CN110452845A - 一种产蔗糖磷酸化酶的大肠杆菌 - Google Patents
一种产蔗糖磷酸化酶的大肠杆菌 Download PDFInfo
- Publication number
- CN110452845A CN110452845A CN201910754470.XA CN201910754470A CN110452845A CN 110452845 A CN110452845 A CN 110452845A CN 201910754470 A CN201910754470 A CN 201910754470A CN 110452845 A CN110452845 A CN 110452845A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- spl0224
- sucrose phosphorylase
- application
- coli
- 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
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 44
- 108020000005 Sucrose phosphorylase Proteins 0.000 title claims abstract description 42
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 11
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 8
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 238000009472 formulation Methods 0.000 claims description 5
- 239000000411 inducer Substances 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- BJRNKVDFDLYUGJ-ZIQFBCGOSA-N alpha-Arbutin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-ZIQFBCGOSA-N 0.000 claims description 4
- 229940033280 alpha-arbutin Drugs 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000002537 cosmetic Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 18
- 238000002703 mutagenesis Methods 0.000 abstract description 6
- 231100000350 mutagenesis Toxicity 0.000 abstract description 6
- 230000003834 intracellular effect Effects 0.000 abstract description 5
- 238000010276 construction Methods 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract description 2
- 229930006000 Sucrose Natural products 0.000 description 13
- 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 13
- 229960004793 sucrose Drugs 0.000 description 13
- 239000005720 sucrose Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 240000000220 Panda oleosa Species 0.000 description 7
- 235000016496 Panda oleosa Nutrition 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 108010073135 Phosphorylases Proteins 0.000 description 3
- 102000009097 Phosphorylases Human genes 0.000 description 3
