、植物和微生物中均发现有果胶酶的存在,但是依靠动物、植物生产果胶酶,存在产量低、提取困难等问题,采用微生物发酵法生产果胶酶,具有微生物分布广、生长速度快、易培养的优点,细菌、真菌、放线菌中均发现有能够产生果胶酶的菌株,如黑曲霉、青霉、嗜热芽孢杆菌、利迪链霉菌、欧文氏菌、米根霉、串珠镰刀菌、灰葡萄孢菌、海栖热袍菌等,其中曲霉是商业果胶酶的主要生产菌。
发明内容
本发明的目的在于提供具有更高酶活的聚半乳糖醛酸酶。
本发明的制备过程如下:
1.从NCBI上获得聚半乳糖醛酸酶的序列(polygalacturonase,XP_011073814.1),交生物公司合成,设计PCR引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTC AACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGA CGTACATTGA-3',PCR反应结束后,加20μl Cloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,采用EcoRⅠ、NotⅠ酶切pPICZαA质粒,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,挑取阳性克隆,送生物公司测序,得到含有野生型序列质粒的基因工程菌;
2.质粒DNA的提取
将上述步骤含有野生型序列质粒的基因工程菌扩大培养,抽提质粒;
3.易错PCR扩增与突变文库的构建
以步骤二获得的质粒DNA为模板,用EcoRⅠ、NotⅠ酶切使质粒线性化,用引物序列引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTCAACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGACGTACATTGA-3',进行易错PCR扩增基因,易错PCR扩增体系(50μl)为10×TaKaRa Taq Buffer,dNTPs M ixture,引物各0.2μmol/L,模板DNA 200ng,Taq DNA聚合酶2.5U,5mmol/l Mn2+,0.5U/μl的Taq DNA聚合酶2.5μl,7mmol/l的Mg2+,PCR反应条件:95℃5min,94℃1min,55℃1min,72℃2min,35个循环,72℃10min,加20μl Cloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,将pPICZαA质粒用EcoRⅠ、NotⅠ酶切线性化,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,涂布于固体LB平板(100μg/ml Amp)抗性筛选阳性转化子,所有转化子构成突变体文库;
4.重组质粒的电击转化
将步骤三所得阳性转化子混合转入20ml氨苄抗性LB培养基中37℃培养过夜,使用质粒提取试剂盒提取全部质粒,使用NotⅠ将所质粒线性化,使用胶回收试剂盒回收纯化酶切产物,取20μl的线性化DNA加入100μl毕赤酵母GS115感受态细胞混匀立即转入预冷的0.2cm电击杯中,冰浴静置5min,用吸水纸擦干电击杯外壁的水分,迅速进行电击转化,电转条件:电压1.5kv,电容25μF,电阻200Ω,电击结束后立即加入1ml冰浴的50%1M山梨,50%YPD到电击杯中,然后转至1.5ml无菌离心管中,28℃,80r/min培养1h复苏;
5.酵母转化子筛选
取100μl步骤四所得的复苏菌液涂布到显色筛选培养基上28℃倒置培养,观察转化子生长状况,将透明圈比野生型对照大的转化子,挑出,进行复筛;
6.将复筛通过的阳性克隆,送生物公司测序,测序结果见表3;
7.酶的生产;
8.酶的纯化,使用阴离子交换树脂纯化蛋白;
9.进行GOD酶活力检测和SDS-PAGE分析;
10.采用Bradford测定蛋白质浓度。
本发明的有益效果是:
获得了比野生型酶活性更高的突变体聚半乳糖醛酸酶。
附图说明
图1聚半乳糖醛酸酶SDS-PAGE电泳图
图2野生型及突变体聚半乳糖醛酸酶三维图
具体实施方式
下面通过实施例对本发明做进一步详细说明,这些实施例仅用来说明本发明,并不限制本发明的范围。
实施例1
本实施例涉及的材料和试剂见表1,实验仪器见表2;
表1实验材料和试剂
考马斯亮蓝R-250 | 北京索莱宝科技有限公司 |
