CN117247915A - An ω-amine transaminase, recombinant bacteria and their applications - Google Patents

An ω-amine transaminase, recombinant bacteria and their applications Download PDF

Info

Publication number
CN117247915A
CN117247915A CN202311030831.9A CN202311030831A CN117247915A CN 117247915 A CN117247915 A CN 117247915A CN 202311030831 A CN202311030831 A CN 202311030831A CN 117247915 A CN117247915 A CN 117247915A
Authority
CN
China
Prior art keywords
amine transaminase
reaction
amine
seq
transaminase
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
Application number
CN202311030831.9A
Other languages
Chinese (zh)
Inventor
金利群
易蒲红
郝玉华
刘梦丹
刘汉林
柳志强
薛亚平
郑裕国
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN202311030831.9A priority Critical patent/CN117247915A/en
Publication of CN117247915A publication Critical patent/CN117247915A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1096Transferases (2.) transferring nitrogenous groups (2.6)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00Preparation of nitrogen-containing organic compounds
    • C12P13/001Amines; Imines
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y206/00Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01Transaminases (2.6.1)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to omega-amine transaminase, recombinant bacteria and application thereof. The amino acid sequence of the omega-amine aminotransferase is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO.2 or SEQ ID NO. 3. The recombinant strain is obtained by transferring recombinant expression plasmid containing coding genes for omega-amine transaminase into host escherichia coli and screening. The invention clones and identifies a novel omega-amine transaminase gene from human pallidobacter Ochrobactrum anthropic ZJB-061 for the first time, and proves that the omega-amine transaminase can improve the problem of the unfavorable equilibrium constant of the ammonia transfer reaction, and the enzyme has great potential to convert byproducts such as keto acid and the like to promote the reaction equilibrium in the similar reactions of preparing L-glufosinate by the ammonia transfer reaction with 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butyric acid as a substrate.

Description

一种ω-胺转氨酶、重组菌及其应用An omega-amine transaminase, recombinant bacteria and their applications

技术领域Technical field

本发明属于生物工程技术领域,具体涉及一种ω-胺转氨酶、重组菌及其应用。The invention belongs to the field of bioengineering technology, and specifically relates to an ω-amine transaminase, recombinant bacteria and their applications.

背景技术Background technique

草铵膦(Phosphinothricin,PPT;化学名为2-氨基-4-[羟基(甲基)膦酰基]丁酸),是世界第二大转基因作物耐受除草剂,能通过抑制植物谷氨酰胺合成酶来发挥除草作用。草铵膦与草甘膦以及百草枯并称世界三大除草剂,目前百草枯因为剧毒已停产,草甘膦也因为长期使用而使植物产生抗性而导致市场逐渐萎缩,而草铵膦具有灭生性广、低毒且对环境友好等特点,具有良好的市场前景。Glufosinate (PPT; chemical name: 2-amino-4-[hydroxy(methyl)phosphono]butyric acid) is the world's second most tolerant herbicide in genetically modified crops and can inhibit plant glutamine synthesis. enzymes to exert herbicidal effect. Glufosinate-ammonium, glyphosate and paraquat are also known as the world's three major herbicides. Paraquat has been discontinued due to its high toxicity. Glyphosate has also caused plant resistance due to long-term use, resulting in a gradual shrinking of the market. Glufosinate-ammonium is one of the three major herbicides in the world. It has the characteristics of broad biocidal properties, low toxicity and environmental friendliness, and has good market prospects.

草铵膦有两种光学异构体,分别为L-草铵膦和D-草铵膦。但只有L-草铵膦具有植物毒性,其除草活性为普通市售外消旋DL-草铵膦的2倍,且在土壤中易分解,对人类和动物的毒性较小,除草谱广,对环境的破坏力小。如果草铵膦产品能以L型的纯光学异构体形式使用,可使草铵膦的使用量降低50%,这对于提高原子经济性、降低使用成本、减轻环境压力都具有重要意义。Glufosinate-ammonium has two optical isomers, namely L-glufosinate-ammonium and D-glufosinate-ammonium. However, only L-glufosinate is phytotoxic. Its herbicidal activity is twice that of ordinary commercially available racemic DL-glufosinate. It is easily decomposed in the soil, is less toxic to humans and animals, and has a broad herbicidal spectrum. Less damaging to the environment. If glufosinate-ammonium products can be used in the form of L-type pure optical isomers, the usage of glufosinate-ammonium can be reduced by 50%, which is of great significance for improving atom economy, reducing use costs, and reducing environmental pressure.

目前制备光学纯L-草铵膦的方法主要有三种:化学合成法、手性拆分法以及生物催化法。There are currently three main methods for preparing optically pure L-glufosinate: chemical synthesis, chiral resolution, and biocatalysis.

化学合成法从手性原料出发合成光学纯L-草铵膦,该方法工艺步骤多、收率低,所用不对称合成试剂大多比较昂贵,导致生产成本偏高,不利于大规模制备L-草铵膦。The chemical synthesis method starts from chiral raw materials to synthesize optically pure L-glufosinate. This method has many process steps and low yield. Most of the asymmetric synthesis reagents used are expensive, resulting in high production costs and is not conducive to the large-scale preparation of L-glufosinate. Ammonium phosphine.

手性拆分法是通过化学合成外消旋DL-草铵膦或其衍生物,再利用手性拆分试剂,进行D型和L型异构体的分离,从而制得光学纯的L-草铵膦。此工艺存在单次拆分收率低、工艺复杂的技术问题。The chiral resolution method is to chemically synthesize racemic DL-glufosinate or its derivatives, and then use chiral resolution reagents to separate the D- and L-isomers to obtain optically pure L- Glufosinate ammonium. This process has technical problems such as low single-split yield and complex process.

相比之下,生物催化法具有立体选择性严格、反应条件温和、收率高及产物易分离纯化等优点,是生产L-草铵膦的潜在优势方法。In contrast, the biocatalytic method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, and easy separation and purification of the product. It is a potentially advantageous method for producing L-glufosinate.

在研究草铵膦在土壤微生物体内的代谢途径时就已经发现,L-草铵膦在转氨酶的作用下,发生转氨反应被分解成一种2-羰基-4-[羟基(甲基)膦酰基]丁酸(简称PPO)。转氨反应是一个可逆反应,PPO可在转氨酶的催化下通过转氨反应生成L-草铵膦。但是该生成L-草铵膦的转氨反应的缺陷在于不利的平衡常数,目前研究多集中于副产物的移除和分解。丙氨酸作为典型的氨基供体,脱氨基产物为丙酮酸,不仅对酶有较强的的抑制能力,也是限制平衡的关键因素之一。目前,通常用来移除丙酮酸的酶有乳酸脱氢酶(LDH)、丙氨酸脱氢酶(AlaDH)、丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)以及偶联GDH或FDH等。When studying the metabolic pathway of glufosinate in soil microorganisms, it has been found that L-glufosinate undergoes a transamination reaction and is decomposed into a 2-carbonyl-4-[hydroxy(methyl)phosphonoyl group under the action of transaminase ]Butyric acid (referred to as PPO). The transamination reaction is a reversible reaction, and PPO can generate L-glufosinate through the transamination reaction under the catalysis of transaminase. However, the shortcoming of the transamination reaction to generate L-glufosinate is the unfavorable equilibrium constant. Current research focuses on the removal and decomposition of by-products. Alanine is a typical amino donor, and the deamination product is pyruvate. It not only has strong inhibitory ability to enzymes, but is also one of the key factors limiting balance. Currently, enzymes commonly used to remove pyruvate include lactate dehydrogenase (LDH), alanine dehydrogenase (AlaDH), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and coupled GDH or FDH et al.