- 108010019589 Staphylococcus aureus glutamic acid-specific endopeptidase Proteins 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000013681 dietary sucrose Nutrition 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 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 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000589779 Pelomonas saccharophila Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012675 alcoholic extract Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种产蔗糖磷酸化酶的大肠杆菌,属于微生物技术领域。本发明将SEQ ID NO.1所示的核苷酸序列,以pET‑28a为载体构建重组质粒pET‑28a‑SPase,转化到大肠杆菌BL21(DE3)中,构建得到重组大肠杆菌,发酵产重组蔗糖磷酸化酶,并通过紫外诱变法对构建好的重组菌进行诱变,筛选出高产蔗糖磷酸化酶的大肠杆菌菌株,其发酵胞内破壁上清酶活为819.16U/mL,比酶活为206.91U/mg,其稳定的产酶性可以为进一步的理论研究和实际生产应用打下基础,实践应用意义重大。
Description
技术领域
本发明涉及一种产蔗糖磷酸化酶的大肠杆菌,属于微生物技术领域。
背景技术
蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,以下简称SPase)主要能够催化两种类型反应:一种是以磷酸化的葡萄糖当作供体转移到不同的物质,如以D-果糖为受体合成蔗糖;另外的一种催化方式是把蔗糖磷酸化酶分解蔗糖得到的葡萄糖基转移至不同类型的受体,如无机磷酸,含酚羟基、醇羟基及羧基的物质,催化合成多种糖苷。
蔗糖磷酸化酶主要分布在细菌中。根据文献报道该酶主要存在于肠膜明串珠菌Leuconostoc mesenteroides、变异链球菌Stococcus mutans、嗜糖假单胞菌Pseudomonassaccharophila、长双歧杆菌Bifidobacterium longum、青春双歧杆菌Bifidobacteriumadolescentis等微生物中。Leuconostoc mescnteroides可以合成蔗糖磷酸化酶[GoedlC.,Schwarz A.,Minain A.,et al.Recombinant sucrose phosphorylase fromLeuconostoc mesenteroides:Characterization,kinetic studies oftransglucosylation,and application of immobilised enzyme for production of d-glucose 1-phosphate[J].Journal of Biotechnol,2007,129(1):77-86.]。
目前,蔗糖磷酸化酶主要分布在细菌微生物中,植物细胞中有少量分布,该酶主要通过生物发酵获得,野生型菌株中的复杂代谢调控机制使得蔗糖磷酸化酶的产量较低,仅通过野生型菌株发酵生产蔗糖磷酸化酶,难以满足工业应用的要求。目前,在大肠杆菌、枯草芽孢杆菌中异源表达蔗糖磷酸化酶均有文献报道,但存在表达量不高,酶活力不高等问题。
因此,提供一种产蔗糖磷酸化酶的大肠杆菌,对于蔗糖磷酸化酶的工业生产具有重要的应用价值。
发明内容
本发明的第一个目的是提供一株大肠杆菌(Escherichia coli),分类命名为Escherichia coliSPL0224,已于2019年5月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2019389,保藏地址为中国武汉,武汉大学。
本发明的第二个目的是提供含有上述大肠杆菌SPL0224的微生物制剂。
在本发明的一种实施方式中,所述微生物制剂中含有活菌数不低于106CFU/g或106CFU/mL的大肠杆菌SPL0224细胞。
在本发明的一种实施方式中,所述大肠杆菌细胞包括大肠杆菌SPL0224干菌体或湿菌体。
本发明的第三个目的是提供一种生产蔗糖磷酸化酶的方法,是利用上述大肠杆菌SPL0224进行发酵。
在本发明的一种实施方式中,以TB培养基为发酵培养基。
在本发明的一种实施方式中,将大肠杆菌SPL0224单菌落接种于LB液体培养基中,于35-39℃,200-220r/min培养8-12h后,按1-5%接种量接入到TB液体培养基中,于35-39℃,200-220r/min培养至菌密度OD600达到0.5-0.7后,加入IPTG诱导剂于23-27℃,200-220r/min诱导20-30h后收集菌液,离心收集菌体,重悬后破碎菌体,离心收集上清,即为蔗糖磷酸化酶粗酶液。
在本发明的一种实施方式中,将大肠杆菌SPL0224单菌落接种于含Kana的LB液体培养基中,于37℃,200r/min过夜培养后,按1%接种量接入到含Kana的TB液体培养基中,于37℃,200r/min培养至菌密度OD600达到0.6后,加入IPTG诱导剂于25℃,200r/min诱导24h后收集菌液。将菌液于4℃,7000r/min的低温冷冻离心机中离心15min,收集菌体。菌体用50mmol/L K2HPO4/KH2PO4缓冲液(pH 6.5)洗两次后收集菌体。将收集好的湿菌体加入50mmol/L K2HPO4/KH2PO4缓冲液(pH 6.5)缓冲液制成菌悬液,放在冰上并固定好,然后用超声波破碎菌体。超声波破碎仪工作时间:2s,间歇时间:4s,总时间:30min。将破碎后的液体于4℃,7000r/min的低温冷冻离心机下离心30min收集上清即为蔗糖磷酸化酶粗酶液。