琼脂糖凝胶DNA回收试剂盒 | 大连宝生物工程有限公司 |
限制性内切酶 | 大连宝生物工程有限公司 |
Taq酶 | 大连宝生生物工程有限公司 |
pPICZαA | Invitrogen公司 |
E.coli DH5α | Invitrogen公司 |
毕赤酵母GS115 | Invitrogen公司 |
无水葡萄糖 | 国药集团化学试剂有限公司 |
表2实验仪器设备
LB培养基配方:胰蛋白胨10g/l,酵母粉5g/l,NaCl 10g/l,pH7.0;
LB平板筛选培养基:蛋白胨1%,酵母膏0.5%,氯化钠1%,琼脂粉1.2%,NaOH调节pH至7.0,高压蒸汽灭菌,室温下冷却至50-60℃左右,加入卡那霉素溶液轻轻摇匀使其终浓度为30ng/ml;
PTM1微量元素母液:CuSO4·5H2O 6.0g/l,NaI 0.08g/l,MnSO4·H2O 3.0g/l,Na2MoO4·2H2O 0.2g/l,HBO3 0.02g/l,CoCl2·6H2O 0.914g/l,ZnCl2 20.0g/l,FeSO4·7H2O65.0g/l,浓H2SO4 5.0ml/l,生物素0.2g/l,配制过程中,先在烧杯内加入一定量的纯水,然后按顺序依次加入上述成分,加入浓H2SO4后,避光搅拌至全部溶解,加入生物素搅拌至溶解,定容后用0.22μm孔径无菌滤膜过滤至适宜的无菌容器,置4℃避光保存;
发酵基础培养基(BSM):甘油40g/l 85%H3PO4 13ml/l,KOH 10.6g/l,CaSO40.82g/l,K2SO4 18.20g/l,MgSO4·7H2O 14.9g/l,(NH4)2SO4 13.2g/l,Antifoam 204 0.33ml/l,PTM1 4.5ml/l,使用时,先不加入PTM1,以甘油为碳源的发酵基础培养基121℃高压湿热灭菌30min,以葡萄糖为碳源的发酵基础培养基115℃高压湿热灭菌30min,降至室温后,将PTM1微量元素母液加入至生物反应器中,搅拌过夜;
碳源补料培养基:50%(w/v)甘油,配置时,甘油补料培养基121℃高压湿热灭菌30min,葡萄糖补料培养基115℃高压湿热灭菌30min,冷却后,每100ml碳源补料培养基加入1.2ml PTM1微量元素母液,避光保存待用;
YND培养基(w/v):1%葡萄糖、0.67%YNB、生物素0.4mg/l,配制方法如下:先配制0.67%YNB,121℃高压湿热灭菌20min,使用时,每100ml 0.67%YNB溶液加2ml 50%葡萄糖溶液和200μl生物素母液(0.2g/l);
YPD液体培养基:1%酵母提取物,2%蛋白胨,2%葡萄糖,高压蒸汽灭菌,室温下冷却后置于4℃冰箱保存;
以上培养基,如果需要使用固体培养基,则在高压湿热灭菌前加入2%琼脂粉;
如需使用抗生素,使用前在超净台加入,毕赤酵母的抗生素使用量如下:Zeocin100μg/ml,G418 250μg/ml,潮霉素750μg/ml,灭瘟素500μg/ml;
0.1%半乳糖醛酸溶液:称取0.1000g于80℃烘至恒重的半乳糖醛酸于烧杯中,加适量水溶解,然后转至100ml容量瓶定容后,储存于4℃冰箱备用;
1.0%果胶溶液:称取1.000果胶于烧杯中,加入适量pH3.5柠檬酸-柠檬酸钠缓冲液,室温下用磁力搅拌器低速搅拌至溶解,用同样的缓冲液定容于100ml容量瓶,放置于2-8℃冰箱中备用;
1.从NCBI上获得聚半乳糖醛酸酶的序列(polygalacturonase,XP_011073814.1),交生物公司合成,设计PCR引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTC AACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGA CGTACATTGA-3',PCR反应结束后,加20μl Cloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,采用EcoRⅠ、NotⅠ酶切pPICZαA质粒,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,挑取阳性克隆,送生物公司测序,得到含有野生型序列质粒的基因工程菌;
2.质粒DNA的提取
将上述步骤含有野生型序列质粒的基因工程菌扩大培养,抽提质粒;
3.易错PCR扩增与突变文库的构建
