CN116064449A - A kind of transaminase mutant and its application - Google Patents

A kind of transaminase mutant and its application Download PDF

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CN116064449A
CN116064449A CN202210845862.9A CN202210845862A CN116064449A CN 116064449 A CN116064449 A CN 116064449A CN 202210845862 A CN202210845862 A CN 202210845862A CN 116064449 A CN116064449 A CN 116064449A
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李想
柳学伟
李娟�
方武宏
巨晓芝
房杰
郭万成
李岩
李洁萍
唐建军
罗香
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Abstract

本发明涉及一种转氨酶突变体及其应用,该转氨酶突变体是由SEQID NO:1所示的氨基酸序列发生突变得到的,所述突变至少包括下列突变位点中的一个:第60位由W突变为A、D或K,第89位由F突变为A、D或K,第121位由S突变为A、D或F,第153位由Y突变为A、D或F,第226位由E突变为A、R或F,第261位由V突变为A、D或Y,第380位由Q突变为A、K或F,第417位由A突变为F、D或K。该转氨酶突变体可应用于大位阻手性胺类化合物的绿色化学合成中,催化活性高,稳定性好。与化学合成方法相比,其所催化的反应简单温和,无需氢化反应,反应选择性高,制备成本低,具有较好的应用前景。The present invention relates to a transaminase mutant and its application. The transaminase mutant is obtained by mutation of the amino acid sequence shown in SEQID NO: 1, and the mutation includes at least one of the following mutation sites: the 60th position is formed by W Mutation to A, D or K, mutation at position 89 from F to A, D or K, mutation at position 121 from S to A, D or F, mutation at position 153 from Y to A, D or F, position 226 E is mutated to A, R or F, the 261st position is mutated from V to A, D or Y, the 380th position is mutated from Q to A, K or F, and the 417th position is mutated from A to F, D or K. The transaminase mutant can be applied to the green chemical synthesis of large-sterically hindered chiral amine compounds, and has high catalytic activity and good stability. Compared with the chemical synthesis method, the reaction catalyzed by it is simple and mild, does not require hydrogenation reaction, has high reaction selectivity, low preparation cost, and has good application prospect.

Description

一种转氨酶突变体及其应用A transaminase mutant and its application

技术领域Technical Field

本发明涉及生物技术领域,具体地说涉及一种转氨酶突变体及其应用。The present invention relates to the field of biotechnology, in particular to a transaminase mutant and application thereof.

背景技术Background Art

转氨酶催化氨基供体(胺供体)和前手性酮底物之间氨基的立体选择性转移,是生成光学纯手性胺的有效生物催化工具。转氨酶(TAs)可以分为两类:α-转氨酶(α-TAs)和ω-转氨酶(ω-TAs),该分类这取决于所转化的底物的类型。α-TAs要求在酮底物羰基官能团的α-位置和胺供体上均存在一个羧基。ω-TAs可以接受脂肪族酮和胺作为底物(即不仅仅是α-酮酸和氨基酸)。ω-TAs可进一步分为两个亚组,β-TAs和胺转氨酶(ATAs),其中ATAs具有工业价值,因为它们能够使用广泛的胺供体和酮受体进行还原胺化反应。ATAs催化胺供体和酮受体的分子间交换,近年来在药物中间体的生产中受到了相当大的关注。Transaminases catalyze the stereoselective transfer of amino groups between amino donors (amine donors) and prochiral keto substrates and are effective biocatalytic tools for the generation of optically pure chiral amines. Transaminases (TAs) can be divided into two categories: α-transaminases (α-TAs) and ω-transaminases (ω-TAs), depending on the type of substrate being transformed. α-TAs require the presence of a carboxyl group both at the α-position of the carbonyl functional group of the keto substrate and on the amine donor. ω-TAs can accept aliphatic ketones and amines as substrates (i.e., not just α-keto acids and amino acids). ω-TAs can be further divided into two subgroups, β-TAs and amine transaminases (ATAs), of which ATAs are of industrial interest because they are able to perform reductive amination reactions using a wide range of amine donors and ketone acceptors. ATAs catalyze the intermolecular exchange of amine donors and ketone acceptors and have received considerable attention in recent years in the production of pharmaceutical intermediates.

虽然转氨酶催化的不对称转氨反应为手性胺化合物的合成提供了一种经济、绿色的合成思路。然而,ATAs在大规模应用中的一些局限性和挑战也已被确定,如底物范围相当窄、不利的热力学反应平衡和底物/产物抑制。一般来说,潜在手性羰基旁边的基团如果比甲基大,转氨酶的催化效果会明显降低。为了克服这一限制,人们已经付出了大量的努力来进化ATAs以改善其底物范围,特别是在所谓的“体积大”化合物。不同的蛋白质工程方法被用于优化可用ATAs。Although transaminase-catalyzed asymmetric transamination reactions provide an economical and green synthetic approach for the synthesis of chiral amine compounds. However, some limitations and challenges of ATAs in large-scale applications have also been identified, such as a rather narrow substrate range, unfavorable thermodynamic reaction equilibrium, and substrate/product inhibition. In general, if the group next to the potential chiral carbonyl is larger than the methyl group, the catalytic effect of the transaminase will be significantly reduced. To overcome this limitation, a lot of efforts have been made to evolve ATAs to improve their substrate range, especially in so-called "bulky" compounds. Different protein engineering approaches have been used to optimize the available ATAs.

CN112980810A公开了一种来源于紫色色杆菌(Chromobaterium violaceum)的转氨酶突变体,旨在使用该转氨酶突变体将潜手性羰基旁边的基团大于甲基的酮类化合物高选择性地转化为手性胺化合物。但该转氨酶突变体的获得需要进行饱和突变及迭代突变,筛选工作量大,操作十分繁琐。CN112980810A discloses a transaminase mutant derived from Chromobaterium violaceum, which is intended to use the transaminase mutant to convert ketone compounds with a group larger than methyl next to the potential chiral carbonyl into chiral amine compounds with high selectivity. However, the acquisition of the transaminase mutant requires saturation mutation and iterative mutation, which requires a large amount of screening work and is very cumbersome.

发明内容Summary of the invention

为了解决化学合成手性胺过程中存在的低效率和低选择性和高环境影响,以及生物合成过程中生物酶催化活性低以及稳定性差的问题,本发明的一方面的目的是提供一种催化活性高、热稳定性好并且可以高效率催化合成大位阻手性胺化合物的转氨酶突变体。为实现本发明的目的,本发明采用以下技术方案:In order to solve the problems of low efficiency, low selectivity and high environmental impact in the chemical synthesis of chiral amines, as well as low catalytic activity and poor stability of biological enzymes in the biosynthesis process, one aspect of the present invention is to provide a transaminase mutant with high catalytic activity, good thermal stability and high efficiency in catalyzing the synthesis of large sterically hindered chiral amine compounds. To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

一种转氨酶突变体,该转氨酶突变体的氨基酸序列是由SEQ ID NO:1所示的氨基酸序列发生突变得到的氨基酸序列,所述突变至少包括下列突变位点中的一个:A transaminase mutant, the amino acid sequence of which is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes at least one of the following mutation sites:

第60位由W突变为A、D或K(W60A/D/K);第89位由F突变为A、D或K(F89A/D/K);第121位由S突变为A、D或F(S121A/F/D);第153位由Y突变为A、D或F(Y153A/D/F);第226位由E突变为A、R或F(E226A/R/F);第261位由V突变为A、D或Y(V261A/D/Y);第380位由Q突变为A、K或F(Q380A/F/K),第417位由A突变为F、D或K(A417F/D/K),或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。Position 60 mutates from W to A, D or K (W60A/D/K); position 89 mutates from F to A, D or K (F89A/D/K); position 121 mutates from S to A, D or F (S121A/F/D); position 153 mutates from Y to A, D or F (Y153A/D/F); position 226 mutates from E to A, R or F (E226A/R/F); position 261 mutates from V to A, D or Y (V261A/D/Y); position 380 mutates from Q to A, K or F (Q380A/F/K), position 417 mutates from A to F, D or K (A417F/D/K), or the amino acid sequence of the amino acid transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence.