发明内容Contents of the invention

在为解决上述技术问题,本发明提供了一种ω-胺转氨酶、重组菌及其应用。In order to solve the above technical problems, the present invention provides an ω-amine transaminase, recombinant bacteria and their applications.

本发明通过以下技术方案实现:The present invention is realized through the following technical solutions:

本发明的第一目的在于,提供一种ω-胺转氨酶,所述ω-胺转氨酶的氨基酸序列如SEQ ID NO.1所示。The first object of the present invention is to provide an ω-amine transaminase, the amino acid sequence of which is shown in SEQ ID NO. 1.

本发明的ω-胺转氨酶基因来源于人苍白杆菌Ochrobactrum anthropic ZJB-061(CN101063090A专利现有菌株,具体保藏单位为中国典型培养物保藏中心,地址为中国.武汉.武汉大学,邮编430072;保藏日期为2006年4月14日;保藏编号为CCTCC NO:M206039),将该菌在NCBI中进行比对后,选择了与其相近的已经全基因组测序的同源菌种Ochrobactrumanthropic(Genbank:CP022605.1)为参照,对其基因组中所有ω-胺转氨酶基因进行分析,从中筛选获得与其同源性最高的基因序列进行克隆引物设计,并发现克隆产物与Ochrobactrumanthropi (WP_011982390.1)来源的ω-转氨酶蛋白序列的相似性仅为69.52%,验证了其ω-胺转氨酶的功能与应用价值。The ω-amine transaminase gene of the present invention is derived from the existing strain of Ochrobactrum anthropic ZJB-061 (CN101063090A) patented. The specific depository unit is the China Typical Culture Collection Center, the address is Wuhan University, Wuhan, China, Postal Code 430072; the deposit date on April 14, 2006; the deposit number is CCTCC NO:M206039). After comparing the bacteria in NCBI, we selected the homologous strain Ochrobactrumanthropic (Genbank: CP022605.1) that is close to it and has been sequenced in its entire genome. As a reference, we analyzed all ω-amine transaminase genes in its genome, screened out the gene sequence with the highest homology to it, and designed cloning primers. We found that the cloned product was identical to the ω-aminase protein sequence derived from Ochrobactrumanthropi (WP_011982390.1). The similarity is only 69.52%, which verifies the function and application value of its ω-amine transaminase.

作为优选,所述ω-胺转氨酶编码基因的核苷酸序列如SEQ ID NO.2所示。Preferably, the nucleotide sequence of the ω-amine transaminase encoding gene is shown in SEQ ID NO. 2.

作为优选,所述ω-胺转氨酶编码基因的核苷酸序列如SEQ ID NO.3所示。Preferably, the nucleotide sequence of the ω-amine transaminase encoding gene is shown in SEQ ID NO. 3.

由于如SEQ ID NO.2所示的ω-胺转氨酶基因可溶性表达量较低,为提高可溶性表达效率,本发明根据大肠杆菌表达系统的偏好性,对ω-胺转氨酶基因原始序列进行了密码子优化,得到了如SEQ ID NO.3所示的序列。Since the soluble expression level of the ω-amine transaminase gene shown in SEQ ID NO. 2 is low, in order to improve the soluble expression efficiency, the present invention codon-coded the original sequence of the ω-amine transaminase gene according to the preference of the E. coli expression system. After optimization, the sequence shown in SEQ ID NO.3 was obtained.

本发明的第二目的在于,提供一种重组表达质粒,所述重组表达质粒包含有编码ω-胺转氨酶的编码基因,所述编码基因的核苷酸序列如SEQ ID NO.2或SEQ ID NO.3所示。The second object of the present invention is to provide a recombinant expression plasmid that contains a coding gene encoding ω-amine transaminase, and the nucleotide sequence of the coding gene is such as SEQ ID NO. 2 or SEQ ID NO. .3 shown.

作为优选,所述重组表达质粒是将编码基因插入pET-28a载体得到。Preferably, the recombinant expression plasmid is obtained by inserting the coding gene into the pET-28a vector.

本发明的第三目的在于,提供一种重组基因工程菌,包含有上述任一种重组表达质粒。The third object of the present invention is to provide a recombinant genetically engineered bacterium containing any of the above recombinant expression plasmids.

作为优选,所述重组工程菌是将重组表达质粒转入宿主大肠杆菌BL21(DE3)或大肠杆菌Top10得到。Preferably, the recombinant engineering bacterium is obtained by transforming the recombinant expression plasmid into the host E. coli BL21 (DE3) or E. coli Top10.

作为进一步优选,所述重组工程菌是将重组表达质粒转入宿主大肠杆菌BL21(DE3)得到。As a further preference, the recombinant engineering bacterium is obtained by transforming the recombinant expression plasmid into the host E. coli BL21 (DE3).

本发明的第四目的在于,提供一种生产ω-胺转氨酶的方法,所述生产方法为:将上述任一种重组基因工程菌进行发酵培养,将培养后的重组基因工程菌破壁后进行分离纯化,得到ω-胺转氨酶。The fourth object of the present invention is to provide a method for producing ω-amine transaminase. The production method is as follows: fermenting and culturing any of the above recombinant genetically engineered bacteria, and breaking the cultured recombinant genetically engineered bacteria. Separate and purify to obtain ω-amine transaminase.

本发明的第五目的在于,提出上述ω-胺转氨酶、重组表达质粒或者重组基因工程菌在转氨反应中进行应用。The fifth object of the present invention is to propose the use of the above-mentioned ω-amine transaminase, recombinant expression plasmid or recombinant genetically engineered bacteria in transamination reactions.

本发明中转氨反应是指催化氨基供体(氨基酸或胺)上的氨基转移到前手性的受体醛、酮或酮酸的反应。所述转氨反应具体可以用于动力学拆分制备或不对称合成制备非天然氨基酸、用于制备脂肪族和芳香族非天然氨基酸或者用于制备手性胺和手性氨基酸。The transamination reaction in the present invention refers to a reaction that catalyzes the transfer of the amino group on the amino donor (amino acid or amine) to the prochiral acceptor aldehyde, ketone or keto acid. The transamination reaction can be specifically used for kinetic resolution preparation or asymmetric synthesis to prepare unnatural amino acids, for preparing aliphatic and aromatic unnatural amino acids, or for preparing chiral amines and chiral amino acids.

作为进一步优选,本发明还提出上述ω-胺转氨酶、重组表达质粒或者重组基因工程菌在通过转氨反应合成L-草铵膦中应用。As a further preference, the present invention also proposes the application of the above-mentioned ω-amine transaminase, recombinant expression plasmid or recombinant genetically engineered bacteria in the synthesis of L-glufosinate through a transamination reaction.