以步骤二获得的质粒DNA为模板,用EcoRⅠ、NotⅠ酶切使质粒线性化,用引物序列引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTCAACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGACGTACATTGA-3',进行易错PCR扩增基因,易错PCR扩增体系(50μl)为10×TaKaRa Taq Buffer,dNTPs M ixture,引物各0.2μmol/L,模板DNA 200ng,Taq DNA聚合酶2.5U,5mmol/l Mn2+,0.5U/μl的Taq DNA聚合酶2.5μl,7mmol/l的Mg2+,PCR反应条件:95℃5min,94℃1min,55℃1min,72℃2min,35个循环,72℃10min,加20μl Cloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,将pPICZαA质粒用EcoRⅠ、NotⅠ酶切线性化,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,涂布于固体LB平板(100μg/ml Amp)抗性筛选阳性转化子,所有转化子构成突变体文库;
4.重组质粒的电击转化
将步骤三所得阳性转化子混合转入20ml氨苄抗性LB培养基中37℃培养过夜,使用质粒提取试剂盒提取全部质粒,使用NotⅠ将所质粒线性化,使用胶回收试剂盒回收纯化酶切产物,取20μl的线性化DNA加入100μl毕赤酵母GS115感受态细胞混匀立即转入预冷的0.2cm电击杯中,冰浴静置5min,用吸水纸擦干电击杯外壁的水分,迅速进行电击转化,电转条件:电压1.5kv,电容25μF,电阻200Ω,电击结束后立即加入1ml冰浴的50%1M山梨醇,50%YPD到电击杯中,然后转至1.5ml无菌离心管中,28℃,80r/min培养1h复苏;
5.酵母转化子筛选
取100μl步骤四所得的复苏菌液涂布到筛选培养基上,培养120h后,观察水解圈比野生型对照大的转化子,挑出,进行复筛;
6.将复筛通过的阳性克隆,送生物公司测序,测序结果见表3;
7.酶的生产
将复选通过的菌株在摇瓶中培养,进行甘油分批及补料培养时种子培养基使用MGY培养基,进行葡萄糖分批及补料培养时种子培养基使用YND培养基,种子培养方法如下:
a.一级种子培养:从-80℃中取出冻存种子,按1:50接入到50ml MGY培养基或者YND培养基中,30℃、200r/min培养至对数生长期;
b.二级种子培养:菌株按1:8比例,将一级种子接入到300ml MGY培养基或者Y ND培养基中,30℃、200r/min培养至对数生长期;
野生菌株和突变菌株的分批及补料培养方法如下:
a.发酵罐灭菌之前校正溶解氧电极和pH电极;
b.待BSM发酵基础培养基灭菌完成并冷却至30℃之后,在火焰保护下按照4.5ml/l发酵培养基的量加入PTM1微量元素母液;
c.取样,用离线pH计测定发酵罐内培养基的pH值,并与在线的pH计读数做比较,计算偏差;
d.逐步加入25%氨水,至发酵罐内培养基pH值接近5.0,30℃、200r/min搅拌过夜;
e.溶解氧100%校正:接种之前,通气量调节至1vvm,搅拌转速调节至200r/min,罐压调节至0.02MPa,稳定30min后将此状态设定为溶解氧100%的状态;
f.接种:将种子培养基在火焰保护下全部接入发酵罐中,培养温度30℃开始分批培养;
g.转速及通气量调节:保持发酵过程中溶解氧始终大于30%,发酵培养过程中可以通过在200-1000r/min范围内改变转速来调节溶解氧,也可以在3-6l/min范围内调节通气量,当搅拌及通气量达到最大值时可以适量向进气中混合通入纯氧来满足溶解氧要求;
h.pH调节:发酵过程中设定pH误差为5.0±0.1,通过发酵罐联动的pH调节蠕动泵自动调节;
i.每隔4h测定OD600或菌体湿重;
补料控制:当发酵过程中溶解氧突然开始急剧上升,视为发酵培养基中的碳源已经被消耗完,开始缓慢补入相应的碳源补料培养基以维持菌株继续生长,8000rpm离心5min收集上清;
8.酶的纯化
将步骤七得到的上清液,使用截流量为100kDa的膜包,在蠕动泵作用下降发酵液浓缩至20ml左右,将浓缩后的发酵液经12000rpm离心15min,将其转移至截留量为30kDa的透析袋中,随后置于合适pH的缓冲液中过夜透析,将透析后的发酵液12000rp m离心15min,使用阴离子交换树脂对目标酶蛋白进行纯化;
