CN105505846A - 表面展示谷氨酸脱氢酶的重组芽孢及其构建方法与应用 - Google Patents
表面展示谷氨酸脱氢酶的重组芽孢及其构建方法与应用 Download PDFInfo
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- CN105505846A CN105505846A CN201610008737.7A CN201610008737A CN105505846A CN 105505846 A CN105505846 A CN 105505846A CN 201610008737 A CN201610008737 A CN 201610008737A CN 105505846 A CN105505846 A CN 105505846A
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- Prior art keywords
- glutamate dehydrogenase
- cotg
- gene
- spore
- seqidno
- Prior art date
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound 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)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01003—Glutamate dehydrogenase (NAD(P)+)(1.4.1.3)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01004—Glutamate dehydrogenase (NADP+) (1.4.1.4)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/60—Vectors containing traps for, e.g. exons, promoters
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种表面展示谷氨酸脱氢酶的重组芽孢,该重组芽孢融合表达枯草芽孢杆菌的锚定蛋白CotG与谷氨酸脱氢酶GDH2,所述的锚定蛋白CotG,其核苷酸序列如SEQ?ID?NO:1所示;所述的谷氨酸脱氢酶GDH2,其核苷酸序列如SEQ?ID?NO:2所示。本发明还提供了该重组芽孢在制备L-2-氨基丁酸中的应用,重组芽孢上锚定的谷氨酸脱氢酶蛋白发挥全细胞催化剂功能,以α-酮丁酸为底物,催化生产L-2-氨基丁酸,发酵液中L-2-氨基丁酸的浓度可达38.72g/L,转化率为95.6%。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一株表面展示谷氨酸脱氢酶的重组芽孢及其构建方法与应用。
背景技术
L-2-氨基丁酸是一种抑制人体神经信息传递的非天然氨基酸,具有加强葡萄糖磷酸酯酶的活性,促进脑细胞代谢的作用。同时,L-2-氨基丁酸也是一种重要的化工原料和很多药物合成的手性中间体,已被广泛用于药物合成,例如,它是抗癫痫药物左乙拉西坦和布瓦西坦的关键生产原料,也是抑菌抗结核药乙胺丁醇盐酸盐的关键手性前体。因此,大量生产L-2-氨基丁酸可以降低这些疾病治疗的成本,同时提高其高效性和安全性。根据文献报道,经过定点突变K92V和T195S的新型谷氨酸脱氢酶能在辅因子NADPH的存在下作用于产L-2-氨基丁酸的代谢过程。
芽孢表面展示技术是新近发展起来的一门基因操作技术。芽孢是枯草芽孢杆菌在营养缺乏等逆境条件下,营养细胞分化形成的休眠体。由于芽孢的特殊结构,使其能忍耐一些极端环境如高温、高压、化学药物等。实现芽孢表面展示技术的关键是需要一种高丰富度、表面结合能力强的芽孢衣壳蛋白。外层的芽胞衣壳主要由疏水的衣壳蛋白构成,具有抗酶解、抗药物功能。目前已报道被成功应用的芽孢衣壳蛋白如CotB、CotC、CotG和CotX,都是来源于外层。从现有的文献报道来看,CotB和CotC常用于锚定抗原制备疫苗,而CotG则锚定酶蛋白发挥全细胞催化剂功能。
发明内容