优选地,上述突变至少包括下列突变位点中的一个:Preferably, the above mutation includes at least one of the following mutation sites:

第60位由W突变为A(W60A),第89位由F突变为K(F89K),第121位由S突变为D(S121D),第153位由Y突变为A(Y153A),第226位由E突变为R(E226R),第261位由V突变为A(V261A),第380位由Q突变为F(Q380F),第417位由A突变为F(A417F),或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。The 60th position mutates from W to A (W60A), the 89th position mutates from F to K (F89K), the 121st position mutates from S to D (S121D), the 153rd position mutates from Y to A (Y153A), the 226th position mutates from E to R (E226R), the 261st position mutates from V to A (V261A), the 380th position mutates from Q to F (Q380F), and the 417th position mutates from A to F (A417F), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence.

更优选地,上述转氨酶突变体包括下列突变位点:More preferably, the above transaminase mutant comprises the following mutation sites:

第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A和第226位由E突变为R,或者,The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A and the 226th position mutates from E to R, or,

第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R和第261位由V突变为A,或者,The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A, the 226th position mutates from E to R and the 261st position mutates from V to A, or,

第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A和第380位由Q突变为F,或者,position 60 from W to A, position 89 from F to K, position 121 from S to D, position 153 from Y to A, position 226 from E to R, position 261 from V to A and position 380 from Q to F, or,

第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A,第380位由Q突变为F和第417位由A突变为F。Position 60 mutated from W to A, position 89 mutated from F to K, position 121 mutated from S to D, position 153 mutated from Y to A, position 226 mutated from E to R, position 261 mutated from V to A, position 380 mutated from Q to F and position 417 mutated from A to F.

更优选地,上述转氨酶突变体包括突变位点第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A,第380位由Q突变为F和第417位由A突变为F,其氨基酸序列如SEQ ID NO.3所示。More preferably, the above-mentioned transaminase mutant includes mutation sites 60 from W to A, 89 from F to K, 121 from S to D, 153 from Y to A, 226 from E to R, 261 from V to A, 380 from Q to F and 417 from A to F, and its amino acid sequence is shown in SEQ ID NO.3.

本发明另一方面的目的是提供编码上述转氨酶突变体的基因。Another object of the present invention is to provide a gene encoding the above-mentioned aminotransferase mutant.

优选地,上述基因的核苷酸序列如SEQ ID NO.4所示,或者上述基因的核苷酸序列为与SEQ ID NO.4所示序列具有95%以上同源性的序列。Preferably, the nucleotide sequence of the above gene is as shown in SEQ ID NO.4, or the nucleotide sequence of the above gene is a sequence having more than 95% homology with the sequence shown in SEQ ID NO.4.

本发明再一方面的目的是提供一种重组表达载体,该重组表达载体包含编码上述转氨酶突变体的基因。Another aspect of the present invention is to provide a recombinant expression vector comprising a gene encoding the above-mentioned transaminase mutant.

优选地,所述重组表达载体选自pET-28a、pET-dute1、pRSF-dute1,更优选pET-28a。Preferably, the recombinant expression vector is selected from pET-28a, pET-dute1, pRSF-dute1, more preferably pET-28a.

本发明再一方面的目的是提供一种基因工程菌,该基因工程菌用于生产上述转氨酶突变体,其包含上述重组表达载体。Another aspect of the present invention is to provide a genetically engineered bacterium for producing the above-mentioned aminotransferase mutant, which comprises the above-mentioned recombinant expression vector.

优选地,上述基因工程菌选自大肠杆菌MG1655或大肠杆菌BL21(DE3)或大肠杆菌BL21(DE3)pLysS。Preferably, the genetically engineered bacteria is selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3), or Escherichia coli BL21 (DE3) pLysS.

更优选地,上述基因工程菌选自大肠杆菌BL21(DE3)。More preferably, the genetically engineered bacteria is selected from Escherichia coli BL21 (DE3).

本发明再一方面的目的是提供上述转氨酶突变体在催化羰基类化合物或氨基供体制备手性胺中的应用。Another aspect of the present invention is to provide the use of the above transaminase mutant in catalyzing the preparation of chiral amines from carbonyl compounds or amino donors.

本发明再一方面的目的是一种生产手性胺的方法,该方法包括利用上述转氨酶突变体对酮类化合物或氨基供体进行催化转氨基反应的步骤。Another aspect of the present invention is a method for producing chiral amines, which comprises the step of using the above-mentioned aminotransferase mutant to catalyze the transamination reaction of a ketone compound or an amino donor.

优选地,上述酮类化合物为

Figure BDA0003752745160000041
其中R1和R2各自独立地为选取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基;R1和R2可单独或两者互相结合形成取代或未被取代的环。Preferably, the ketone compound is
Figure BDA0003752745160000041
Wherein R 1 and R 2 are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon group; R 1 and R 2 can be alone or in combination with each other to form a substituted or unsubstituted ring.

所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。The substitution refers to substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

本发明再一方面的目的是一种式(II)所示化合物的制备方法,该制备方法包括将式(I)所示化合物利用上述转氨酶突变体催化转化形成式(II)所示化合物的步骤,其反应方程式如下:Another aspect of the present invention is a method for preparing a compound of formula (II), which comprises the step of catalytically converting the compound of formula (I) using the above-mentioned aminotransferase mutant to form a compound of formula (II), and the reaction equation is as follows:

Figure BDA0003752745160000042
Figure BDA0003752745160000042

本发明的上述转氨酶突变体是在SEQ ID NO:1所示的转氨酶的基础上,通过定点突变的方法进行突变,从而改变其氨基酸序列,实现蛋白质结构和功能的改变,再通过定向筛选的方法,得到具有上述突变位点的转氨酶,本发明的转氨酶突变体具有酶活性大幅度提高的优势,其酶活性相对于转氨酶母本提高了多倍,稳定性也大幅度提高,用于手性胺生产时,大幅度降低了手性胺工业生产的成本。The above-mentioned transaminase mutant of the present invention is mutated on the basis of the transaminase shown in SEQ ID NO: 1 by a site-directed mutagenesis method, thereby changing its amino acid sequence and realizing changes in protein structure and function, and then a directed screening method is used to obtain a transaminase having the above-mentioned mutation site. The transaminase mutant of the present invention has the advantage of greatly improved enzyme activity, and its enzyme activity is increased by several times compared with the transaminase parent, and its stability is also greatly improved. When used for the production of chiral amines, the cost of industrial production of chiral amines is greatly reduced.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为实施例1的转氨酶重组表达载体的图。FIG. 1 is a diagram of the transaminase recombinant expression vector of Example 1.

图2a为实施例3得到的摇瓶中小规模生产的转氨酶突变体(i)、(ii)、(iii)、(iv)、(v)和(vi)蛋白的聚丙烯酰胺凝胶电泳结果,其中从左至右的泳道分别为Marker、空白、野生型、突变体(i)、突变体(ii)、突变体(iii)、突变体(iv)、突变体(v)和突变体(vi)的聚丙烯酰胺凝胶电泳结果。Figure 2a is the polyacrylamide gel electrophoresis results of the small-scale production of transaminase mutants (i), (ii), (iii), (iv), (v) and (vi) proteins in shake flasks obtained in Example 3, wherein the lanes from left to right are the polyacrylamide gel electrophoresis results of Marker, blank, wild type, mutant (i), mutant (ii), mutant (iii), mutant (iv), mutant (v) and mutant (vi), respectively.

图2b为实施例3得到的摇瓶中小规模生产的转氨酶突变体(vi)(其氨基酸序列为SEQ ID NO:3)的聚丙烯酰胺凝胶电泳图,其中从左至右的泳道分别为Marker和突变体(vi)的聚丙烯酰胺凝胶电泳结果。Figure 2b is a polyacrylamide gel electrophoresis diagram of the small-scale production of the transaminase mutant (vi) (whose amino acid sequence is SEQ ID NO: 3) in a shake flask obtained in Example 3, wherein the lanes from left to right are the polyacrylamide gel electrophoresis results of the Marker and the mutant (vi), respectively.