ω-胺转氨酶能高效转化丙酮酸,可以用来催化丙酮酸来辅助生成L-丙氨酸,而L-丙氨酸又是以2-羰基-4-[羟基(甲基)膦酰基]丁酸为底物转氨反应制备L-草铵膦的常用氨基供体,经反应后生成丙酮酸,因此本发明的ω-胺转氨酶可以改善以2-羰基-4-[羟基(甲基)膦酰基]丁酸为底物转氨反应制备L-草铵膦的过程中,副产物丙酮酸对该转氨反应的正向抑制作用,缓解转氨反应的不利平衡常数问题。ω-Amine transaminase can efficiently convert pyruvate and can be used to catalyze pyruvate to assist in the production of L-alanine, and L-alanine is based on 2-carbonyl-4-[hydroxy(methyl)phosphono]butanol Acid is a commonly used amino donor for preparing L-glufosinate in the substrate transamination reaction, and pyruvate is generated after the reaction. Therefore, the ω-amine transaminase of the present invention can improve the conversion of 2-carbonyl-4-[hydroxy(methyl)phosphine In the process of preparing L-glufosinate through a transamination reaction using acyl]butyric acid as a substrate, the by-product pyruvate has a positive inhibitory effect on the transamination reaction and alleviates the problem of unfavorable equilibrium constants in the transamination reaction.

与现有技术相比,本发明具有以下有益效果:Compared with the prior art, the present invention has the following beneficial effects:

本发明首次从人苍白杆菌Ochrobactrum anthropic ZJB-061中克隆、鉴定了一种新型ω-胺转氨酶基因,经验证发现该ω-胺转氨酶可以改善转氨反应的不利平衡常数问题,在以2-羰基-4-[羟基(甲基)膦酰基]丁酸为底物转氨作用制备L-草铵膦等类似可逆转氨反应中该酶具有转化酮酸等副产物来推动反应平衡的巨大潜力。The present invention clones and identifies a new type of ω-amine transaminase gene from Ochrobactrum anthropic ZJB-061 for the first time. After verification, it is found that the ω-amine transaminase can improve the unfavorable equilibrium constant problem of the transamination reaction. -4-[Hydroxy(methyl)phosphono]butyric acid is used as the substrate for transamination to prepare L-glufosinate and other similar reversible ammonia reactions. This enzyme has great potential to convert keto acids and other by-products to promote reaction balance.

其余有益效果将在实施例中展开说明。The remaining beneficial effects will be explained in the examples.

序列描述sequence description

SEQ ID NO.1为ω-胺转氨酶ata-Oc3氨基酸序列;SEQ ID NO.1 is the amino acid sequence of ω-amine transaminase ata-Oc3;

SEQ ID NO.2为ω-胺转氨酶ata-Oc核苷酸序列;SEQ ID NO.2 is the nucleotide sequence of ω-amine transaminase ata-Oc;

SEQ ID NO.3为ω-胺转氨酶ata-Oc优化后的ω-胺转氨酶ata-Oc3的核苷酸序列;SEQ ID NO.3 is the nucleotide sequence of the optimized ω-amine transaminase ata-Oc3;

SEQ ID NO.4为Ochrobactrum anthropic ZJB-061中克隆基因正向引物ata-OcF;SEQ ID NO.4 is the forward primer ata-OcF of the cloned gene in Ochrobactrum anthropic ZJB-061;

SEQ ID NO.5为Ochrobactrum anthropic ZJB-061中克隆基因反向引物ata-OcR。SEQ ID NO.5 is the reverse primer ata-OcR of the cloned gene in Ochrobactrum anthropic ZJB-061.

附图说明Description of drawings

下面将就附图进行简单介绍:The following is a brief introduction to the attached drawings:

图1为ω-胺转氨酶催化反应示意图;Figure 1 is a schematic diagram of the ω-amine transaminase catalyzed reaction;

图2为ω-胺转氨酶的凝胶电泳图:泳道M为核酸分子量Marker,泳道1为ω-胺转氨酶pET28a-ata-Oc,泳道2为ω-胺转氨酶ata-Oc;Figure 2 shows the gel electrophoresis pattern of ω-amine transaminase: lane M is the nucleic acid molecular weight marker, lane 1 is ω-amine transaminase pET28a-ata-Oc, and lane 2 is ω-amine transaminase ata-Oc;

图3为ω-胺转氨酶的SDS-PAGE图:泳道M为蛋白质分子量Marker,泳道1ω-胺转氨酶破碎细胞上清液,泳道2为ω-胺转氨酶破碎细胞沉淀;Figure 3 is the SDS-PAGE image of ω-amine transaminase: lane M is the protein molecular weight marker, lane 1 is the supernatant of cells disrupted by ω-amine transaminase, and lane 2 is the pellet of cells disrupted by ω-amine transaminase;

图4为ω-胺转氨酶催化合成L-丙氨酸的反应示意图;Figure 4 is a schematic diagram of the reaction catalyzed by ω-amine transaminase to synthesize L-alanine;

图5为ω-胺转氨酶催化合成L-草铵膦的反应示意图。Figure 5 is a schematic diagram of the reaction catalyzed by ω-amine transaminase to synthesize L-glufosinate.

具体实施方式Detailed ways

下面结合说明书附图以及具体实施例对本发明做进一步描述。本领域普通技术人员在基于这些说明的情况下将能够实现本发明。此外,下述说明中涉及到的本发明的实施例通常仅是本发明一部分的实施例,而不是全部的实施例。因此,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。The present invention will be further described below with reference to the accompanying drawings and specific embodiments. A person of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention mentioned in the following description are generally only some embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

实施例1 :ω-胺转氨酶基因ata-Oc的获得Example 1: Obtainment of ω-amine transaminase gene ata-Oc

本发明的ω-胺转氨酶基因来源于人苍白杆菌Ochrobactrum anthropic ZJB-061,经16S rDNA测序后,基于NCBI中blastn 进行同源比对,发现其与Ochrobactrumanthropic序列一致性最高。并对已经全基因组测序的Ochrobactrumanthropic(Genbank:CP022605.1)中所有转氨酶进行分析,从中筛选获得与其同源性最高的ω-转氨酶基因序列进行克隆引物设计,并发现克隆产物与苍白杆菌属Ochrobactrum anthropi (WP_011982390.1)来源的ω-转氨酶蛋白序列(5GHF)的相似性为69.52%,并验证了其ω-转氨酶功能与应用价值。The ω-amine transaminase gene of the present invention is derived from Ochrobactrum anthropic ZJB-061. After 16S rDNA sequencing, homology alignment based on blastn in NCBI found that it has the highest sequence consistency with Ochrobactrum anthropic. All transaminase enzymes in Ochrobactrumanthropic (Genbank: CP022605.1), which has been sequenced in its entirety, were analyzed and the ω-aminase gene sequence with the highest homology was screened and cloned primers were designed. The cloned product was found to be closely related to Ochrobactrum anthropi. The similarity of the ω-aminase protein sequence (5GHF) derived from (WP_011982390.1) is 69.52%, and its ω-aminase function and application value are verified.

首先使用DNA快速提取试剂盒(FastDNA™ SPIN, MP)提取Ochrobactrumanthropic ZJB-061的基因组DNA,并根据ω-转氨酶基因序列设计特异性引物。First, a DNA fast extraction kit (FastDNA™ SPIN, MP) was used to extract the genomic DNA of Ochrobactrumanthropic ZJB-061, and specific primers were designed based on the ω-aminase gene sequence.