本发明的第四个目的是提供上述大肠杆菌SPL0224在生产α-熊果苷或含α-熊果苷的产品中的应用,是以蔗糖和氢醌为底物,以大肠杆菌SPL0224为生物催化剂。
本发明的第五个目的是提供上述大肠杆菌SPL0224在生产1-磷酸葡萄糖或含1-磷酸葡萄糖的产品中的应用。
本发明的第六个目的是提供上述大肠杆菌SPL0224在生产D-果糖或含D-果糖的产品中的应用。
本发明的第七个目的是提供上述大肠杆菌SPL0224在食品、化妆品或制药领域中的应用。
本发明的有益效果
本发明将SEQ ID NO.1所示的核苷酸序列,以pET-28a为载体构建重组质粒pET-28a-SPase,转化到大肠杆菌BL21(DE3)中,构建得到重组大肠杆菌,发酵产重组蔗糖磷酸化酶,并通过紫外诱变法对构建好的重组菌进行诱变,筛选出高产蔗糖磷酸化酶的大肠杆菌菌株,其发酵胞内破壁上清酶活为819.16U/mL,比酶活为206.91U/mg,其稳定的产酶性可以为进一步的理论研究和实际生产应用打下基础,实践应用意义重大。
生物材料保藏
一株大肠杆菌(Escherichia coli),分类命名为Escherichia coli SPL022,已于2019年5月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2019389,保藏地址为中国武汉,武汉大学。
附图说明
图1为重组菌Escherichia coli SPL0224摇瓶发酵胞内破壁上清SDS-PAGE电泳图,第1泳道为marker,第2泳道为空载菌摇瓶发酵胞内破壁上清,第3、4泳道为重组菌摇瓶发酵胞内破壁上清,可以清楚的看到图中55KDa处有明显的条带与蔗糖磷酸化酶一致,表明可以获得蔗糖磷酸化酶。
具体实施方式
实施例1Escherichia coli SPL02基因工程菌的构建
(1)基因的优化:来源于Leuconostoc mesenteroides ATCC 12291的Sucrosephosphorylase(GenBank Accession NO.D90314)进行分析优化,获得SEQ ID NO.1所示基因序列,并将两个酶切位点NcoI、XhoI加到经过优化后的蔗糖磷酸化酶两端,经过合成得到SPase基因。
(2)基因工程菌的构建:将合成的SPase基因和pET-28a载体用限制性内切酶NcoI和XhoI双酶切,酶切后产物用Solution I连接,然后将重组载体pET-28a-SPase转入大肠杆菌BL21(DE3)中表达。
实施例2重组蔗糖磷酸化酶的制备
将重组菌株单菌落接种于含Kana的LB液体培养基中,于37℃,200r/min过夜培养后,按1%接种量接入到含Kana的TB液体培养基中,于37℃,200r/min培养至菌密度OD600达到0.6后,加入IPTG诱导剂于25℃,200r/min诱导24h后收集菌液。将菌液于4℃,7000r/min的低温冷冻离心机中离心15min,收集菌体。菌体用50mmol/L K2HPO4/KH2PO4缓冲液(pH6.5)洗两次后收集菌体。将收集好的湿菌体加入50mmol/L K2HPO4/KH2PO4缓冲液(pH 6.5)缓冲液制成菌悬液,放在冰上并固定好,然后用超声波破碎菌体。超声波破碎仪工作时间:2s,间歇时间:4s,总时间:30min。将破碎后的液体于4℃,7000r/min的低温冷冻离心机下离心30min收集上清即为蔗糖磷酸化酶粗酶液。
SDS-PAGE检测:吸取10μL粗酶液样品用SDS-PAGE检测(12%分离胶,5%浓缩胶),55KDa处有明显的条带与蔗糖磷酸化酶一致,表明可以获得蔗糖磷酸化酶。
实施例3重组蔗糖磷酸化酶的酶活测定
在磷酸缓冲液中,蔗糖磷酸化酶能够催化蔗糖和无机磷酸生成葡萄糖-1-磷酸和D-果糖,可以先进行蔗糖水解反应,然后用DNS检测生成的D-果糖的含量,从而测定蔗糖磷酸化酶的活力[Choi H.C.,Seo D.H.,Jung J.H.,et al.Developmemt of new assay forsucrosephosphorylase and its application to the characterization ofBifidobacterium longumSJ32sucrosephosphorylase[J].Food Science andBiotechnology,2011,20(2):513-518.]。
酶活测定方法参照[吴敬,吴丹,朱洁,等.一种表达L.mesenteroides来源的蔗糖磷酸化酶重组枯草芽孢杆菌.中国发明专利申请,申请号:201710637427.6,公开号:CN107236696A]。测定步骤包括:5%蔗糖溶液500μL,蔗糖磷酸化酶粗酶液50μL,50mmol/L磷酸盐缓冲液(pH 6.5)450μL,30℃准确反应10min后,加入1.5mL DNS煮沸15min后迅速置于冷水中冷却,540nm处测定吸光值,以空载为对照。