图3为标准品与实施例6得到的手性胺产物的1HNMR图谱。上半部分为标准品的1HNMR图谱,下半部分为实施例6得到的产物的1HNMR图谱。3 is the 1 HNMR spectrum of the standard product and the chiral amine product obtained in Example 6. The upper part is the 1 HNMR spectrum of the standard product, and the lower part is the 1 HNMR spectrum of the product obtained in Example 6.

图4为标准品与实施例6得到的手性胺产物的氟谱,上半部分为标准品的氟谱,下半部分为实施例6得到的产物的氟谱。FIG4 is the fluorine spectrum of the standard product and the chiral amine product obtained in Example 6, the upper half is the fluorine spectrum of the standard product, and the lower half is the fluorine spectrum of the product obtained in Example 6.

具体实施方式DETAILED DESCRIPTION

来源于Xenophilus sp.AP218F的氨基转氨酶可以选择性催化羰基的转化,但是其活性较低,稳定性较差。发明人通过理性设计的方法提高来源于Xenophilus sp.AP218F的转氨酶的活性与稳定性。通过全质粒PCR的方式在来源于Xenophilus sp.AP218F的氨基转移酶上引入突变位点,以及对突变体进行活性和稳定性检测,挑选活性和稳定性提高的突变体。The aminotransferase derived from Xenophilus sp.AP218F can selectively catalyze the conversion of carbonyl groups, but its activity is low and its stability is poor. The inventors improved the activity and stability of the aminotransferase derived from Xenophilus sp.AP218F by a rationally designed method. Mutation sites were introduced into the aminotransferase derived from Xenophilus sp.AP218F by means of whole plasmid PCR, and the activity and stability of the mutants were tested to select mutants with improved activity and stability.

本发明所提供的转氨酶突变体基因来源于Xenophilus sp.AP218F的野生型基因。该野生型基因的氨基酸序列如SEQ ID NO.1所示,针对大肠杆菌进行密码子优化后的基因序列如SEQ ID NO.2所示。其中,“野生型”是指在自然界发现的形式。例如,天然存在的或野生型的多肽或多核苷酸序列是存在于生物体中的序列,能够从自然界来源中分离并且没有被人为操作有意修饰。这些基因表达后得到的酶对于某些底物的催化活力低,热稳定性较差。The transaminase mutant gene provided by the present invention is derived from the wild-type gene of Xenophilus sp.AP218F. The amino acid sequence of the wild-type gene is shown in SEQ ID NO.1, and the gene sequence after codon optimization for Escherichia coli is shown in SEQ ID NO.2. Wherein, "wild type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism, which can be isolated from a natural source and has not been intentionally modified by human manipulation. The enzymes obtained after expression of these genes have low catalytic activity for certain substrates and poor thermal stability.

本发明通过在线蛋白结构预测工具获得来源于Xenophilus sp.AP218F的氨基转移酶的三维结构,并通过PDB获取与其结构相似度最高的转氨酶的三维结构(6S4G,同源性87.58%)做结构比对,然后通过AutoDock进行式I底物与转氨酶蛋白质三维结构的进行结合模拟,最后通过Pymol分析,选择有可能与底物结合相关的氨基酸作为突变氨基酸。根据Pymol分析结果,设计了多对定点突变(W60A/D/K;F89A/D/K;S121A/F/D;Y153A/D/F,E226A/R/F,V261A/D/Y;Q380A/F/K;A417F/D/K)引物,利用定点突变手段,以pET-28a为表达载体,获得带有目的基因的突变质粒。其中,定点突变:是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组,也可以是质粒)中引入所需变化(通常是表征有利方向的变化),包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征,是基因研究工作中一种非常有用的手段。The invention obtains the three-dimensional structure of the aminotransferase derived from Xenophilus sp.AP218F through an online protein structure prediction tool, obtains the three-dimensional structure of the transaminase with the highest structural similarity (6S4G, homology 87.58%) through PDB for structural comparison, then performs binding simulation of the substrate of formula I and the three-dimensional structure of the transaminase protein through AutoDock, and finally selects the amino acid that may be related to the substrate binding as the mutant amino acid through Pymol analysis. According to the Pymol analysis results, multiple pairs of site-directed mutation (W60A/D/K; F89A/D/K; S121A/F/D; Y153A/D/F, E226A/R/F, V261A/D/Y; Q380A/F/K; A417F/D/K) primers are designed, and a mutant plasmid with a target gene is obtained by using a site-directed mutation method and taking pET-28a as an expression vector. Among them, site-directed mutagenesis refers to the introduction of desired changes (usually changes that characterize favorable directions) into target DNA fragments (which can be genomes or plasmids) through methods such as polymerase chain reaction (PCR), including addition, deletion, and point mutation of bases. Site-directed mutagenesis can quickly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.

利用全质粒PCR引入定点突变是目前使用比较多的手段。其原理是,一对包含突变位点的引物(正、反向),和模版质粒退火后用聚合酶“循环延伸”,所谓的循环延伸是指聚合酶按照模版延伸引物,一圈后回到引物5’端终止,再经过反复加热退火延伸的循环,这个反应区别于滚环扩增,不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。Dpn I酶切延伸产物,由于原来的模版质粒来源于常规大肠杆菌,是经dam甲基化修饰的,对Dpn I敏感而被切碎,而体外合成的带突变序列的质粒由于没有甲基化而不被切开,因此在随后的转化中得以成功转化,即可得到突变质粒的克隆。将突变质粒转化至大肠杆菌细胞内,在大肠杆菌中过量表达。然后离心分离获得湿细胞,通过超声破碎细胞的方法获得粗酶。The introduction of site-directed mutations using whole plasmid PCR is a method that is currently used more frequently. The principle is that a pair of primers (forward and reverse) containing the mutation site are annealed with the template plasmid and then "circularly extended" by the polymerase. The so-called circular extension refers to the polymerase extending the primer according to the template, returning to the 5' end of the primer after one circle to terminate, and then undergoing repeated heating annealing and extension cycles. This reaction is different from rolling circle amplification and will not form multiple tandem copies. After annealing, the extension products of the forward and reverse primers are paired to form an open-circular plasmid with a nick. The extension product is cut by Dpn I. Since the original template plasmid is derived from conventional Escherichia coli and is modified by dam methylation, it is sensitive to Dpn I and is chopped up. The plasmid with a mutation sequence synthesized in vitro is not methylated and is not cut open. Therefore, it can be successfully transformed in the subsequent transformation, and the mutant plasmid clone can be obtained. The mutant plasmid is transformed into Escherichia coli cells and overexpressed in Escherichia coli. Then, the wet cells are obtained by centrifugation, and the crude enzyme is obtained by ultrasonic cell disruption.