本发明要解决的技术问题是,提供一株表面展示谷氨酸脱氢酶的重组芽孢。
本发明还要解决的技术问题是,提供上述表面展示谷氨酸脱氢酶的重组芽孢的构建方法。
本发明最后要解决的技术问题是,提供上述表面展示谷氨酸脱氢酶的重组芽孢在产L-2-氨基丁酸中的应用。
为解决上述技术问题,本发明采用如下技术方案:
表面展示谷氨酸脱氢酶的重组芽孢,其特征在于,该重组芽孢导入了表达枯草芽孢杆菌的锚定蛋白CotG基因与谷氨酸脱氢酶GDH2基因,
所述的锚定蛋白CotG基因的核苷酸序列如SEQIDNO:1所示;所述的谷氨酸脱氢酶GDH2基因的核苷酸序列如SEQIDNO:2所示。
其中,锚定蛋白CotG基因的N端为CotG基因的启动子,所述的CotG基因的启动子,其核苷酸序列如SEQIDNO:3所示。
其中,锚定蛋白CotG基因与谷氨酸脱氢酶GDH2基因之间的连接区域的氨基酸序列如SEQIDNO:4所示。
上述表面展示谷氨酸脱氢酶的重组芽孢的构建方法,包括如下步骤:
(1)将SEQIDNO:3、SEQIDNO:1、SEQIDNO:4、SEQIDNO:2所示的核苷酸序列依次连接,得到整合片段CotG-gdhA;
(2)将整合片段CotG-gdhA克隆到pEB03质粒的多克隆位点处,得到重组质粒pEB03-CotG-gdh2;
(3)将重组质粒pEB03-CotG-gdhA转化至枯草芽孢杆菌WB600,得到重组枯草芽孢杆菌pEB03-CotG-GDH2;
(4)将步骤(3)得到的重组枯草芽孢杆菌在GYS培养基中培养,使其产生芽孢,然后收集菌体,用溶菌酶裂解,得到表面展示谷氨酸脱氢酶的重组芽孢。
步骤(2)中,所述的pEB03质粒,其核苷酸序列如SEQIDNO:5所示
步骤(4)中,将重组枯草芽孢杆菌在GYS培养基中,37℃培养24h,然后离心收集菌体,菌体用100mM的pH8.0磷酸盐缓冲悬浮,用50mg/L的溶菌酶在37℃裂解1h,然后12000rpm离心30min,沉淀用含有1mol/LNaCl、1mol/LKCl和的0.1mol/L、pH8.0磷酸盐缓充液冲洗,即可得纯化的表面展示谷氨酸脱氢酶的重组芽孢。
具体的重组枯草芽孢杆菌的构建方法如下:
(1)以大肠杆菌谷氨酸棒杆菌的基因组为模板,F-GDH(BamHⅠ):CGCGGATCCATGGATCAGACATATTCTCTGGAGT;R-GDH(XhoⅠ):CCGCTCGAGTAAATCACACCCTGCGCCAGC为引物,扩增得到谷氨酸脱氢酶,该基因片段全长1344bp,编码蛋白包含447个氨基酸,通过基因突变,将第92位氨基K酸突变为V,第195为氨基酸T突变为S,得到gdhA片段;
(2)以Bacillussubtilis168基因组为模板,cotG-F:GTCGACGGTATCGATAAGCTTAGTGTCCCTAGCTCCGAG,cotG-R:TGAACCCCCACCTCCTTTGTATTTCTTTTTGACTACCCAG为引物,扩增得到锚定蛋白CotG基因及启动子片段;
(3)将谷氨酸脱氢酶GDH2的基因片段连接到锚定蛋白CotG基因的C端,得到整合片段CotG-gdhA,将CotG-gdhA片段克隆到pEB03质粒上,得到重组质粒pEB03-CotG-gdhA;
(4)将重组质粒pEB03-CotG-gdhA转化枯草芽孢杆菌WB600,得到重组芽孢杆菌pEB03-CotG-GDH2。
(5)芽孢的纯化:将含有重组质粒的B.subtilisWB600在37℃条件下,用GYS培养基((NH4)2SO42g/L、酵母膏2g/L、K2HPO40.5g/L、葡萄糖1g/L、MgSO40.41g/L、CaCl2*H2O0.08g/L、MnSO4*5H2O0.07g/L)培养24h,然后离心收集菌体。菌体用100mM的PH8.0磷酸盐缓冲悬浮,用0.5%的溶菌酶在37℃裂解1h,然后12000rpm离心30min,随后用1MNaCl,1MKCl和磷酸酸盐缓冲冲洗,即可得纯化的芽孢细胞,
上述表面展示谷氨酸脱氢酶的重组芽孢的构建方法构建得到的表面展示谷氨酸脱氢酶的重组芽孢在制备L-2-氨基丁酸中的应用。
催化体系如下:取1g纯化后重组芽孢加入100ml催化反应液(0.1mol/LpH8.0PBS,0.2mol/LL-2-酮丁酸,0.2mol/LNH4Cl,0.2×10-2mol/LNADPH)
催化条件如下:温度37℃,260rpm,时间12h。离心留取上清。