将每分钟水解蔗糖生成1μmol的果糖所需酶量定义为蔗糖磷酸化酶的一个酶活力单位(U)。
酶活计算方法:
其中,A:吸光值、b:截距、n:稀释倍数、M:果糖分子质量、k:斜率。
蛋白含量测定:Brandford法测定所制备酶液中蛋白质的含量。
酶活测定结果显示,酶活为504.19U/mL,比酶活为145.19U/mg。
实施例4将构建好的Escherichia coli SPL02基因工程菌进行紫外诱变
⑴制作菌悬液:挑取Escherichia coli SPL02单克隆菌株接种于含50mL LB液体培养基(Kana浓度100μg/mL)的250mL装量三角瓶中,37℃震荡培养12~18h,将活化的菌体菌液取5m L倒入离心管中,3500r/min离心10min,弃去上清液,吸入5m L无菌生理盐水冲洗两三次,加入15mL生理盐水吹打均匀制成菌悬液。
⑵紫外线处理:打开紫外灯,预热20min。取2mL菌悬液均匀的涂布于无菌培养基中,制作6份,置于紫外灯下,距灯管28.5cm处,先连盖在紫外灯下灭菌l min,分别在紫外灯下照射15、30、45、60、75、90s和105s,照射后37℃避光培养24h。一般认为紫外致死率在80%时正突变率最高。
⑶初筛:经过紫外诱变后得到大量突变大肠杆菌菌株,挑选菌落大的突变大肠杆菌分别接种斜面培养基及液体培养基中进行培养筛选。37℃、120r/min条件下震荡培养8~10h后测定其600nm处吸光值,选取吸光值大的菌株进行复筛。
⑷复筛:将初筛获得的优良菌株接入含有50mL LB培养基(Kana浓度100μg/mL)中,37℃、200r/min条件下震荡培养10h后按1%接种量接入到含Kana的TB液体培养基中,于37℃,200r/min培养至菌密度OD600达到0.6后,加入IPTG诱导剂于25℃,200r/min诱导24h后收集菌液制备蔗糖磷酸化酶粗酶液。检测其蔗糖磷酸化酶活性,筛选出蔗糖磷酸化酶活力较高的优良大肠杆菌菌株,并经过3~5代传代培养,最后获得了蔗糖磷酸化酶酶活力有较大提高且遗传稳定性较好的突变株,将其命名为Escherichia coli SPL0224,该菌株于2019年5月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2019389,保藏地址为中国武汉,武汉大学。
采用实施例2~3相同策略制备重组蔗糖磷酸化酶并测定其酶活,区别在于重组菌为Escherichia coli SPL0224基因工程菌,粗酶液样品用SDS-PAGE检测(12%分离胶,5%浓缩胶),如图1所示,可以清楚的看到图中55KDa处有明显的条带与蔗糖磷酸化酶一致,表明可以获得蔗糖磷酸化酶。酶活测定结果显示,酶活为819.16U/mL,比酶活为206.91U/mg。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种产蔗糖磷酸化酶的大肠杆菌
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1473
<212> DNA
<213> 人工序列
<400> 1
atggaaatcc agaacaaagc tatgctgatc acctacgctg actctctggg taaaaacctg 60
aaagacgttc accaggttct gaaagaagac atcggtgacg ctatcggtgg tgttcacctg 120
ctgccgttct tcccgtctac cggtgaccgt ggtttcgctc cggctgacta cacccgtgtt 180
gacgctgctt tcggtgactg ggctgacgtt gaagctctgg gtgaagaata ctacctgatg 240
ttcgacttca tgatcaacca catctctcgt gaatctgtta tgtaccagga cttcaaaaaa 300
aaccacgacg actctaaata caaagacttc ttcatccgtt gggaaaaatt ctgggctaaa 360
gctggtgaaa accgtccgac ccaggctgac gttgacctga tctacaaacg taaagacaaa 420
gctccgaccc aggaaatcac cttcgacgac ggtactaccg aaaacctgtg gaacaccttc 480
ggtgaagaac agatcgacat cgacgttaac tctgctatcg ctaaagaatt tatcaaaacc 540
accctggaag acatggttaa acacggtgct aacctgatcc gtctggacgc tttcgcttac 600
gctgttaaaa aagttgacac caacgacttc ttcgttgaac cggaaatctg ggacaccctg 660
aacgaagttc gtgaaatcct gaccccgctg aaagctgaaa tcctgccgga aatccacgaa 720