作为本发明的第一方面,本发明提供的转氨酶突变体是由SEQ ID NO:1所示的氨基酸序列发生突变得到的氨基酸序列,所述突变至少包括下列突变位点中的一个:第60位由W突变为A、D或K(W60A/D/K);第89位由F突变为A、D或K(F89A/D/K);第121位由S突变为A、D或F(S121A/F/D);第153位由Y突变为A、D或F(Y153A/D/F);第226位由E突变为A、R或F(E226A/R/F);第261位由V突变为A、D或Y(V261A/D/Y);第380位由Q突变为A、K或F(Q380A/F/K),第417位由A突变为F、D或K(A417F/D/K),或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。在本发明的一个优选具体实施例中,所述突变至少包括下列突变位点中的一个:第60位由W突变为A(W60A),第89位由F突变为K(F89K),第121位由S突变为D(S121D),第153位由Y突变为A(Y153A),第226位由E突变为R(E226R),第261位由V突变为A(V261A),第380位由Q突变为F(Q380F),第417位由A突变为F(A417F),或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。其中“同源性”是指两个氨基酸序列之间的同一度。本发明用不同程度同源性限定的序列还必须要同时具有改进的转氨酶活性。本领域技术人员可以在本申请公开内容的教导下获得转氨酶突变体的氨基酸序列具有上述发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。As a first aspect of the present invention, the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is mutated, and the mutation includes at least one of the following mutation sites: the 60th position is mutated from W to A, D or K (W60A/D/K); the 89th position is mutated from F to A, D or K (F89A/D/K); the 121st position is mutated from S to A, D or F (S121A/F/D); the 153rd position is mutated from Y to A, D or F (Y153A/D/F); the 226th position is mutated from E ... R or F (E226A/R/F); position 261 mutates from V to A, D or Y (V261A/D/Y); position 380 mutates from Q to A, K or F (Q380A/F/K), position 417 mutates from A to F, D or K (A417F/D/K), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. In a preferred embodiment of the present invention, the mutation includes at least one of the following mutation sites: position 60 mutates from W to A (W60A), position 89 mutates from F to K (F89K), position 121 mutates from S to D (S121D), position 153 mutates from Y to A (Y153A), position 226 mutates from E to R (E226R), position 261 mutates from V to A (V261A), position 380 mutates from Q to F (Q380F), position 417 mutates from A to F (A417F), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology with the mutated amino acid sequence. Wherein "homology" refers to the identity between two amino acid sequences. The sequences defined by different degrees of homology in the present invention must also have improved transaminase activity at the same time. Under the guidance of the disclosure of the present application, a person skilled in the art can obtain an amino acid sequence of a transaminase mutant having the mutation site in the above-mentioned mutated amino acid sequence and having an amino acid sequence with more than 80% homology with the mutated amino acid sequence.

在本发明的一个更优选具体实施例中,上述转氨酶突变体包括突变位点:W60A,F89K,S121D,Y153A和E226R。在本发明的一个更优选具体实施例中,上述转氨酶突变体包括突变位点:W60A,F89K,S121D,Y153A,E226R和V261A。在本发明的一个更优选具体实施例中,上述转氨酶突变体包括突变位点:W60A,F89K,S121D,Y153A,E226R,V261A和Q380F。In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A and E226R. In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A, E226R and V261A. In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A, E226R, V261A and Q380F.

在本发明的一个更优选具体实施例中,上述转氨酶突变体包括突变位点:W60A,F89K,S121D,Y153A,E226R,V261A,Q380F和A417F,其氨基酸序列如SEQ ID NO.3所示。In a more preferred embodiment of the present invention, the above-mentioned transaminase mutant includes mutation sites: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F and A417F, and its amino acid sequence is shown in SEQ ID NO.3.

本发明的上述转氨酶突变体是在如SEQ ID NO:1所示的转氨酶序列的基础上,通过定点突变的方法进行突变,从而改变其氨基酸序列,实现蛋白质结构和功能的改变,再通过定向筛选的方法,得到具有上述突变位点的转氨酶,本发明的转氨酶突变体具有酶活性大幅度提高的优势,其同等条件下对底物的转化率相对于转氨酶母本提高了95倍,并且酶稳定性也有相应提高,从而大幅度降低了手性胺工业生产中的成本。The above-mentioned transaminase mutant of the present invention is mutated by site-directed mutagenesis on the basis of the transaminase sequence shown in SEQ ID NO: 1, thereby changing its amino acid sequence and realizing changes in protein structure and function, and then obtaining a transaminase having the above-mentioned mutation site by a directed screening method. The transaminase mutant of the present invention has the advantage of greatly improved enzyme activity. Its conversion rate of the substrate is increased by 95 times compared with the transaminase parent under the same conditions, and the enzyme stability is also correspondingly improved, thereby greatly reducing the cost in the industrial production of chiral amines.

作为本发明的第二方面,本发明提供了编码上述转氨酶突变体的基因。本发明通过理性设计(定点突变或其它方法改变蛋白质分子中的个别氨基酸)以及重叠延伸PCR、无缝克隆等方法对野生型转氨酶的基因进行突变,获得了上述转氨酶突变体的目的基因。在本发明的一个优选具体实施例中,该基因的核苷酸序列如SEQ ID NO.4所示,或者该基因的核苷酸序列为与SEQ ID NO.4所示序列具有具有95%以上同源性的序列。其中“同源性”是指两个核苷酸序列之间的同一度。As a second aspect of the present invention, the present invention provides a gene encoding the above-mentioned aminotransferase mutant. The present invention mutates the gene of the wild-type aminotransferase by rational design (site-directed mutagenesis or other methods to change individual amino acids in the protein molecule) and overlap extension PCR, seamless cloning and other methods to obtain the target gene of the above-mentioned aminotransferase mutant. In a preferred embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.4, or the nucleotide sequence of the gene is a sequence with more than 95% homology with the sequence shown in SEQ ID NO.4. Wherein "homology" refers to the degree of identity between two nucleotide sequences.

本发明的基因编码得到的转氨酶,提高了酶活性和酶的稳定性。使得手性胺的工业生产效率更高,成本更低。The transaminase obtained by the gene encoding of the present invention improves the enzyme activity and enzyme stability, making the industrial production efficiency of chiral amines higher and the cost lower.

作为本发明的第三方面,本发明提供了一种重组表达载体。该重组表达载体含有编码本发明的转氨酶突变体的基因。该基因位于重组表达载体的适当位置,使得上述基因能够正确地、顺利地复制、转录或表达。为了满足重组操作的要求,在该重组表达载体中,上述基因序列的两端可添加合适的限制性内切酶的酶切位点,或者额外增加启动密码子、终止密码子等。该重组表达载体可以是原核表达载体也可以是真核表达载体。在本发明中,该重组表达载体包括但不局限于pET-28a、pET-dute1或pRSF-dute1。As a third aspect of the present invention, the present invention provides a recombinant expression vector. The recombinant expression vector contains a gene encoding the transaminase mutant of the present invention. The gene is located at an appropriate position in the recombinant expression vector so that the above gene can be correctly and smoothly replicated, transcribed or expressed. In order to meet the requirements of the recombination operation, in the recombinant expression vector, both ends of the above gene sequence can be added with suitable restriction endonuclease sites, or additional start codons, stop codons, etc. can be added. The recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. In the present invention, the recombinant expression vector includes but is not limited to pET-28a, pET-dute1 or pRSF-dute1.

作为本发明的第四方面,本发明提供了一种基因工程菌,该基因工程菌用于生产上述转氨酶突变体,其包上述重组表达载体。在本发明中,该基因工程菌包括但不局限于大肠杆菌MG1655或大肠杆菌BL21(DE3)或大肠杆菌BL21(DE3)pLysS。As a fourth aspect of the present invention, the present invention provides a genetically engineered bacterium for producing the above-mentioned aminotransferase mutant, which comprises the above-mentioned recombinant expression vector. In the present invention, the genetically engineered bacterium includes but is not limited to Escherichia coli MG1655 or Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

通过发酵培养该基因工程菌可制备上述转氨酶突变体。例如在一定生产罐发酵条件下,进行工业化制备上述转氨酶突变体。所述生产罐发酵条件优选:DO 20%以上,温度20℃。The above transaminase mutant can be prepared by fermenting and culturing the genetically engineered bacteria. For example, the above transaminase mutant can be prepared industrially under certain production tank fermentation conditions. The production tank fermentation conditions are preferably: DO 20% or more, temperature 20°C.

作为本发明的第五方面,本发明提供了上述转氨酶突变体在催化羰基类化合物制备手性胺中的应用。As a fifth aspect of the present invention, the present invention provides the use of the above-mentioned aminotransferase mutant in catalyzing the preparation of chiral amines from carbonyl compounds.

作为本发明的第六方面,本发明提供了一种生产手性胺的方法,该方法包括利用上述转氨酶突变体对酮类化合物进行催化转氨基反应的步骤。As a sixth aspect of the present invention, the present invention provides a method for producing chiral amines, which comprises the step of utilizing the above-mentioned aminotransferase mutant to catalyze the transamination reaction of a ketone compound.