以Ochrobactrum anthropic ZJB-061的基因组DNA为模板,ata-OcF和ata-OcR为引物进行PCR后,得到1374 bp的产物片段,将产物进行测序后于NCBI库中比对,发现其与苍白杆菌属Ochrobactrum anthropi来源的ω-转氨酶蛋白序列5GHF的相似性为69.52%。After PCR was performed using the genomic DNA of Ochrobactrum anthropic ZJB-061 as the template and ata-OcF and ata-OcR as primers, a 1374 bp product fragment was obtained. The product was sequenced and compared in the NCBI library, and it was found that it was closely related to the genus Ochrobacterium The similarity of the ω-aminase protein sequence 5GHF derived from Ochrobactrum anthropi is 69.52%.

其中,设计引物ata-OcF (序列如SEQ ID NO.4所示)和ata-OcR (序列如SEQ IDNO.5所示),利用PCR向Ochrobactrum anthropic ZJB-061 ω-转氨酶的DNA编码框5’和3’两侧分别引入NcoI和XhoI限制性酶切位点,酶切位点前为保护碱基,酶切位点后为有效序列;Among them, the primers ata-OcF (sequence shown in SEQ ID NO. 4) and ata-OcR (sequence shown in SEQ ID NO. 5) were designed, and PCR was used to construct the DNA coding frame 5' of Ochrobactrum anthropic ZJB-061 ω-aminase. NcoI and XhoI restriction enzyme sites are introduced on both sides of and 3' respectively. There are protective bases before the restriction site and effective sequences after the restriction site;

ata-OcF:5'ATCCATGGATGACGAGACAGCCCAATTC3'ata-OcF:5'ATCCATGGATGACGAGACAGCCCAATTC3'

ata-OcR:5'TACTCGAGTTAAGAGCGACCGATTGCGGTC3'ata-OcR:5'TACTCGAGTTAAGAGCGACCGATTGCGGTC3'

以上述分离的Ochrobactrum anthropic ZJB-061基因组DNA为模板,采用上面设计的引物进行PCR扩增,得到ω-胺转氨酶基因ata-Oc。Using the above isolated Ochrobactrum anthropic ZJB-061 genomic DNA as a template, PCR amplification was performed using the primers designed above to obtain the ω-amine transaminase gene ata-Oc.

其中50 μL PCR反应体系构成为:25μL10×DNA Polymerase Buffer,2μL10 mMdNTP mixture (dATP、dCTP、dGTP和dTTP各2.5 mM),1μLDNA Polymerase,1μL10 μM ata-OcF,1μL10 μM ata-OcR,1μL模板DNA,19μLdd H2O。PCR的反应条件为:95 ℃,5 min预变性;95 ℃,30 s,变性;60 ℃,30 s,退火;72 ℃,1 min,延伸,第二步到第四步进行30个循环;72 ℃, 5 min,16 ℃,彻底延伸。The 50 μL PCR reaction system consists of: 25 μL 10×DNA Polymerase Buffer, 2 μL 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), 1 μL DNA Polymerase, 1 μL 10 μM ata-OcF, 1 μL 10 μM ata-OcR, 1 μL template DNA, 19 μL dd H 2 O. The reaction conditions of PCR were: 95°C, 5 min pre-denaturation; 95°C, 30 s, denaturation; 60°C, 30 s, annealing; 72°C, 1 min, extension, and 30 cycles from the second to the fourth step; 72°C, 5 min, 16°C, complete extension.

使用凝胶电泳检测PCR扩增产物,获得大小为1374 bp的DNA片段(详见图2),利用DNA回收纯化试剂盒(AxyPrep PCR Cleanup Kit)对PCR产物进行纯化回收,具体步骤参照纯化试剂盒说明书。PCR产物通过琼脂糖凝胶电泳验证大小以及进行胶回收纯化。具体步骤按照Trelief®DNA Gel Extraction Kit凝胶回收试剂盒说明书回收目的基因片段。Use gel electrophoresis to detect the PCR amplification product and obtain a DNA fragment with a size of 1374 bp (see Figure 2 for details). Use the DNA recovery and purification kit (AxyPrep PCR Cleanup Kit) to purify and recover the PCR product. For specific steps, refer to the purification kit. manual. PCR products were size verified by agarose gel electrophoresis and purified by gel recovery. For specific steps, follow the instructions of the Trelief® DNA Gel Extraction Kit to recover the target gene fragment.

实施例2 :含有ω-胺转氨酶基因ata-Oc的重组质粒的构建Example 2: Construction of recombinant plasmid containing ω-amine transaminase gene ata-Oc

本发明的含有ω-胺转氨酶基因ata-Oc的重组质粒是通过将ω-胺转氨酶基因ata-Oc与可选的质粒pET-28a (表达载体) 连接构建而成。回收的目的基因片段和pET-28a质粒在37 ℃下,利用NcoI和XhoI限制性内切酶(TaKaRa)对扩增片段进行双酶切处理;回收酶切片段并利用T4DNA连接酶(TaKaRa)并于16 ℃连接过夜,获得将连接产物。取10 μL连接产物加入感受态细胞DH5α中,置于冰上30 min,42 ℃热激60 s后立即放在冰上,放置5 min后,加入600 μL不含抗生素的LB培养基,37 ℃摇床培养1 h,将转化的菌液涂于LB (Amp)蓝白板上,37 ℃培养过夜。在平板上分别挑取多个菌落做PCR鉴定。The recombinant plasmid containing the ω-amine transaminase gene ata-Oc of the present invention is constructed by connecting the ω-amine transaminase gene ata-Oc with the optional plasmid pET-28a (expression vector). The recovered target gene fragment and pET-28a plasmid were double-digested using NcoI and XhoI restriction endonucleases (TaKaRa) at 37°C; the digested fragments were recovered and digested using T4 DNA ligase (TaKaRa). Ligation was performed overnight at 16°C to obtain the ligation product. Add 10 μL of the ligation product to the competent cells DH5α, place on ice for 30 min, heat shock at 42°C for 60 s and immediately place on ice. After leaving for 5 min, add 600 μL of LB medium without antibiotics, and incubate at 37°C. Incubate on a shaking table for 1 hour, spread the transformed bacterial solution on an LB (Amp) blue and white plate, and culture at 37°C overnight. Pick multiple colonies on the plate for PCR identification.

质粒提取:用Trelief® Plasmid Mini Kit质粒抽提试剂盒提取菌落PCR鉴定为阳性的克隆质粒,经核酸测序获得人苍白杆菌ω-胺转氨酶蛋白编码基因的核酸序列。该蛋白的氨基酸序列及核苷酸序列见SEQ ID NO.1和SEQ ID NO.2。Plasmid extraction: Use the Trelief® Plasmid Mini Kit plasmid extraction kit to extract the clone plasmids identified as positive by colony PCR, and obtain the nucleic acid sequence of the ω-amine transaminase protein encoding gene of Ochibacterium hominis through nucleic acid sequencing. The amino acid sequence and nucleotide sequence of this protein are shown in SEQ ID NO.1 and SEQ ID NO.2.

根据大肠杆菌蛋白表达的偏好性,对得到的基因序列做选择性改造,经密码子优化后的ω-胺转氨酶基因ata-Oc命名为ata-Oc3,其基因序列如SEQ ID NO.3 所示,优化后的序列由生物公司合成。According to the preference of E. coli protein expression, the obtained gene sequence was selectively modified. The codon-optimized ω-amine transaminase gene ata-Oc was named ata-Oc3, and its gene sequence is shown in SEQ ID NO.3 , the optimized sequence was synthesized by a biological company.