9.进行GOD酶活力检测和SDS-PAGE分析;
10.蛋白质浓度测定
配制一组浓度分别为0.10mg/ml,0.08mg/ml,0.06mg/ml,0.04mg/ml,0.02mg/ml,0mg/ml的牛血清蛋白(BSA)溶液,取1ml Bradford染液与100μl不同浓度BSA溶液反应30min,在595nm处测定这组溶液的吸光度值,得到蛋白质浓度对吸光度值的一条标准曲线,将纯化蛋白稀释至适宜浓度,按照上述方法测定吸光度值,根据标准曲线即可得到蛋白浓度;
野生型和突变型聚半乳糖醛酸酶在生产水平上没有明显差异,纯化酶的回收率约为46.1-52.7%,纯化后突变酶的纯度与分子量通过SDS-PAGE分析估计,突变酶分子量与野生型大小相当约42KD,野生型及突变体聚半乳糖醛酸酶三维图见图2。
表3突变体的测序结果
突变体编号 | 原始核酸序列 | 突变后核酸序列 | 原始氨基酸序列 | 突变后氨基酸序列 |
突变体308-309 | TGCCCT | TGGCGT | CP | WR |
实验一:酶活的测定方法
取1%果胶溶液0.4ml于试管中,加入pH 5的0.04mol/l Na2HPO4 0.02mol/l柠檬酸缓冲液1.0ml,45℃水浴平衡5min,加入0.1ml适当稀释的酶液,45℃反应30min(空白对照以煮沸失活的酶液代替),加入3.0ml DNS溶液终止反应,沸水浴5min,冷却,定容至15ml,于分光光度计540nm波长下测吸光度,得到反应体系中半乳糖醛酸的量,酶活力单位:1ml酶液在45℃、pH 5.0的条件下,每1min分解底物产生1μg半乳糖醛酸的酶量定义为1单位聚半乳糖醛酸酶(PG)酶活,以U/ml表示,酶活力计算公式:
U=(C实验-C对照)×N×1000×(V总/V酶)/t
式中,N为酶液稀释倍数,V总为反应总体积,V酶为酶液体积,t为反应时间(min)
原理是根据半乳糖醛酸能将3,5-二硝基水杨酸分子中的硝基还原成橙黄色的3-胺基5-硝基水杨酸,溶液橙黄色强度与半乳糖醛酸量成正比。
实验结果见表4。
表4酶活测定结果
名称 | 酶活(U/mg) | 提高比率 |
野生型 | 640.7±2.37 | - |
突变体308-309 | 697.2±3.65 | 8.82% |
Claims (2)
1.高活性的聚半乳糖醛酸酶,其特征在于,所述酶氨基酸序列(polygalacturonase,XP_011073814.1)在第308位、第309位上发生突变,由原来的CP突变为WR,构建方法包括以下步骤:
1)从NCBI上获得聚半乳糖醛酸酶的序列(polygalacturonase,XP_011073814.1),交生物公司合成,设计PCR引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTC AACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGA CGTACATTGA-3',PCR反应结束后,加20μlCloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,采用EcoRⅠ、NotⅠ酶切pPICZαA质粒,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,挑取阳性克隆,送生物公司测序,得到含有野生型序列质粒的基因工程菌;
2)质粒DNA的提取
将上述步骤含有野生型序列质粒的基因工程菌扩大培养,抽提质粒;
3)易错PCR扩增与突变文库的构建
以步骤二获得的质粒DNA为模板,用EcoRⅠ、NotⅠ酶切使质粒线性化,用引物序列引物F:5'-AGAGAGGCTGAAGCTGAATTCACCACCATTCAACAACGCACA-3',R:5'-TGTTCTAGAAAGCTGGCGGCCGCTAATGCCAAGAAAGACGTACATTGA-3',进行易错PCR扩增基因,易错PCR扩增体系(50μl)为10×TaKaRa Taq Buffer,dNTPs M ixture,引物各0.2μmol/L,模板DNA 200ng,Taq DNA聚合酶2.5U,5mmol/l Mn2+,0.5U/μl的Taq DNA聚合酶2.5μl,7mmol/l的Mg2+,PCR反应条件:95℃5min,94℃1min,55℃1min,72℃2min,35个循环,72℃10min,加20μl Cloning Enhancer到PCR体系中,37℃孵育15min,80℃孵育15min,将pPICZαA质粒用EcoRⅠ、NotⅠ酶切线性化,酶切产物经过0.75%琼脂糖凝胶电泳后回收,溶于灭菌双蒸水中,将孵育后的PCR产物与纯化的线性载体混合均匀,加入In-Fusion酶,50℃反应15min,转化大肠杆菌DH5α,涂布于固体LB平板(100μg/ml Amp)抗性筛选阳性转化子,所有转化子构成突变体文库;