优选地,上述酮类化合物为

Figure BDA0003752745160000081
其中R1和R2各自独立地为取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基;或R1和R2可单独或两者互相结合形成取代或未被取代的环。所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。Preferably, the ketone compound is
Figure BDA0003752745160000081
Wherein R1 and R2 are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon; or R1 and R2 may be alone or in combination with each other to form a substituted or unsubstituted ring. The substitution refers to substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

更优选地,R1和R2各自独立地为碳原子数1~20的取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基,或R1和R2可单独或两者互相结合形成取代或未被取代的碳原子数1~20的环。More preferably, R1 and R2 are each independently a substituted or unsubstituted alkyl group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heteroaromatic hydrocarbon group having 1 to 20 carbon atoms, or R1 and R2 may be combined alone or in combination to form a substituted or unsubstituted ring having 1 to 20 carbon atoms.

更优选地,R1和R2各自独立地为碳原子数1~10的取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基,或R1和R2可单独或两者互相结合形成取代或未被取代的碳原子数1~10的环。More preferably, R1 and R2 are each independently a substituted or unsubstituted alkyl group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heteroaromatic hydrocarbon group having 1 to 10 carbon atoms, or R1 and R2 may be combined alone or in combination to form a substituted or unsubstituted ring having 1 to 10 carbon atoms.

上述烷基,例如,可选自甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基和环庚基。The above-mentioned alkyl group can be selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl, for example.

上述不饱和烃基,例如,可选自乙烯基、1-甲基乙烯基、1-乙基乙烯基、1-丙烯基、2-丙烯基、2-甲基-1-丙烯基、1-丁烯基、2-丁烯基、2-甲基-1-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、4-甲基-1-戊烯基、1-己烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、1,5-己二烯基、2-庚烯基、2-辛烯基、2-壬烯基、和2-癸烯基、乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-甲基-1-丁炔基、3,3-二甲基-1-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、1-己炔基、2-己炔基、3-己炔基、4-己炔基、1-甲基-3-戊炔基、1-甲基-3-己炔基、2-庚炔基、2-辛炔基、2-壬炔基和2-癸炔基等。The unsaturated hydrocarbon group may be selected from vinyl, 1-methylvinyl, 1-ethylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 2-methyl-1-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 4-methyl-1-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1,5-hexadienyl, 2-heptenyl , 2-octenyl, 2-nonenyl, and 2-decenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-methyl-1-butynyl, 3,3-dimethyl-1-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 1-methyl-3-pentynyl, 1-methyl-3-hexynyl, 2-heptynyl, 2-octynyl, 2-nonynyl and 2-decynyl, etc.

上述芳烃基,例如,可选自苯基、萘基。The aromatic hydrocarbon group may be selected from, for example, a phenyl group and a naphthyl group.

上述杂环芳烃基,例如,可选自嘧啶基、呋喃基、吡咯基、噻吩基、咪唑基、噻唑基、吡啶基、苯并呋喃基、吲哚基。The heterocyclic aromatic hydrocarbon group may be selected from, for example, pyrimidinyl, furanyl, pyrrolyl, thienyl, imidazolyl, thiazolyl, pyridyl, benzofuranyl and indolyl.

所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。The substitution refers to substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

更优选地,上述酮类化合物选自具有下列结构的化合物:More preferably, the ketone compound is selected from compounds having the following structures:

Figure BDA0003752745160000091
Figure BDA0003752745160000091

利用上述转氨酶突变体对酮类化合物进行催化转氨基反应的步骤中,催化转氨基反应的反应体系中,pH优选为7~10,更优选为8~9,最优选为8.5;反应体系的温度优选为40℃~60℃,更优选为45~55℃,最优选为50℃。In the step of using the above-mentioned transaminase mutant to catalyze the transamination reaction of ketone compounds, the pH of the reaction system for the catalytic transamination reaction is preferably 7-10, more preferably 8-9, and most preferably 8.5; the temperature of the reaction system is preferably 40°C to 60°C, more preferably 45-55°C, and most preferably 50°C.

作为本发明的第七方面,本发明提供了一种式(II)所示化合物的制备方法,该制备方法包括将式(I)所示化合物利用上述转氨酶突变体催化转化形成式(II)所示化合物的步骤,其反应方程式如下:As a seventh aspect of the present invention, the present invention provides a method for preparing a compound represented by formula (II), the preparation method comprising the step of catalytically converting the compound represented by formula (I) using the above-mentioned aminotransferase mutant to form a compound represented by formula (II), and the reaction equation is as follows:

Figure BDA0003752745160000101
Figure BDA0003752745160000101

优选地,上述制备方法中,反应体系中,pH优选为7~10,更优选为8~9,最优选为8.5;反应体系的温度优选为40℃~60℃,更优选为45~55℃,最优选为50℃。Preferably, in the above preparation method, in the reaction system, the pH is preferably 7-10, more preferably 8-9, most preferably 8.5; the temperature of the reaction system is preferably 40°C-60°C, more preferably 45-55°C, most preferably 50°C.

本发明同现有技术相比具有以下优点及效果:Compared with the prior art, the present invention has the following advantages and effects:

1.本发明的如SEQ ID NO.3所示的氨基酸序列的转氨酶突变体,对于大位阻底物有较高的催化活力,使底物(式(I)所示化合物)在12h的转化率为60%,而野生型酶则对这类底物的酶活小于1%。此外,本发明的转氨酶突变体在50℃时,半衰期大于48小时,而野生型酶在相同条件下半衰期只有12小时。1. The aminotransferase mutant of the present invention, as shown in SEQ ID NO.3, has a higher catalytic activity for a large sterically hindered substrate, so that the conversion rate of the substrate (the compound shown in formula (I)) is 60% in 12 hours, while the enzyme activity of the wild-type enzyme for such substrate is less than 1%. In addition, the aminotransferase mutant of the present invention has a half-life of more than 48 hours at 50°C, while the half-life of the wild-type enzyme under the same conditions is only 12 hours.

2.本发明所提供的转氨酶突变体在碱性条件下催化活性高、耐热性好,可应用于制备手性胺的生物合成;与化学合成方法相比,其所催化的转氨反应简单温和,反应选择性高,制备成本低,具有较好的应用前景。2. The transaminase mutant provided by the present invention has high catalytic activity and good heat resistance under alkaline conditions, and can be used in the biosynthesis of chiral amines; compared with chemical synthesis methods, the transamination reaction catalyzed by it is simple and mild, has high reaction selectivity, low preparation cost, and has good application prospects.

3.本发明的转氨酶突变体对酮类底物的催化活性相比野生型有大幅提高,实现了利用转氨酶催化合成大位阻的手性胺化合物,不需要使用重金属催化剂,实现绿色化学合成,且使用活性提高的转氨酶进行转氨反应,容易得到高选择性的手性胺化合物。3. The catalytic activity of the transaminase mutant of the present invention towards ketone substrates is greatly improved compared to the wild type, and the synthesis of large sterically hindered chiral amine compounds by transaminase catalysis is realized without the need for heavy metal catalysts, thereby achieving green chemical synthesis. Moreover, the use of transaminase with enhanced activity for transamination reactions can easily obtain highly selective chiral amine compounds.