实施例3:含有ω-胺转氨酶基因ata-Oc3的重组基因工程菌株的构建与培养Example 3: Construction and cultivation of recombinant genetically engineered strains containing ω-amine transaminase gene ata-Oc3

将依照实施例2的方法获得的重组质粒pET28a-ata-Oc3转入大肠杆菌BL21(DE3)感受态细胞(北京擎科生物有限公司),均匀涂布在卡那霉素抗性平板上,37 ℃倒置培养16h,挑取单菌落验证为阳性克隆的为重组基因工程菌株E.coli BL21(DE3)/pET28a-ata-Oc3。挑取阳性单菌落接种至含有50μg/mL卡那霉素的10 mL LB液体培养基中,37 ℃,200rpm震荡培养12 h,获得种子液。将种子液按1%接种量转入100 mL新鲜培养基中,在37℃、180 rpm条件下震荡培养至OD600=0.4-0.6,加入0.1% 浓度为100 mM的IPTG进行诱导,在28℃,180 rpm条件下培养诱导表达12 h后收集菌体。The recombinant plasmid pET28a-ata-Oc3 obtained according to the method of Example 2 was transferred into E. coli BL21 (DE3) competent cells (Beijing Qingke Biotechnology Co., Ltd.), and evenly spread on the kanamycin-resistant plate, 37 Incubate at ℃ for 16 hours upside down, pick a single colony and verify that the positive clone is the recombinant genetically engineered strain E.coli BL21(DE3)/pET28a-ata-Oc3. Pick a positive single colony and inoculate it into 10 mL LB liquid medium containing 50 μg/mL kanamycin, and culture it at 37°C and 200 rpm for 12 h to obtain the seed liquid. Transfer the seed solution into 100 mL of fresh culture medium according to the inoculum amount of 1%, and culture with shaking at 37°C and 180 rpm until OD 600 = 0.4-0.6. Add 0.1% IPTG with a concentration of 100 mM for induction, and incubate at 28°C. , cultured at 180 rpm to induce expression for 12 h and then collected the cells.

实施例4:ω-胺转氨酶蛋白的表达和纯化Example 4: Expression and purification of omega-amine transaminase protein

将收集的菌体用质量分数为0.85%的生理盐水(NaCl)清洗一次,4℃、8000 rpm/min离心10 min,再次收集菌体。将收集到的菌体用含有0.1 mM PLP的20 mM PB磷酸盐缓冲液(pH为8.0)进行重悬,于低温冰浴中超声破碎(40 W,持续2 s,间歇4 s,连续破碎40min),将破碎后的细胞液12000 rpm离心10 min后,上清液即工程菌粗酶液。使用亲和镍柱(40×12.6 mm,Bio-Rad,USA)进行纯化,透析脱盐后,获得目的蛋白,即ω-胺转氨酶。该ω-胺转氨酶的氨基酸序列如SEQ ID NO.1所示,共457个氨基酸,其理论分子量为49.7 kDa。SDS-PAGE电泳图如图3所示。The collected bacterial cells were washed once with physiological saline (NaCl) with a mass fraction of 0.85%, centrifuged at 4°C and 8000 rpm/min for 10 min, and the bacterial cells were collected again. The collected bacterial cells were resuspended in 20 mM PB phosphate buffer (pH 8.0) containing 0.1 mM PLP, and ultrasonic disrupted in a low-temperature ice bath (40 W, lasting for 2 s, with an interval of 4 s, and continuously disrupted for 40 min. ), centrifuge the broken cell solution at 12,000 rpm for 10 minutes, and the supernatant is the crude enzyme solution of the engineering bacteria. An affinity nickel column (40×12.6 mm, Bio-Rad, USA) was used for purification, and after dialysis and desalting, the target protein, namely ω-amine transaminase, was obtained. The amino acid sequence of the ω-amine transaminase is shown in SEQ ID NO. 1, with a total of 457 amino acids, and its theoretical molecular weight is 49.7 kDa. The SDS-PAGE electrophoresis pattern is shown in Figure 3.

实施例5:ω-胺转氨酶ata-Oc3酶活、最适温度及最适pH测定Example 5: Determination of ω-amine transaminase ata-Oc3 enzyme activity, optimal temperature and optimal pH

测定ω-胺转氨酶酶活、比活力,具体测定方法如下:Determine the enzyme activity and specific activity of ω-amine transaminase. The specific measurement methods are as follows:

标准酶活检测体系(1 mL):1 mL的50 mM Tris-HCl 9.0中,含有20 mM丙酮酸、30mM 异丙胺、0.1 mM 磷酸吡哆醛(简称为PLP),35 ℃保温10 min,然后加入适量纯酶液,于50 ℃、600 rpm反应10 min。反应结束后立即取出反应液置于冰上,加入10μL浓度为6 M的HCl终止反应,反应液经离心(12000 rpm, 1 min),取上清液检测产物L-丙氨酸浓度。Standard enzyme activity detection system (1 mL): 1 mL of 50 mM Tris-HCl 9.0 contains 20 mM pyruvate, 30mM isopropylamine, and 0.1 mM pyridoxal phosphate (PLP), incubated at 35°C for 10 min, and then Add an appropriate amount of pure enzyme solution and react at 50°C and 600 rpm for 10 minutes. Immediately after the reaction, take out the reaction solution and place it on ice. Add 10 μL of HCl with a concentration of 6 M to terminate the reaction. The reaction solution is centrifuged (12000 rpm, 1 min), and the supernatant is taken to detect the concentration of product L-alanine.

酶活单位(U)定义:在酶活测定条件下,每分钟生成1 μmol L-丙氨酸所需的酶量。Enzyme activity unit (U) definition: The amount of enzyme required to generate 1 μmol L-alanine per minute under enzyme activity measurement conditions.

比活力定义:每毫克蛋白所具有的酶活单位数,记为U/mg。Specific activity definition: The number of enzyme activity units per milligram of protein, recorded as U/mg.

丙氨酸检测方法:采用配备荧光检测器的赛默飞U3000液相色谱仪,色谱柱Unitary® C18柱(4.6×250mm,Acchrom,China);流动相为甲醇:50 mM乙酸铵(pH为5.7),体积比为30:70;流速1.0 mL/min;检测波长Ex=350 nm、Em=460 nm;进样量10 μL;柱温35℃。L-丙氨酸、D-丙氨酸的保留时间分别为:13.5分钟,14.6分钟。Alanine detection method: Use a Thermo Fisher U3000 liquid chromatograph equipped with a fluorescence detector, a Unitary® C18 column (4.6×250mm, Acchrom, China); the mobile phase is methanol: 50 mM ammonium acetate (pH 5.7 ), the volume ratio is 30:70; the flow rate is 1.0 mL/min; the detection wavelength Ex=350 nm, Em=460 nm; the injection volume is 10 μL; the column temperature is 35°C. The retention times of L-alanine and D-alanine are: 13.5 minutes and 14.6 minutes respectively.