4)重组质粒的电击转化
将步骤三所得阳性转化子混合转入20ml氨苄抗性LB培养基中37℃培养过夜,使用质粒提取试剂盒提取全部质粒,使用NotⅠ将所质粒线性化,使用胶回收试剂盒回收纯化酶切产物,取20μl的线性化DNA加入100μl毕赤酵母GS115感受态细胞混匀立即转入预冷的0.2cm电击杯中,冰浴静置5min,用吸水纸擦干电击杯外壁的水分,迅速进行电击转化,电转条件:电压1.5kv,电容25μF,电阻200Ω,电击结束后立即加入1ml冰浴的50%1M山梨醇,50%YPD到电击杯中,然后转至1.5ml无菌离心管中,28℃,80r/min培养1h复苏;
5)酵母转化子筛选
取100μl步骤四所得的复苏菌液涂布到显色筛选培养基上28℃倒置培养,观察转化子生长状况,将透明圈比野生型对照大的转化子,挑出,进行复筛;
6)将复筛通过的阳性克隆,送生物公司测序,测序结果见表3;
7)酶的生产;
8)酶的纯化,使用阴离子交换树脂纯化蛋白;
9)进行GOD酶活力检测和SDS-PAGE分析;
10)采用Bradford测定蛋白质浓度。
2.根据权利要求1所述的高活性的聚半乳糖醛酸酶的制备和应用,其特征在于,所述酶用于化工、食品、医药领域。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010333827.XA CN111500553A (zh) | 2020-04-24 | 2020-04-24 | 高活性的聚半乳糖醛酸酶的制备和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010333827.XA CN111500553A (zh) | 2020-04-24 | 2020-04-24 | 高活性的聚半乳糖醛酸酶的制备和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111500553A true CN111500553A (zh) | 2020-08-07 |
Family
ID=71867911
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010333827.XA Pending CN111500553A (zh) | 2020-04-24 | 2020-04-24 | 高活性的聚半乳糖醛酸酶的制备和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111500553A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111926001A (zh) * | 2020-10-15 | 2020-11-13 | 中国农业科学院北京畜牧兽医研究所 | 具有高热稳定性的多聚半乳糖醛酸酶突变体t316c/g344c及其基因和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103289975A (zh) * | 2013-05-26 | 2013-09-11 | 中国农业科学院饲料研究所 | 一种突变的聚半乳糖醛酸酶8fnA及其编码基因和应用 |
CN105838728A (zh) * | 2016-05-13 | 2016-08-10 | 中国农业科学院生物技术研究所 | 聚半乳糖醛酸酶优化基因及其表达载体和应用 |
-
2020
- 2020-04-24 CN CN202010333827.XA patent/CN111500553A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103289975A (zh) * | 2013-05-26 | 2013-09-11 | 中国农业科学院饲料研究所 | 一种突变的聚半乳糖醛酸酶8fnA及其编码基因和应用 |