有益效果:
本发明在枯草芽孢杆菌WB600中表面展示谷氨酸脱氢酶,构建并筛选得到一株重组芽孢杆菌,此重组芽孢上锚定的谷氨酸脱氢酶蛋白发挥全细胞催化剂功能,以α-酮丁酸为底物,催化生产L-2-氨基丁酸,发酵液中L-2-氨基丁酸的浓度可达38.72g/L,转化率为95.6%。枯草芽孢杆菌是GRAS菌种之一,因此它产生的芽孢是无毒的,同时操作简单,稳定性好,安全性高;并且L-2-氨基丁酸的产量较高,底物转化率高,发酵工艺简单高效,没有副产物,易于分离,且易于工业化大生产,具有良好的产业化应用前景。
附图说明
图1谷氨酸脱氢酶基因的PCR图。
图2谷氨酸脱氢酶基因与T载体连接后提取质粒与酶切验证图。
图3谷氨酸脱氢酶基因与pET-28a载体连接后提取质粒与酶切验证图。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:谷氨酸脱氢酶基因的克隆与表达。
(1)谷氨酸脱氢酶基因片段扩增:
扩增谷氨酸脱氢酶引物如下:
F-GDH(BamHⅠ):CGCGGATCCATGGATCAGACATATTCTCTGGAGT
R-GDH(XhoⅠ):CCGCTCGAGTAAATCACACCCTGCGCCAGC
以谷氨酸棒杆菌基因组大肠杆菌DH5α基因组为模板,扩增的目的片段全长1344bp,该基因编码蛋白包含447个氨基酸。图为PCR之后所得到的产物,再进行切胶回收目的片段,如图1所示。
(2)目的片段与T-载体连接转化并进行双酶切(BamHⅠ和EcoRⅠ)验证:
PCR获得的谷氨酸脱氢酶基因片段与T-载体连接,转化大肠杆菌,以质粒小提试剂盒提取质粒,双酶切鉴定并进行琼脂糖凝胶电泳,双酶切电泳图显示有两条带,通过酶切位点分析,证实片段成功插入到载体(图2)。
(3)测序正确后,酶切下片段与载体pET-28a连接转化提质粒并进行酶切验证:
含目的基因的T载与载体pET-28a分别经BamHⅠ和XhoⅠ双酶切,胶回收的目的基因片段与线性化的载体连接,转化成功后提质粒进行酶切验证连接成功(图3)。
(4)选出连接成功的一管样品pET-28a-GDH送金唯智公司定点突变为将第92位氨基K酸突变为V,第195为氨基酸T突变为S,得到pET-28a-GDH2质粒。
(5)酶活检测:
蛋白表达:
酶切鉴定正确后,将pET28a-GDH2重组质粒转化至表达宿主E.coliRosetta(DE3)感受态细胞,涂布于LB/(Kan+cl)抗性平板培养过夜。从平板中挑取单菌落于5mlLB中37℃200rpm培养过夜(由于Rosetta具有Cl抗性,pET-28a具有kan抗性,为保证质粒不丢失,液体培养基中均加入千分之一的Cl和Kan抗性)。按照1%的量转接入200mlLB液体中,同时加入抗生素,37℃200rpm培养至OD=0.8左右加入0.4mMIPTG,30℃200rpm诱导10h,集菌。
粗酶液的获得:
用100mmol/LPH8.0的Tris-HCl缓冲将菌泥洗两遍,具体操作为:用缓冲将菌泥重悬,7000rpm5min,重复一遍。用10ml缓冲重悬,超声(超3秒,停5秒,60次),4℃离心15min,取上清,即获得的粗酶液。
酶纯化:
A、将树脂中的20%的乙醇从纯化管下方滴出;
B、用10ml超纯水过柱子;
C、用10ml500mM咪唑过柱子(目的是洗去上一个人未洗下来的蛋白);
D、10mlTris-HCl缓冲过柱子;
E、重复D;
F、加粗酶液、摇晃、重悬,将柱子在冰上放置30min左右后弃酶液;
G、10ml20mM咪唑洗涤,洗3-4次,在最后快洗完的时候接一管测蛋白浓度;
H、2ml500mM咪唑洗脱蛋白,用5ml离心管接住;
I、用10ml500mM咪唑继续洗去剩余蛋白,洗2次;
J、用10ml20%的乙醇保存。
蛋白鉴定:
取上清液20μL按比例加入2×SDS-PAGE上样缓冲液,重悬菌液,煮沸5~10min,进行SDS-PAGE检测。重组蛋白样品通过SDS-PAGE分析,约在51KD处出现显著的特异性条带。
蛋白质浓度测定采用Bradford法,以牛血清蛋白为标准样。取3.5ml考马斯亮蓝+0.1ml酶(稀释液),测OD595nm的吸光值,并根据标曲计算蛋白浓度。蛋白浓度为5.93mg/ml。
谷氨酸脱氢酶(GDH2)酶活测定:
2×10-2mol/Lα-酮丁酸,0.2mol/LNH4Cl,0.2×10-3mol/LNADPH,0.1mol/LPH8.0的Trisbuffer,及适量酶液混合后于室温下反应,用酶标仪在340nm测定反应开始1min内的光吸收变化值。NAD(P)H的摩尔消光系数以6.22计。
酶单位定义:每分钟产生1μmolNADPH变化所需的酶量为1个酶活动单位,以NADPHμmol/min表示。
粗酶液测得GDH2比酶活为0.511U/mg。经纯化后,其比酶活为20.03U/mg。
实施例2:pEB03-CotG-gdh2质粒的构建。
菌株:Bacillussubtilis168、B.subtilisWB600。
质粒:PET-28a-GDH2,抗性kanamycin;
pEB03:抗性Spectinomycin(终浓度100ug/ml,用水配成100mg/mL的储备液,按千分之一的添加量)
酶切位点:HindⅢ、BamHⅠ。
以Bacillussubtilis168基因组为模板,cotG-F:
GTCGACGGTATCGATAAGCTTAGTGTCCCTAGCTCCGAG、cotG-R:
TGAACCCCCACCTCCTTTGTATTTCTTTTTGACTACCCAG为引物,用PCR方法从枯草芽孢杆菌W168基因组中扩增出锚定蛋白CotG基因及启动子片段,同时双酶切出GDH2基因片段,并将外源蛋白GDH2片段连接于CotG基因片段的C端,并插入穿梭质粒PEB03中,构建整合型表达载体PEB03-CotG-gdhA。在CotG基因与GDH2片段之间设有linker(Gly-Gly-Gly-Gly-Ser)。
实施例3:枯草芽孢杆菌感受态制备和转化。
试剂配制:
SP盐:0.2%(NH4)2SO4,1.4%K2HPO4,0.6%KH2PO4,0.02%MgSO4·7H2O,0.1%柠檬酸钠。
CAYE(100×):2%Casaminoacid,10%酵母膏。
SPI培养基:SP盐溶液加入1%体积浓度为50%葡萄糖溶液,1%体积100×CAYE溶液。
SPII培养基:SPI培养基加入1%体积50mmol/LCaCl2溶液,1%体积250mmol/LMgCl2溶液。
SP-ASaltsSolution(500ml):(NH4)2SO42g、K2HPO4·3H2O14g、1.2%KH2PO46g、TrisodiumCitrateDihydrate1g,121℃灭菌20min。
SP-BSaltsSolution(500ml):MgSO4·7H2O0.2g,121℃灭菌20min。
100×CAYESolution(100ml):Casaminoacid2g、YeastExtract10g;121℃灭菌20min。
SPIMedium(20mL):SP-ASaltsSolution9.8mL、SP-BSaltsSolution9.8mL、(1%V)Glucose(50%w/v,115℃灭菌20min)200μL、(1%V)100×CAYE200μL。
SPIIMedium(6mL):SPIMedium:5.88mL、(1%V)50mMCaCl260μL、(1%V)250mMMgCl260μL。
100×EGTASolution:10mmol/LEGTA溶液,溶解时需加少量NaOH至pH8.0。
(1)准备新鲜的枯草芽孢杆菌(WB600)单克隆平板,取一满环枯草芽孢杆菌甘油菌划LB平板,37℃培养箱培养12h。
(2)转化前一天晚间挑单菌落至3mlLB培养基中,37℃,250r/min培养过夜。
(3)第二天上午取160μl培养液转接至8mlSPI培养基中,37℃,250r/min培养至对数生长末期(168约4-5h)。
(4)取0.2ml生长至对数期末的培养液至2mlSPII培养基中,37℃,100r/min培养90分钟。
(5)在上述SPII培养基的菌体中加入20μl10mmol/LEGTA,再于37℃,100r/min培养10分钟。
(6)将上述处理后的菌液分装成0.5ml每管,各加入5μlDNA(DNA量不能过高,不超过5μg),再于37℃,250r/min培养90分钟,取菌液涂布筛选平板。
实施例4:芽孢的纯化。