下面结合实施例对本发明做进一步的详细说明,以下实施例是对本发明的解释而本发明并不局限于以下实施例。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The present invention is further described in detail below in conjunction with the examples, which are explanations of the present invention and are not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

实施例1:野生转氨酶基因工程菌的建立Example 1: Establishment of wild transaminase genetically engineered bacteria

根据NCBI收录的Xenophilus sp.AP218F转氨酶野生型基因序列(GenBank:OWY40557.1)进行序列优化后人工合成全基因片段(核苷酸序列如SEQ ID NO.2所示),并经基因合成公司通过NdeI和XhoI内切酶将基因插入pET-28a质粒中(图1),将连接后的载体转入大肠杆菌BL21(DE3)中建立野生型转氨酶基因工程菌,卡那霉素抗性筛选后测序验证。The whole gene fragment (nucleotide sequence as shown in SEQ ID NO.2) was artificially synthesized after sequence optimization based on the wild-type gene sequence of Xenophilus sp.AP218F transaminase included in NCBI (GenBank: OWY40557.1), and the gene was inserted into the pET-28a plasmid (Figure 1) by a gene synthesis company through NdeI and XhoI endonucleases. The ligated vector was transferred into Escherichia coli BL21 (DE3) to establish the wild-type transaminase gene engineering bacteria, and the kanamycin resistance was screened and sequenced for verification.

实施例2:转氨酶突变体及目标基因的获得Example 2: Obtaining transaminase mutants and target genes

通过在线蛋白结构预测工具获得SEQ ID NO.1三维结构,并通过PDB获取与其结构相似度最高的转氨酶的三维结构(6S4G,同源性84.97%)做结构比对,然后通过AutoDock进行式I底物与转氨酶蛋白质三维结构的进行结合模拟,最后通过Pymol分析,选择有可能与底物结合相关的氨基酸作为突变氨基酸。The three-dimensional structure of SEQ ID NO.1 was obtained through an online protein structure prediction tool, and the three-dimensional structure of the transaminase with the highest structural similarity (6S4G, homology 84.97%) was obtained through PDB for structural comparison. Then, the binding simulation of the substrate of formula I and the three-dimensional structure of the transaminase protein was performed through AutoDock. Finally, amino acids that may be related to substrate binding were selected as mutant amino acids through Pymol analysis.

根据上述Pymol分析结果,使SEQ ID NO.1所示野生型转氨酶氨基酸序列中的至少1个位点发生突变:第60位、第89位、第121位、第153位、第226位、第261位、第380位和第417位。且第60位W突变为A、D或K;第89位F突变为A、D或K;第121位的S突变为A、D或F;第153位的Y突变为A、D或F;第226位的E突变为A、R或F;第261位V突变为A、D或Y;第380位Q突变为A、K或F。根据对接结果分析底物结合区域与底物距离

Figure BDA0003752745160000111
疏水氨基酸,优选使SEQ ID NO.1所示野生型转氨酶氨基酸序列中的至少1个位点发生突变:W60A,F89K,S121D,Y153A,E226R,V261A,Q380F,A417F,根据Pymol分析,最终设计从组合突变位点:(i)W60A,F89K,S121D;(ii)W60A,F89K,S121D,Y153A;(iii)W60A,F89K,S121D,Y153A,E226R;(iv)W60A,F89K,S121D,Y153A,E226R,V261A;(v)W60A,F89K,S121D,Y153A,E226R,V261A,Q380F;(vi)W60A,F89K,S121D,Y153A,E226R,V261A,Q380F,A417F中筛选高活性突变株。其中,在对转氨酶基进行突变时,采用的是无缝克隆的方式,pET28a质粒上的引物分别位于转氨酶基因的上下游。突变位点处设置两端同源臂15bp的引物,PCR反应条件为:95℃预变性5min;94℃变性30s,53-60℃退火15s及72℃延伸50s进行30个循环;于72℃下继续延伸10min,冷却至4℃。According to the above Pymol analysis results, at least one site in the wild-type aminotransferase amino acid sequence shown in SEQ ID NO.1 is mutated: position 60, position 89, position 121, position 153, position 226, position 261, position 380 and position 417. And the W at position 60 is mutated to A, D or K; the F at position 89 is mutated to A, D or K; the S at position 121 is mutated to A, D or F; the Y at position 153 is mutated to A, D or F; the E at position 226 is mutated to A, R or F; the V at position 261 is mutated to A, D or Y; the Q at position 380 is mutated to A, K or F. According to the docking results, the distance between the substrate binding region and the substrate is analyzed.
Figure BDA0003752745160000111
Hydrophobic amino acids, preferably mutating at least one site in the wild-type aminotransferase amino acid sequence shown in SEQ ID NO.1: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F. According to Pymol analysis, the final design is from the combination of mutation sites: (i) W60A, F89K, S121D; (ii) W60A, F89K, S121D, Y153A; (iii) W60A, F89K, S121D, Y153A, E226R; (iv) W60A, F89K, S121D, Y153A, E226R, V261A; (v) W60A, F89K, S121D, Y153A, E226R, V261A, Q380F; (vi) W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F were selected for high-activity mutants. When the aminotransferase base was mutated, a seamless cloning method was used, and the primers on the pET28a plasmid were located at the upstream and downstream of the aminotransferase gene, respectively. Primers with 15 bp homology arms at both ends were set at the mutation site, and the PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 53-60°C for 15 s, and extension at 72°C for 50 s for 30 cycles; extension was continued at 72°C for 10 min and cooled to 4°C.

按照上述方法PCR扩增后片段与pET28a质粒载体,用无缝克隆试剂盒连接,将连接后载体转入之大肠杆菌BL21(DE3)中建立转氨酶基因突变体利用大肠杆菌BL21(DE3)为宿主,pET28a质粒为载体,表达扩展突变的转氨酶。The fragment after PCR amplification according to the above method was connected with the pET28a plasmid vector using a seamless cloning kit, and the connected vector was transferred into Escherichia coli BL21 (DE3) to establish the transaminase gene mutant. Escherichia coli BL21 (DE3) was used as the host and the pET28a plasmid was used as the vector to express the extended mutant transaminase.

本发明以下面的实施例5所述的酶活力检测方法筛选高活性突变株,对突变后高活性转氨酶基因进行鉴定。检测结果见下表1:The present invention uses the enzyme activity detection method described in Example 5 below to screen high-activity mutants and identify the high-activity transaminase gene after mutation. The detection results are shown in Table 1 below:

表1.Table 1.

Figure BDA0003752745160000121
Figure BDA0003752745160000121

注:上表中,+代表底物转化率大于等于0%且小于20%,++代表底物转化率大于等于20%且小于40%,+++代表底物转化率大于等于40%且小于80%,++++代表底物转化率大于等于80%。Note: In the above table, + represents that the substrate conversion rate is greater than or equal to 0% and less than 20%, ++ represents that the substrate conversion rate is greater than or equal to 20% and less than 40%, +++ represents that the substrate conversion rate is greater than or equal to 40% and less than 80%, and ++++ represents that the substrate conversion rate is greater than or equal to 80%.

对突变后高活性转氨酶的基因进行鉴定,得到具有组合突变位点(vi)的突变体(其氨基酸序列如SEQ ID NO.3所示)的核苷酸序列如SEQ ID NO.4所示。The gene of the mutated high-activity transaminase was identified, and the nucleotide sequence of the mutant having the combined mutation site (vi) (whose amino acid sequence is shown in SEQ ID NO.3) was obtained as shown in SEQ ID NO.4.