CN105838728A (zh) * | 2016-05-13 | 2016-08-10 | 中国农业科学院生物技术研究所 | 聚半乳糖醛酸酶优化基因及其表达载体和应用 |
Non-Patent Citations (1)
Title |
---|
齐凤坤等: "番茄芝麻斑病原菌产生的细胞壁降解酶种类及其活性变化", 《植物保护》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111926001A (zh) * | 2020-10-15 | 2020-11-13 | 中国农业科学院北京畜牧兽医研究所 | 具有高热稳定性的多聚半乳糖醛酸酶突变体t316c/g344c及其基因和应用 |
CN111926001B (zh) * | 2020-10-15 | 2021-01-15 | 中国农业科学院北京畜牧兽医研究所 | 具有高热稳定性的多聚半乳糖醛酸酶突变体t316c/g344c及其基因和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11680255B2 (en) | Glucoamylase TLGA15 and gene and application thereof | |
CN107760700B (zh) | β-甘露聚糖酶基因、重组表达载体、菌株、β-甘露聚糖酶及其制备方法及应用 | |
CN110713996B (zh) | 一种海藻糖酶及其载体与应用 | |
US20210388336A1 (en) | Mutant of Nitrile Hydratase Derived from Caldalkalibacillus thermarum | |
CN107142225A (zh) | 一种强化表达Streptomyces sp. FA1来源木聚糖酶的毕氏酵母重组菌 | |
CN110643620B (zh) | 一种高活性茯苓纤维素内切酶基因及其蛋白和重组载体 | |
CN111363731A (zh) | 高活性的葡萄糖氧化酶的制备和应用 | |
CN111500553A (zh) | 高活性的聚半乳糖醛酸酶的制备和应用 | |
CN113874498A (zh) | 一种热稳定性葡萄糖氧化酶 | |
CN107384989A (zh) | 一种分支酶及其在抗性糊精制备中的应用 | |
CN113493799B (zh) | 一株高产酸性乳糖酶的黑曲霉菌株 | |
JP2022542737A (ja) | β-1,6-グルカナーゼを用いたゲンチオオリゴ糖の製造方法及びその使用 | |
CN116200279A (zh) | 一种里氏木霉重组菌株、其制备方法及其应用 | |
Schiraldi et al. | Innovative fermentation strategies for the production of extremophilic enzymes | |
CN116200323A (zh) | 一株产蛋白质谷氨酰胺酶的菌株及其应用 | |
CN110592121B (zh) | 一种高表达的纤维素内切酶基因及其重组载体和蛋白 | |
CN110577959B (zh) | 一种纤维素外切酶基因及其蛋白和重组载体 | |
CN107849592A (zh) | 使用耐热性芽孢杆菌属细菌发酵来生产乳酸或其盐的方法 | |
CN107916268B (zh) | 聚半乳糖醛酸裂解酶基因、重组表达载体、菌株、聚半乳糖醛酸裂解酶及其制备方法 | |
CN110804620A (zh) | 一种麦芽糖生产用重组异淀粉酶高效表达及应用 | |
CN114891641B (zh) | 一种产淀粉酶的菌株 | |
CN118147036B (zh) | 一种可稳定表达目标产物的枯草芽孢杆菌及其应用 | |
CN105331597A (zh) | 一种高纯度琼胶酶的制备工艺 | |
CN108410841B (zh) | 一种嗜热杜邦菌α-淀粉酶的高效制备及其应用 | |
CN116904468A (zh) | 一种磷酸盐响应的启动子及其在d-乳酸生产中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200807 |
|
WD01 | Invention patent application deemed withdrawn after publication |