将含有重组质粒的B.subtilisWB600在37℃条件下,用GYS培养基((NH4)2SO42g/L、酵母膏2g/L、K2HPO40.5g/L、葡萄糖1g/L、MgSO40.41g/L、CaCl2*H2O0.08g/L、MnSO4*5H2O0.07g/L)培养24h,然后离心收集菌体。菌体用100mM的PH8.0磷酸盐缓冲悬浮,用0.5%的溶菌酶在37℃裂解1h,然后12000rpm离心30min,随后用1MNaCl,1MKCl和磷酸酸盐缓冲冲洗,即可得纯化的芽孢细胞,用磷酸盐缓冲悬浮放4℃保存备用。
表面展示GDH2重组菌在温度37℃,pH8.0时酶活最高,酶活单位定义为测定条件下每分钟生成1μmolL-2-氨基丁酸所需要的酶量,达到87.15U/g。这说明通过CotG的N端絮凝功能区,以非共价键的方式将GDH2展示于枯草芽孢杆菌WB600细胞表面。
实施例5:催化反应。
催化体系:取1g纯化后重组芽孢加入100ml催化反应液(0.1MpH8.0PBS,0.2mol/Lα-酮丁酸,0.2mol/LNH4Cl,0.2×10-2mol/LNADPH)
催化条件:温度37℃,260rpm,时间12h。离心留取上清。
HPLC检测条件:
流动相的制备:流动相A40MmNa2HP04用NaOH溶液调PH至7.8,流动相B为乙腈:甲醇:水(45:45:10,V/V/V);
L-2-氨基丁酸测定:OPA衍生后,进样测定L-2-氨基丁酸含量。
色谱柱采用安捷伦ZORBAXEclipse—AAA柱(4.6×150mm),流速为1mL/min,检测波长338nm,柱温40℃。
HPLC检测L-2-氨基丁酸的产量,反应液中L-2-氨基丁酸的含量为19.6g/L,转化率达到95%。
实施例6:催化反应。
选择改造后的基因工程菌重组芽孢发酵,进行催化生产氨基丁酸,转化反应器中加入各物质至终浓度如下:20g/L的重组芽孢,2mMNADPH,0.2MNH4Cl,加入无菌水至1L,控制温度37℃,搅拌转数260r/min,分批补料加入α-酮丁酸,控制反应体系pH在8.0左右。发酵24h反应结束。离心取上清液,过滤后,HPLC检测,发酵液中L-2-氨基丁酸产量达到38.72g/L,转化率为95.6%,且产物易于分离。
Claims (7)
1.表面展示谷氨酸脱氢酶的重组芽孢,其特征在于,该重组芽孢导入了表达枯草芽孢杆菌的锚定蛋白CotG基因与谷氨酸脱氢酶GDH2基因,
所述的锚定蛋白CotG基因的核苷酸序列如SEQIDNO:1所示;所述的谷氨酸脱氢酶GDH2基因的核苷酸序列如SEQIDNO:2所示。
2.根据权利要求1所述的表面展示谷氨酸脱氢酶的重组芽孢,其特征在于,锚定蛋白CotG基因的N端为CotG基因的启动子,所述的CotG基因的启动子,其核苷酸序列如SEQIDNO:3所示。
3.根据权利要求1所述的表面展示谷氨酸脱氢酶的重组芽孢,其特征在于,锚定蛋白CotG基因与谷氨酸脱氢酶GDH2基因之间的连接区域的核苷酸序列如SEQIDNO:4所示。
4.权利要求1表面展示谷氨酸脱氢酶的重组芽孢的构建方法,其特征在于,包括如下步骤:
(1)将SEQIDNO:3、SEQIDNO:1、SEQIDNO:4、SEQIDNO:2所示的核苷酸序列依次连接,得到整合片段CotG-gdhA;
(2)将整合片段CotG-gdhA克隆到pEB03质粒的多克隆位点处,得到重组质粒pEB03-CotG-gdhA;
(3)将重组质粒pEB03-CotG-gdh2转化至枯草芽孢杆菌WB600,得到重组枯草芽孢杆菌;
(4)将步骤(3)得到的重组枯草芽孢杆菌在GYS培养基中培养,使其产生芽孢,然后收集菌体,用溶菌酶裂解,得到表面展示谷氨酸脱氢酶的重组芽孢。
5.根据权利要求3所述表面展示谷氨酸脱氢酶的重组芽孢的构建方法,其特征在于,步骤(2)中,所述的pEB03质粒,其核苷酸序列如SEQIDNO:5所示。
6.根据权利要求2所述表面展示谷氨酸脱氢酶的重组芽孢的构建方法,其特征在于,步骤(4)中,将重组枯草芽孢杆菌在GYS培养基中,37℃培养24h,然后离心收集菌体,菌体用100mM的pH8.0磷酸盐缓冲悬浮,用50mg/L的溶菌酶在37℃裂解1h,然后12000rpm离心30min,沉淀用含有1mol/LNaCl、1mol/LKCl和的0.1mol/L、pH8.0磷酸盐缓充液冲洗,即可得纯化的表面展示谷氨酸脱氢酶的重组芽孢。
7.权利要求1~3任一项所述表面展示谷氨酸脱氢酶的重组芽孢在制备L-2-氨基丁酸中的应用。
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