实施例3:摇瓶中小规模生产转氨酶突变体蛋白Example 3: Small-scale production of transaminase mutant proteins in shake flasks

将单个大肠杆菌转氨酶突变体((i)、(ii)、(iii)、(iv)、(v)、(vi))的重组细胞在LB液体培养基(100μg/ml卡那霉素)中培养,37℃和220rpm下过夜培养;将培养物以1:100比例转接入50mL新鲜LB培养基(250mL摇瓶)中,并在37℃下生长。当600nm处的光密度(OD600)达到约0.6时,加入异丙基硫代半乳糖苷(IPTG),使其终浓度为1mM,细胞在25℃下生长16小时。12000rpm、4℃离心10min,弃上清,细胞沉淀以预冷的100mM Tris-HCl缓冲液(pH8.5)按200g/L重悬后,超声破碎,然后12000rpm、4℃离心30min,收集上清,即粗酶液,储存于-20℃。对粗酶液进行聚丙烯酰胺凝胶电泳,结果见图2a,其中图2a中从左至右的泳道分别为Marker、空白、野生型、突变体(i)、突变体(ii)、突变体(iii)、突变体(iv)、突变体(v)和突变体(vi)的聚丙烯酰胺凝胶电泳结果。图2b是突变体(vi)的聚丙烯酰胺凝胶电泳结果,其中从左至右的泳道分别为Marker和突变体(vi)的聚丙烯酰胺凝胶电泳结果。The recombinant cells of the single E. coli aminotransferase mutants ((i), (ii), (iii), (iv), (v), (vi)) were cultured in LB liquid medium (100 μg/ml kanamycin) at 37°C and 220 rpm overnight; the culture was transferred into 50 mL of fresh LB medium (250 mL shake flask) at a ratio of 1:100 and grown at 37°C. When the optical density at 600 nm (OD600) reached about 0.6, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown at 25°C for 16 hours. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in pre-cooled 100 mM Tris-HCl buffer (pH 8.5) at 200 g/L, ultrasonically disrupted, and then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C. The crude enzyme solution was subjected to polyacrylamide gel electrophoresis, and the results are shown in Figure 2a, wherein the lanes from left to right in Figure 2a are the polyacrylamide gel electrophoresis results of Marker, blank, wild type, mutant (i), mutant (ii), mutant (iii), mutant (iv), mutant (v) and mutant (vi). Figure 2b is the polyacrylamide gel electrophoresis result of mutant (vi), wherein the lanes from left to right are the polyacrylamide gel electrophoresis results of Marker and mutant (vi).

实施例4:转氨酶突变体蛋白的高密度发酵制备Example 4: High-density fermentation preparation of transaminase mutant protein

接种实施例2所得的重组突变菌株于3mL液体LB培养基中,37℃、220rpm震荡过夜培养后,按1%左右的比例接种于400mL液体LB培养基中,培养至OD600达到4时作为种子液,接入2L的发酵培养基中进行高密度发酵。初始温度37℃,搅拌速度300rpm,通气量1.5vvm/L/min,pH为6.8,之后不断提高搅拌转速最大至1000rpm。发酵培养分为两个阶段,第一阶段接种后,培养4小时左右,培养基中的碳源消耗完全,按照DO进行反馈补料。补料后温度降为25℃,溶氧保持30%以上,补料8小时后加入异丙基硫代半乳糖苷(IPTG)进行诱导,诱导12小时后放罐。使用离心机8000rpm离心10min弃上清得到的菌体湿细胞,用100mM Tris-HCl缓冲液(pH8.5)悬浮,利用高压均质破碎机对悬浮细胞进行破碎,得到高密度发酵转氨酶的粗酶液。The recombinant mutant strain obtained in Example 2 was inoculated into 3mL liquid LB medium, and after overnight culture at 37°C and 220rpm, it was inoculated into 400mL liquid LB medium at a ratio of about 1%, and cultured until OD600 reached 4 as seed liquid, and then accessed into 2L fermentation medium for high-density fermentation. The initial temperature was 37°C, the stirring speed was 300rpm, the ventilation volume was 1.5vvm/L/min, and the pH was 6.8, and then the stirring speed was continuously increased to a maximum of 1000rpm. The fermentation culture was divided into two stages. After the first stage was inoculated, it was cultured for about 4 hours, the carbon source in the culture medium was completely consumed, and the feeding was fed back according to DO. After feeding, the temperature dropped to 25°C, the dissolved oxygen was maintained at more than 30%, and isopropylthiogalactoside (IPTG) was added for induction after 8 hours of feeding, and the tank was released after 12 hours of induction. The wet cells were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were suspended in 100 mM Tris-HCl buffer (pH 8.5). The suspended cells were crushed by a high-pressure homogenizer to obtain a crude enzyme solution of high-density fermentation transaminase.

实施例5:转氨酶的全细胞催化反应体系Example 5: Whole-cell catalytic reaction system of transaminase

本实施例使用的底物为下面的式(I)所示的化合物:该底物经本发明的转氨酶催化反应形成式(II)所示化合物。The substrate used in this example is a compound represented by the following formula (I): the substrate is catalyzed by the transaminase of the present invention to form a compound represented by formula (II).

Figure BDA0003752745160000141
Figure BDA0003752745160000141

室温下将分别含有上述转氨酶突变体(i)、(ii)、(iii)、(iv)、(v)和(vi)的湿细胞48g(发酵液离心后固体)与23mL 6M异丙胺、100mg磷酸吡哆醛和72mL的100mM Tris-HCl缓冲液(pH8.5)加入到250mL玻璃反应器中。以100rpm搅拌20min以混匀所有固体,加入10%氢氧化钠溶液调节pH至8.5。随后,使用循环水浴将温度提高到50℃。然后将3g底物(前述化合物I)溶解在5mL的DMSO中,在250mL的反应器中以500rpm缓慢加入搅拌混合物中。在反应过程中监测pH控制在8.5,并对混合物取样进行HPLC分析。12h后,突变体(i)、(ii)、(iii)、(iv)、(v)和(vi)催化条件下的底物(化合物(I))的转化率分别为4%、9%、12%、16%、45%和85.9%;24h后,突变体(i)、(ii)、(iii)、(iv)、(v)和(vi)催化条件下的底物的转化率分别为17%、19%、26%、31%、60%和99.9%。反应完全后,将含有突变体(vi)的体系用盐酸调至pH=2-3,过滤。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH=12,再用50mL二氯甲烷萃取2次。合并有机相用无水硫酸钠干燥后于T<40℃条件下浓缩。得到目标产物经HPLC检测,纯度>98%,de值>99%,收率90%。产物的1HNMR图谱图3,与标准品的1HNMR图谱一致。At room temperature, 48 g of wet cells (solids after centrifugation of the fermentation broth) containing the above-mentioned transaminase mutants (i), (ii), (iii), (iv), (v) and (vi) were added to a 250 mL glass reactor with 23 mL of 6 M isopropylamine, 100 mg of pyridoxal phosphate and 72 mL of 100 mM Tris-HCl buffer (pH 8.5). Stir at 100 rpm for 20 min to mix all the solids, and add 10% sodium hydroxide solution to adjust the pH to 8.5. Subsequently, a circulating water bath was used to increase the temperature to 50° C. Then 3 g of substrate (the aforementioned compound I) was dissolved in 5 mL of DMSO and slowly added to the stirred mixture at 500 rpm in a 250 mL reactor. The pH was monitored during the reaction and controlled at 8.5, and the mixture was sampled for HPLC analysis. After 12 hours, the conversion rates of the substrate (compound (I)) under the catalytic conditions of mutants (i), (ii), (iii), (iv), (v) and (vi) were 4%, 9%, 12%, 16%, 45% and 85.9%, respectively; after 24 hours, the conversion rates of the substrate under the catalytic conditions of mutants (i), (ii), (iii), (iv), (v) and (vi) were 17%, 19%, 26%, 31%, 60% and 99.9%, respectively. After the reaction was complete, the system containing mutant (vi) was adjusted to pH = 2-3 with hydrochloric acid and filtered. After filtration, the filtrate was extracted with 50 mL of methyl tert-butyl ether. The aqueous phase was adjusted to pH = 12 and then extracted twice with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated at T < 40 ° C. The target product was obtained and tested by HPLC, with a purity of > 98%, a de value of > 99%, and a yield of 90%. The 1 H NMR spectrum of the product is shown in Figure 3, which is consistent with the 1 H NMR spectrum of the standard product.

实施例6:转氨酶和突变体的温度稳定性及催化效果Example 6: Temperature stability and catalytic effect of transaminases and mutants

在实施例5的催化反应体系下,分别取野生型和突变体(vi)(其氨基酸序列如SEQID NO:3所示)的转氨酶粗酶液,在不同温度(30~50℃,温度间隔10℃)孵育12、24、48小时,然后按照如下方法测定残余酶活力(酶活力定义为:在上述条件下,每分钟生成1μmol的产物(前述化合物II)所需的酶量定义为1个酶活单位)。In the catalytic reaction system of Example 5, crude enzyme solutions of wild-type and mutant (vi) (whose amino acid sequence is shown in SEQ ID NO: 3) were taken respectively, incubated at different temperatures (30-50° C., with a temperature interval of 10° C.) for 12, 24, and 48 hours, and then the residual enzyme activity was determined according to the following method (enzyme activity is defined as: under the above conditions, the amount of enzyme required to generate 1 μmol of product (the aforementioned compound II) per minute is defined as 1 enzyme activity unit).