ω-胺转氨酶ata-Oc3的最适反应pH值分析Analysis of optimal reaction pH value of ω-amine transaminase ata-Oc3

本发明的ω-胺转氨酶ata-Oc3的最适反应pH在6.0-10范围内测定。检测的反应体系(1 mL)为:50 mM反应缓冲液中,含有20 mM丙酮酸、30 mM 异丙胺、0.1 mM PLP,35 ℃保温10 min,然后加入适量的纯酶液,于35 ℃、600 rpm反应10 min。反应结束后立即取出反应液置于冰上,加入10 μL浓度为6 M的HCl终止反应,反应液经离心后(12000 rpm,1 min),取上清液检测产物L-丙氨酸浓度。测定所使用的反应缓冲液为:磷酸钠缓冲液(pH6.0-8.0), Tris-HCl缓冲液(pH 8.0-9.0),甘氨酸-NaOH缓冲液(pH9.0-10.0),其中pH为8.0时为磷酸钠缓冲液,pH为9.0时为Tris-HCl缓冲液。测定结果如表1所示。The optimal reaction pH of the ω-amine transaminase ata-Oc3 of the present invention is measured in the range of 6.0-10. The reaction system (1 mL) tested is: 50 mM reaction buffer containing 20 mM pyruvate, 30 mM isopropylamine, and 0.1 mM PLP. Incubate at 35°C for 10 min. Then add an appropriate amount of pure enzyme solution and incubate at 35°C. React at 600 rpm for 10 minutes. Immediately after the reaction, the reaction solution was taken out and placed on ice. 10 μL of HCl with a concentration of 6 M was added to terminate the reaction. After the reaction solution was centrifuged (12000 rpm, 1 min), the supernatant was taken to detect the concentration of the product L-alanine. The reaction buffers used in the measurement are: sodium phosphate buffer (pH6.0-8.0), Tris-HCl buffer (pH 8.0-9.0), glycine-NaOH buffer (pH9.0-10.0), of which the pH is 8.0 When the pH is 9.0, it is sodium phosphate buffer, and when the pH is 9.0, it is Tris-HCl buffer. The measurement results are shown in Table 1.

表1、ω-胺转氨酶ata-Oc3最适反应pH测定Table 1. Determination of optimum reaction pH of ω-amine transaminase ata-Oc3

通过观察上表可知,ω-胺转氨酶在pH 8.5-9.0范围内均具有较高活性,其中pH为9.的Tris-HCl缓冲溶液中ω-胺转氨酶的相对酶活最高。By observing the above table, we can see that ω-amine transaminase has high activity in the pH range of 8.5-9.0. Among them, the relative enzyme activity of ω-amine transaminase in the Tris-HCl buffer solution with pH 9. is the highest.

ω-胺转氨酶ata-Oc3的最适反应温度分析Analysis of the optimal reaction temperature of ω-amine transaminase ata-Oc3

本发明的ω-胺转氨酶的最适反应温度在30-60 ℃范围内测定。检测的反应体系(1 mL)为:50 mM磷酸盐反应缓冲液中,含有20 mM丙酮酸、30 mM 异丙胺、0.1 mM PLP,恒温下保温10 min,然后加入适量的纯酶液,于不同温度下反应10min。反应结束后立即取出反应液置于冰上,加入10μL浓度为6 M的HCl终止反应,反应液经离心后(12000 rpm, 1 min),取上清液检测产物L-丙氨酸浓度。水浴反应温度分别为30 ℃、35 ℃、40 ℃、45 ℃、50 ℃、55 ℃、60 ℃。测定结果如表2所示。The optimal reaction temperature of the ω-amine transaminase of the present invention is measured in the range of 30-60°C. The reaction system (1 mL) tested is: 50 mM phosphate reaction buffer, containing 20 mM pyruvate, 30 mM isopropylamine, and 0.1 mM PLP. Incubate at constant temperature for 10 min. Then add an appropriate amount of pure enzyme solution and mix at different times. React at room temperature for 10 minutes. Immediately after the reaction, take out the reaction solution and place it on ice. Add 10 μL of HCl with a concentration of 6 M to terminate the reaction. After the reaction solution is centrifuged (12000 rpm, 1 min), take the supernatant to detect the concentration of the product L-alanine. The water bath reaction temperatures were 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃ respectively. The measurement results are shown in Table 2.

表2、ω-胺转氨酶ata-Oc3最适反应温度测定Table 2. Determination of optimal reaction temperature of ω-amine transaminase ata-Oc3

通过观察上表可知,ω-胺转氨酶在40-55 ℃范围内均具有较高活性,其中温度为50 ℃时ω-胺转氨酶的相对酶活最高。By observing the above table, we can see that ω-amine transaminase has high activity in the range of 40-55 ℃, and the relative enzyme activity of ω-amine transaminase is the highest at 50 ℃.

在标准酶活条件下,测定了ω-胺转氨酶ata-Oc3的比活力大小为9.74 U/mg,e.e.值大于99%。Under standard enzyme activity conditions, the specific activity of ω-amine transaminase ata-Oc3 was determined to be 9.74 U/mg, and the ee value was greater than 99%.

实施例6 :ω-胺转氨酶ata-Oc3的热稳定性分析Example 6: Thermal stability analysis of ω-amine transaminase ata-Oc3

将ω-胺转氨酶纯酶液用50 mM Tris-HCl ( pH为9.0)稀释后分装于2 mLep管中,然后置于不同温度下进行保温。温度设置为:35 ℃条件下分别保温4 h、6 h、10 h、12 h;45℃条件下分别保温2 h、4 h、6 h、8 h。在保温时间达到后立即取出置于冰上待用。在标准酶活检测条件下进行反应,以加入不保温的纯酶液为对照,计算相对酶活大小。结果分别如表3、表4所示。Dilute the pure enzyme solution of ω-amine transaminase with 50 mM Tris-HCl (pH 9.0), distribute it into 2 mLep tubes, and then incubate it at different temperatures. The temperature settings were: 4 h, 6 h, 10 h, and 12 h at 35 °C; 2 h, 4 h, 6 h, and 8 h at 45 °C. After the holding time is up, take it out immediately and place it on ice until use. Carry out the reaction under standard enzyme activity detection conditions, add pure enzyme solution without incubation as a control, and calculate the relative enzyme activity. The results are shown in Table 3 and Table 4 respectively.

表3、ω-胺转氨酶ata-Oc3在35 ℃条件下的热稳定性Table 3. Thermal stability of ω-amine transaminase ata-Oc3 at 35 ℃

表4、ω-胺转氨酶ata-Oc3在45 ℃条件下的热稳定性Table 4. Thermal stability of ω-amine transaminase ata-Oc3 at 45 ℃

通过观察上表可知,ω-胺转氨酶ata-Oc3的热稳定性良好。From the above table, it can be seen that ω-amine transaminase ata-Oc3 has good thermal stability.

实施例7 :ω-胺转氨酶ata-Oc3的底物耐受性分析Example 7: Substrate tolerance analysis of omega-amine transaminase ata-Oc3

测定ω-胺转氨酶对异丙胺的耐受性。在1 mL标准反应体系中:控制丙酮酸终浓度为20 mM,其他条件不变,改变异丙胺浓度分别为:5 mM、10 mM、20 mM、50 mM、100 mM、150mM、200 mM、300 mM,35 ℃保温10 min后加入适量纯酶液,于35 ℃、600 rpm反应10 min。反应结束后立即取出反应液置于冰上,加入10μL浓度为6 M的HCl终止反应,反应液经离心(12000 rpm, 1 min),取上清液检测产物L-丙氨酸浓度,计算相对酶活,结果如表5所示。Determination of the tolerance of omega-amine transaminase to isopropylamine. In the 1 mL standard reaction system: control the final concentration of pyruvate to 20 mM, keep other conditions unchanged, and change the concentration of isopropylamine to: 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150mM, 200 mM, 300 mM, incubate at 35°C for 10 minutes, add an appropriate amount of pure enzyme solution, and react at 35°C and 600 rpm for 10 minutes. Immediately after the reaction, take out the reaction solution and place it on ice. Add 10 μL of HCl with a concentration of 6 M to terminate the reaction. The reaction solution is centrifuged (12000 rpm, 1 min). The supernatant is taken to detect the concentration of product L-alanine and calculate the relative concentration. Enzyme activity, the results are shown in Table 5.