cactactcta tcccgaaaaa aatcaacgac cacggttact tcacctacga cttcgctctg 780
ccgatgacca ccctgtacac cctgtactct ggtaaaacca accagctcgc taaatggctg 840
aaaatgtctc cgatgaaaca gttcaccacc ctggacaccc acgacggtat cggtgttgtt 900
gacgctcgtg acatcctgac cgacgacgaa atcgactacg cttctgaaca gctctacaaa 960
gttggtgcta acgttaaaaa aacctactct tctgcttctt acaacaacct ggacatctac 1020
cagatcaact ctacctacta ctctgctctg ggtaacgacg acgctgctta cctgctgtct 1080
cgtgttttcc aggttttcgc tccgggtatc ccgcagatat actacgttgg tctgctggct 1140
ggtgaaaacg acatcgctct gctggaatct accaaagaag gtcgtaacat caaccgtcac 1200
tactacaccc gtgaagaagt taaatctgaa gttaaacgtc cggttgttgc taacctgctg 1260
aaactgctgt cttggcgtaa cgaatctccg gctttcgacc tggctggttc tatcaccgtt 1320
gacaccccga ccgacaccac catcgttgtt acccgtcagg acgaaaacgg tcagaacaaa 1380
gctgttctga ccgctgacgc tgctaacaaa accttcgaaa tcgttgaaaa cggtcagacc 1440
gttatgtctt ctgacaacct gacccagaac taa 1473
Claims (10)
1.一株大肠杆菌(Escherichia coli)SPL0224,已于2019年5月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2019389,保藏地址为中国武汉,武汉大学。
2.含有权利要求1所述的大肠杆菌SPL0224的微生物制剂。
3.如权利要求2所述的微生物制剂,其特征在于,所述微生物制剂中含有活菌数不低于106CFU/g或106CFU/mL的大肠杆菌SPL0224细胞。
4.一种生产蔗糖磷酸化酶的方法,其特征在于,是利用权利要求1所述的大肠杆菌SPL0224进行发酵。
5.如权利要求4所述的方法,其特征在于,以TB培养基为发酵培养基。
6.如权利要求4所述的方法,其特征在于,将大肠杆菌SPL0224单菌落接种于LB液体培养基中,于35-39℃,200-220r/min培养8-12h后,按1-5%接种量接入到TB液体培养基中,于35-39℃,200-220r/min培养至菌密度OD600达到0.5-0.7后,加入IPTG诱导剂于23-27℃,200-220r/min诱导20-30h后收集菌液,离心收集菌体,重悬后破碎菌体,离心收集上清,即为蔗糖磷酸化酶粗酶液。
7.权利要求1所述的大肠杆菌SPL0224在生产α-熊果苷或含α-熊果苷的产品中的应用。
8.权利要求1所述的大肠杆菌SPL0224在生产1-磷酸葡萄糖或含1-磷酸葡萄糖的产品中的应用。
9.权利要求1所述的大肠杆菌SPL0224在生产D-果糖或含D-果糖的产品中的应用。
10.权利要求1所述的大肠杆菌SPL0224在食品、化妆品或制药领域中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910754470.XA CN110452845B (zh) | 2019-08-15 | 2019-08-15 | 一种产蔗糖磷酸化酶的大肠杆菌 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910754470.XA CN110452845B (zh) | 2019-08-15 | 2019-08-15 | 一种产蔗糖磷酸化酶的大肠杆菌 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110452845A true CN110452845A (zh) | 2019-11-15 |
CN110452845B CN110452845B (zh) | 2021-03-02 |
Family
ID=68486853
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910754470.XA Active CN110452845B (zh) | 2019-08-15 | 2019-08-15 | 一种产蔗糖磷酸化酶的大肠杆菌 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110452845B (zh) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110343654A (zh) * | 2019-08-15 | 2019-10-18 | 江南大学 | 一种产蔗糖磷酸化酶的基因工程菌 |