表5、ω-胺转氨酶在不同浓度异丙胺条件下的耐受性Table 5. Tolerance of ω-amine transaminase under different concentrations of isopropylamine

通过观察上表可知,本发明的ω-胺转氨酶ata-Oc3在不同浓度异丙胺反应条件下的耐受性较高。It can be seen from the above table that the ω-amine transaminase ata-Oc3 of the present invention has high tolerance under the reaction conditions of different concentrations of isopropylamine.

实施例8:ω-胺转氨酶ata-Oc3对丙酮酸的转化分析Example 8: Analysis of the conversion of pyruvate by ω-amine transaminase ata-Oc3

反应一:10 mL的反应体系中含有100 mM 丙酮酸,100 mM 异丙胺,0.1mM PLP和50mM磷酸盐缓冲液,用NaOH将反应体系的pH调节至8.0,加入10 g/L重组大肠杆菌E.coliBL21(DE3)/pET28a-ata-Oc3 湿菌体。反应条件为:设定恒温水浴锅温度为35 ℃,转速600rpm/min。反应每隔2 h取样一次(200μL),加入5μL 6 M 盐酸混匀终止反应,4 ℃, 12000rpm/min离心1 min后取上清备用。通过HPLC检测L-丙氨酸的转化情况。Reaction 1: The 10 mL reaction system contains 100 mM pyruvate, 100 mM isopropylamine, 0.1mM PLP and 50mM phosphate buffer. Use NaOH to adjust the pH of the reaction system to 8.0, and add 10 g/L recombinant E. coli E. .coliBL21(DE3)/pET28a-ata-Oc3 wet thalli. The reaction conditions are: set the temperature of the constant temperature water bath to 35°C and the rotation speed to 600 rpm/min. Sample (200 μL) of the reaction was taken every 2 h, add 5 μL of 6 M hydrochloric acid and mix well to terminate the reaction, centrifuge at 4°C, 12000 rpm/min for 1 min, and take the supernatant for later use. The conversion of L-alanine was detected by HPLC.

反应二:10 mL的反应体系中含有500 mM 丙酮酸,500 mM 异丙胺,0.1 mM PLP和50 mM磷酸盐缓冲液,用NaOH将反应体系的pH调节至8.0,加入50 g/L重组大肠杆菌E.coliBL21(DE3)/pET28a-ata-Oc3 湿菌体。反应条件为:设定恒温水浴锅温度为35 ℃,转速600rpm/min。反应每隔2 h取样一次(200μL),加入5μL 6 M 盐酸混匀终止反应,4 ℃, 12000rpm/min离心1 min后取上清备用。通过HPLC检测L-丙氨酸的转化情况。结果表明,在反应一和反应二中L-丙氨酸产率分别为29.07%和81.13%。Reaction 2: The 10 mL reaction system contains 500 mM pyruvate, 500 mM isopropylamine, 0.1 mM PLP and 50 mM phosphate buffer. Use NaOH to adjust the pH of the reaction system to 8.0, and add 50 g/L recombinant E. coli E.coliBL21(DE3)/pET28a-ata-Oc3 wet thalli. The reaction conditions are: set the temperature of the constant temperature water bath to 35°C and the rotation speed to 600 rpm/min. A sample (200 μL) of the reaction was taken every 2 hours, and 5 μL of 6 M hydrochloric acid was added and mixed to terminate the reaction. Centrifuge at 4°C, 12000 rpm/min for 1 min, and the supernatant was taken for later use. The conversion of L-alanine was detected by HPLC. The results showed that the yields of L-alanine in reaction one and reaction two were 29.07% and 81.13% respectively.

实施例9 :ω-胺转氨酶ata-Oc3在合成L-草铵膦中的应用Example 9: Application of ω-amine transaminase ata-Oc3 in the synthesis of L-glufosinate-ammonium

10 mL的反应体系中含有15 mM PPO,50 mM丙酮酸,50 mM异丙胺,0.1 mM PLP和50mM磷酸盐缓冲液,用NaOH将反应体系的pH调节至8.0,分别加入10 g/L重组大肠杆菌E.coliBL21(DE3)/pET28a-ata-Oc3和E.coliBL21(DE3)/ pET28b-seTA(专利CN 114921432 A)湿菌体。水浴35 ℃ ,磁力搅拌器转速600 rpm/min,反应时间为24 h,设置取样时间为2 h、4h、6 h、8 h、10 h、12 h、24 h;每次取样量为200μL,并加入5μL 6 M 盐酸终止反应,4 ℃,12000 rpm/min离心1 min后取上清于4 ℃冷藏备用。目标检测物为L-丙氨酸、L-草铵膦。L-草铵膦检测方法:采用配备荧光检测器的赛默飞U3000液相色谱仪,色谱柱Unitary® C18柱(4.6×250mm,Acchrom,China);流动相为甲醇:50 mM乙酸铵(pH 5.7),体积比为10:90;流速1.0 mL/min;检测波长Ex=350 nm、Em=460 nm;进样量10μL;柱温35 ℃。L-草铵膦、D-草铵膦的保留时间分别为:10.6分钟,12.6分钟。The 10 mL reaction system contains 15 mM PPO, 50 mM pyruvate, 50 mM isopropylamine, 0.1 mM PLP and 50mM phosphate buffer. Use NaOH to adjust the pH of the reaction system to 8.0, and add 10 g/L recombinant large intestine respectively. Bacillus E.coliBL21(DE3)/pET28a-ata-Oc3 and E.coliBL21(DE3)/pET28b-seTA (Patent CN 114921432 A) wet cells. The water bath is 35 ℃, the magnetic stirrer speed is 600 rpm/min, the reaction time is 24 h, and the sampling time is set to 2 h, 4h, 6 h, 8 h, 10 h, 12 h, and 24 h; each sampling volume is 200 μL. Add 5 μL of 6 M hydrochloric acid to terminate the reaction, centrifuge at 12000 rpm/min for 1 min at 4°C, and then take the supernatant and refrigerate at 4°C for later use. The target detection substances are L-alanine and L-glufosinate. L-glufosinate detection method: Use a Thermo Fisher U3000 liquid chromatograph equipped with a fluorescence detector, a Unitary® C18 column (4.6×250mm, Acchrom, China); the mobile phase is methanol: 50 mM ammonium acetate (pH 5.7), the volume ratio is 10:90; the flow rate is 1.0 mL/min; the detection wavelength Ex=350 nm, Em=460 nm; the injection volume is 10 μL; the column temperature is 35 °C. The retention times of L-glufosinate-ammonium and D-glufosinate-ammonium are: 10.6 minutes and 12.6 minutes respectively.

反应中能同时检测到L-丙氨酸和L-草铵膦,意味着丙酮酸与异丙胺在ω-胺转氨酶催化下产生的L-丙氨酸可以催化PPO合成L-草铵膦,此外该反应同时能将L-草铵膦合成中的副产物丙酮酸进行移除。反应结果如表6所示。L-alanine and L-glufosinate can be detected simultaneously in the reaction, which means that L-alanine produced by pyruvate and isopropylamine under the catalysis of omega-amine transaminase can catalyze the synthesis of L-glufosinate from PPO. In addition This reaction can also remove pyruvate, a by-product in the synthesis of L-glufosinate. The reaction results are shown in Table 6.