CN110656077A (zh) * | 2019-11-07 | 2020-01-07 | 江南大学 | 一种生产蔗糖磷酸化酶的方法及其应用 |
CN112300977A (zh) * | 2020-11-18 | 2021-02-02 | 江南大学 | 合成α-熊果苷的基因工程菌株及其构建方法与应用 |
CN112375724A (zh) * | 2020-11-18 | 2021-02-19 | 江南大学 | 高效合成α-熊果苷的基因工程菌及其构建方法与应用 |
CN113956999A (zh) * | 2021-08-06 | 2022-01-21 | 广西科技师范学院 | 一种产蔗糖磷酸化酶的贝莱斯芽孢杆菌及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6255072B1 (en) * | 1996-09-30 | 2001-07-03 | Smithkline Beecham Corporation | spsA polynucleotides |
EP2069519A2 (en) * | 2006-09-21 | 2009-06-17 | Technische Universität Graz | Method for producing 2-o-glyceryl-alpha-d-glucopyranoside |
CN102174454A (zh) * | 2011-01-17 | 2011-09-07 | 南京工业大学 | 一种表达重组蔗糖磷酸化酶的大肠杆菌工程菌 |
CN102925418A (zh) * | 2012-11-21 | 2013-02-13 | 南京工业大学 | 一种α-熊果苷生产过程中蔗糖磷酸化酶的回收方法 |
CN103025874A (zh) * | 2010-07-12 | 2013-04-03 | 根特大学 | 用于生产附加值生物产品的代谢改造的生物 |
-
2019
- 2019-08-15 CN CN201910754470.XA patent/CN110452845B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6255072B1 (en) * | 1996-09-30 | 2001-07-03 | Smithkline Beecham Corporation | spsA polynucleotides |
EP2069519A2 (en) * | 2006-09-21 | 2009-06-17 | Technische Universität Graz | Method for producing 2-o-glyceryl-alpha-d-glucopyranoside |
CN103025874A (zh) * | 2010-07-12 | 2013-04-03 | 根特大学 | 用于生产附加值生物产品的代谢改造的生物 |
CN102174454A (zh) * | 2011-01-17 | 2011-09-07 | 南京工业大学 | 一种表达重组蔗糖磷酸化酶的大肠杆菌工程菌 |
CN102925418A (zh) * | 2012-11-21 | 2013-02-13 | 南京工业大学 | 一种α-熊果苷生产过程中蔗糖磷酸化酶的回收方法 |
Non-Patent Citations (2)
Title |
---|
万月佳等: "重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷 ", 《生物工程学报》 * |
叶慧等: "蔗糖磷酸化酶在大肠杆菌中的表达及优化 ", 《食品科技》 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110343654A (zh) * | 2019-08-15 | 2019-10-18 | 江南大学 | 一种产蔗糖磷酸化酶的基因工程菌 |
CN110343654B (zh) * | 2019-08-15 | 2021-03-30 | 江南大学 | 一种产蔗糖磷酸化酶的基因工程菌 |
CN110656077A (zh) * | 2019-11-07 | 2020-01-07 | 江南大学 | 一种生产蔗糖磷酸化酶的方法及其应用 |
CN110656077B (zh) * | 2019-11-07 | 2021-10-08 | 江南大学 | 一种生产蔗糖磷酸化酶的方法及其应用 |
CN112300977A (zh) * | 2020-11-18 | 2021-02-02 | 江南大学 | 合成α-熊果苷的基因工程菌株及其构建方法与应用 |
CN112375724A (zh) * | 2020-11-18 | 2021-02-19 | 江南大学 | 高效合成α-熊果苷的基因工程菌及其构建方法与应用 |
CN112375724B (zh) * | 2020-11-18 | 2022-12-16 | 江南大学 | 高效合成α-熊果苷的基因工程菌及其构建方法与应用 |
CN112300977B (zh) * | 2020-11-18 | 2024-01-12 | 江南大学 | 合成α-熊果苷的基因工程菌株及其构建方法与应用 |
CN113956999A (zh) * | 2021-08-06 | 2022-01-21 | 广西科技师范学院 | 一种产蔗糖磷酸化酶的贝莱斯芽孢杆菌及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110452845B (zh) | 2021-03-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110452845A (zh) | 一种产蔗糖磷酸化酶的大肠杆菌 | |
Canale-Parola | Biology of the sugar-fermenting Sarcinae | |
CN105802897B (zh) | 一种d-阿洛酮糖-3-差向异构酶生产菌株及其应用 | |
Zhao et al. | Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001 | |
Chi et al. | Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu | |
CN109679864B (zh) | 一种产转糖基活性β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法 | |
Chellapandian et al. | Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ‘pulque’, a traditional Aztec alcoholic beverage | |
CN104046586B (zh) | 一株基因工程菌及其在生产(2r,3r)-2,3-丁二醇中的应用 | |
Li et al. | Newly isolated Bacillus gibsonii S-2 capable of using sugar beet pulp for alkaline pectinase production | |
CN109536564B (zh) | 特异性菌株及其应用 | |
CN104911135B (zh) | 一种海藻糖合酶生产菌株及其应用 | |
JP2003523203A (ja) | Klebsiella属の細菌単離体およびそれから単離されたイソマルツロース・シンターゼ遺伝子 | |
CN110343654A (zh) | 一种产蔗糖磷酸化酶的基因工程菌 | |
Mei et al. | Cold stress promoting a psychrotolerant bacterium Pseudomonas fragi P121 producing trehaloase | |
CN109576239A (zh) | 耐热磷酸化酶及其应用 | |
CN107151640A (zh) | 一种产乳糖酶的嗜冷杆菌菌株及使用该菌株制备低温乳糖酶的方法 | |
CN105154352B (zh) | 一种海洋微生物菌株Y112及其产α-环糊精葡萄糖基转移酶 | |
Santos et al. | Probiotic cell cultivation | |
SU1124889A3 (ru) | Способ получени производных @ -карбамилфенилглицина | |
CN109988778A (zh) | 一种蔗糖磷酸化酶基因及其应用 | |
CN102533607A (zh) | 一株产β-半乳糖苷酶的菌株及用该酶生产低聚半乳糖的方法 | |
CN109929863A (zh) | 一种利用全细胞转化生产异麦芽酮糖的方法 | |
CN102199643A (zh) | 胞二磷胆碱的制备方法 | |
Sakellaris et al. | Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production | |
CN112501049B (zh) | 产转糖基活性β-半乳糖苷酶的开菲尔乳杆菌及制备的β-半乳糖苷酶生产低聚半乳糖的方法 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20191115 Assignee: SHANGHAI HUAMAO PHARMACEUTICAL Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2023980050989 Denomination of invention: An Escherichia coli that produces sucrose phosphorylase Granted publication date: 20210302 License type: Common License Record date: 20231209 |
|
EE01 | Entry into force of recordation of patent licensing contract |