表6、ω-胺转氨酶以异丙胺为间接供体催化PPO合成L-草铵膦的反应进程Table 6. The reaction process of ω-amine transaminase to catalyze the synthesis of L-glufosinate from PPO using isopropylamine as an indirect donor.

Claims (9)

1.一种ω-胺转氨酶,其特征在于,所述ω-胺转氨酶的氨基酸序列如SEQ ID NO.1所示。1. An ω-amine transaminase, characterized in that the amino acid sequence of the ω-amine transaminase is shown in SEQ ID NO.1. 2.根据权利要求1所述的ω-胺转氨酶,其特征在于,所述ω-胺转氨酶编码基因的核苷酸序列如SEQ ID NO.2所示。2. The ω-amine transaminase according to claim 1, characterized in that the nucleotide sequence of the ω-amine transaminase encoding gene is shown in SEQ ID NO. 2. 3.根据权利要求1所述的ω-胺转氨酶,其特征在于,所述ω-胺转氨酶编码基因的核苷酸序列如SEQ ID NO.3所示。3. The ω-amine transaminase according to claim 1, characterized in that the nucleotide sequence of the ω-amine transaminase encoding gene is shown in SEQ ID NO. 3. 4.一种重组表达质粒,其特征在于,所述重组表达质粒包含有编码ω-胺转氨酶的编码基因,所述编码基因的核苷酸序列如SEQ ID NO.2或SEQ ID NO.3所示。4. A recombinant expression plasmid, characterized in that the recombinant expression plasmid contains a coding gene encoding ω-amine transaminase, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2 or SEQ ID NO.3 Show. 5.根据权利要求4所述的重组表达质粒,其特征在于,所述重组表达质粒是将编码基因插入 pET-28a载体得到。5. The recombinant expression plasmid according to claim 4, characterized in that the recombinant expression plasmid is obtained by inserting the coding gene into the pET-28a vector. 6.一种重组基因工程菌,其特征在于,包含有权利要求4或5所述的重组表达质粒。6. A recombinant genetically engineered bacterium, characterized in that it contains the recombinant expression plasmid according to claim 4 or 5. 7.根据权利要求 6所述的重组基因工程菌,其特征在于,所述重组工程菌是将重组表达质粒转入宿主大肠杆菌BL21(DE3)或大肠杆菌Top10得到。7. The recombinant genetically engineered bacterium according to claim 6, characterized in that the recombinantly engineered bacterium is obtained by transferring the recombinant expression plasmid into the host E. coli BL21 (DE3) or E. coli Top10. 8.一种生产ω-胺转氨酶的方法,其特征在于,将权利要求6或7所述的重组基因工程菌进行发酵培养,将培养后的重组基因工程菌破壁后进行分离纯化,得到ω-胺转氨酶。8. A method for producing ω-amine transaminase, which is characterized in that the recombinant genetically engineered bacteria according to claim 6 or 7 are fermented and cultured, and the cultured recombinant genetically engineered bacteria are separated and purified after wall breaking to obtain ω. -Amine transaminase. 9.权利要求1-3任一项所述的ω-胺转氨酶或者权利要求4或5所述的重组表达质粒或者权利要求6或7所述的重组基因工程菌在转氨反应中进行应用。9. The ω-amine transaminase of any one of claims 1 to 3 or the recombinant expression plasmid of claim 4 or 5 or the recombinant genetically engineered bacterium of claim 6 or 7 is used in a transamination reaction.
CN202311030831.9A 2023-08-16 2023-08-16 An ω-amine transaminase, recombinant bacteria and their applications Pending CN117247915A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311030831.9A CN117247915A (en) 2023-08-16 2023-08-16 An ω-amine transaminase, recombinant bacteria and their applications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311030831.9A CN117247915A (en) 2023-08-16 2023-08-16 An ω-amine transaminase, recombinant bacteria and their applications

Publications (1)

Publication Number Publication Date
CN117247915A true CN117247915A (en) 2023-12-19

Family

ID=89128438

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311030831.9A Pending CN117247915A (en) 2023-08-16 2023-08-16 An ω-amine transaminase, recombinant bacteria and their applications

Country Status (1)

Country Link
CN (1) CN117247915A (en)

Similar Documents

Publication Publication Date Title
CN109609474B (en) Amino acid dehydrogenase mutant and application thereof in synthesis of L-glufosinate-ammonium
CN110791484B (en) Glufosinate-ammonium dehydrogenase mutant and application thereof in production of L-glufosinate-ammonium
WO2022228505A1 (en) D-amino acid oxidase mutant and application thereof in preparing l-glufosinate
CN109750009B (en) A kind of glufosinate-ammonium dehydrogenase mutant and its application
CN111321193B (en) Method for asymmetrically preparing L-glufosinate-ammonium by redox through biological multi-enzyme coupling method
CN106916857B (en) A kind of method of producing L-glufosinate-ammonium
CN103710321B (en) Nicotinamide mononucleotide adenylyltransferase (Nmnat) mutant as well as coding gene and application thereof
CN108660122A (en) A kind of application of transaminase, mutant and its production L-glufosinate-ammonium
CN113088501B (en) A kind of glutamate dehydrogenase mutant for producing L-glufosinate-ammonium and the production method of L-glufosinate-ammonium
CN113897382B (en) Coenzyme self-sufficient escherichia coli and construction method and application thereof
CN110592036A (en) A kind of glufosinate-ammonium dehydrogenase mutant and its application in oxidative-reduction multi-enzyme coupling production of L-glufosinate-ammonium
CN108893455A (en) A kind of transaminase mutant and its application for producing L-glufosinate-ammonium
CN105238807A (en) Construction of coenzyme efficient regeneration system and application thereof
CN109609478B (en) α-Transaminases and mutants and their application in asymmetric synthesis of L-glufosinate
WO2022228506A1 (en) Glu/leu/phe/val dehydrogenase mutant and application thereof in preparation of l-phosphinothricin
CN115197954B (en) Recombinant DNA for fermentative production of 1, 5-pentanediamine, strain and use thereof
CN102304490B (en) Recombinant bacterium for efficiently expressing orotate phosphoribosyl transferase and orotidylic acid decarboxylase and construction method thereof
CN114921432B (en) A kind of transaminase mutant and its engineering bacteria and application
CN115960736B (en) A kind of engineering yeast of saccharomyces cerevisiae producing vanillylamine and capsaicin and its construction method and application
CN111876396A (en) Double-coenzyme-dependent glufosinate-ammonium dehydrogenase mutant and application thereof in catalytic synthesis of L-glufosinate-ammonium
CN117210431A (en) Heat stability aminotransferase mutant, engineering bacteria thereof and application
CN117247915A (en) An ω-amine transaminase, recombinant bacteria and their applications
CN112779233B (en) Recombinant glufosinate dehydrogenase, genetically engineered bacterium and application thereof in preparation of L-glufosinate
CN117070410A (en) Novel omega-amine transaminase, recombinant bacterium and application thereof
CN115786298A (en) D-transaminase mutant and application thereof in preparation of L-glufosinate-ammonium

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