10.2ml100mMTris-Hcl,4.8ml 6M异丙胺,20mg磷酸吡哆醛,0.4g底物(前述化合物I)溶于1mlDMSO,4ml粗酶液(1.6g湿细胞)。45℃/200rpm反应3小时取样测定。对照为不进行温度处理的酶,其活性定为100%。残余酶活力降至原始酶活50%左右的时间,为酶在该温度下的半衰期,以此确定转氨酶的温度稳定性。10.2ml100mMTris-Hcl, 4.8ml6Misopropylamine, 20mgpyridoxal phosphate, 0.4gsubstrate (the aforementioned compound I) dissolved in 1mlDMSO, 4mlcrude enzyme solution (1.6gwet cells). 45℃/200rpm for 3 hours and then sampled for determination. The control is the enzyme without temperature treatment, and its activity is set as 100%. The time when the residual enzyme activity drops to about 50% of the original enzyme activity is the half-life of the enzyme at this temperature, which is used to determine the temperature stability of the transaminase.

温度temperature 野生型转氨酶Wild-type transaminase 突变体转氨酶Mutant transaminase 30℃30℃ 大于48hMore than 48 hours 大于48hMore than 48 hours 40℃40℃ 24h24h 大于48hMore than 48 hours 50℃50℃ 12h12h 大于48hMore than 48 hours

在实施例5的反应体系下,分别取野生和突变的转氨酶粗酶液,于50℃反应,液相色谱分析测定底物转化率。In the reaction system of Example 5, crude enzyme solutions of wild-type and mutant transaminase were respectively taken and reacted at 50° C. The substrate conversion rate was determined by liquid chromatography analysis.

Figure BDA0003752745160000151
Figure BDA0003752745160000151

以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为落入本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as falling within the scope of protection of the present invention.

Claims (13)

1.一种转氨酶突变体,其特征在于,所述转氨酶突变体的氨基酸序列是由SEQ ID NO:1所示的氨基酸序列发生突变得到的氨基酸序列,所述突变至少包括下列突变位点中的一个:1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes at least one of the following mutation sites: 第60位由W突变为A、D或K,第89位由F突变为A、D或K,第121位由S突变为A、D或F,第153位由Y突变为A、D或F,第226位由E突变为A、R或F,第261位由V突变为A、D或Y,第380位由Q突变为A、K或F,第417位由A突变为F、D或K,或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。The 60th position mutates from W to A, D or K, the 89th position mutates from F to A, D or K, the 121st position mutates from S to A, D or F, the 153rd position mutates from Y to A, D or F, the 226th position mutates from E to A, R or F, the 261st position mutates from V to A, D or Y, the 380th position mutates from Q to A, K or F, and the 417th position mutates from A to F, D or K, or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. 2.根据权利要求1所述转氨酶突变体,其特征在于,所述突变至少包括下列突变位点中的一个:2. The aminotransferase mutant according to claim 1, characterized in that the mutation comprises at least one of the following mutation sites: 第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A,第380位由Q突变为F,第417位由A突变为F,或者所述转氨酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点,且与发生突变的氨基酸序列具有80%以上同源性的氨基酸序列。Position 60 mutates from W to A, position 89 mutates from F to K, position 121 mutates from S to D, position 153 mutates from Y to A, position 226 mutates from E to R, position 261 mutates from V to A, position 380 mutates from Q to F, and position 417 mutates from A to F, or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. 3.根据权利要求2所述的转氨酶突变体,其特征在于,所述转氨酶突变体包括下列突变位点:3. The transaminase mutant according to claim 2, characterized in that the transaminase mutant comprises the following mutation sites: 第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A和第226位由E突变为R,或者The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A and the 226th position mutates from E to R, or 第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R和第261位由V突变为A,或者The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A, the 226th position mutates from E to R and the 261st position mutates from V to A, or 第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A和第380位由Q突变为F,或者position 60 from W to A, position 89 from F to K, position 121 from S to D, position 153 from Y to A, position 226 from E to R, position 261 from V to A and position 380 from Q to F, or 第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A,第380位由Q突变为F和第417位由A突变为F。Position 60 mutated from W to A, position 89 mutated from F to K, position 121 mutated from S to D, position 153 mutated from Y to A, position 226 mutated from E to R, position 261 mutated from V to A, position 380 mutated from Q to F and position 417 mutated from A to F. 4.根据权利要求3所述的转氨酶突变体,其特征在于,所述转氨酶突变体包括突变位点:第60位由W突变为A,第89位由F突变为K,第121位由S突变为D,第153位由Y突变为A,第226位由E突变为R,第261位由V突变为A,第380位由Q突变为F,第417位由A突变为F,所述转氨酶突变体的氨基酸序列如SEQ ID NO.3所示。4. The transaminase mutant according to claim 3, characterized in that the transaminase mutant includes mutation sites: position 60 mutates from W to A, position 89 mutates from F to K, position 121 mutates from S to D, position 153 mutates from Y to A, position 226 mutates from E to R, position 261 mutates from V to A, position 380 mutates from Q to F, and position 417 mutates from A to F, and the amino acid sequence of the transaminase mutant is shown in SEQ ID NO.3. 5.一种编码权利要求1至4任一项所述的转氨酶突变体的基因。5. A gene encoding the transaminase mutant according to any one of claims 1 to 4. 6.根据权利要求5所述的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.4所示,或者6. The gene according to claim 5, characterized in that the nucleotide sequence of the gene is as shown in SEQ ID NO.4, or 所述基因的核苷酸序列为与SEQ ID NO.4所示的核苷酸序列具有95%以上同源性的序列。The nucleotide sequence of the gene is a sequence having more than 95% homology with the nucleotide sequence shown in SEQ ID NO.4. 7.一种重组表达载体,其特征在于,所述重组表达载体包含权利要求5或6所述的基因。7. A recombinant expression vector, characterized in that the recombinant expression vector comprises the gene according to claim 5 or 6. 8.根据权利要求7所述的重组表达载体,其特征在于,所述重组表达载体选自pET-28a、pET-dute1或pRSF-dute1,更优选pET-28a。8 . The recombinant expression vector according to claim 7 , characterized in that the recombinant expression vector is selected from pET-28a, pET-dute1 or pRSF-dute1, more preferably pET-28a. 9.一种基因工程菌,其特征在于,所述基因工程菌用于生产权利要求1至4任一项所述的转氨酶突变体,其包含权利要求7或8所述的重组表达载体。9. A genetically engineered bacterium, characterized in that the genetically engineered bacterium is used to produce the transaminase mutant according to any one of claims 1 to 4, and comprises the recombinant expression vector according to claim 7 or 8. 10.根据权利要求9所述的基因工程菌,其特征在于,所述基因工程菌选自大肠杆菌MG1655或大肠杆菌BL21(DE3)或大肠杆菌BL21(DE3)pLysS,更优选大肠杆菌BL21(DE3)。10. The genetically engineered bacterium according to claim 9, characterized in that the genetically engineered bacterium is selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS, more preferably Escherichia coli BL21 (DE3). 11.一种权利要求1至4任一项所述的转氨酶突变体在催化羰基类化合物制备手性胺中的应用。11. Use of the aminotransferase mutant according to any one of claims 1 to 4 in catalyzing the preparation of chiral amines from carbonyl compounds. 12.一种生产手性胺的方法,其特征在于,所述方法包括利用权利要求1至4任一项所述的转氨酶突变体将酮类化合物进行催化转氨基。12. A method for producing chiral amines, characterized in that the method comprises catalyzing the transamination of ketone compounds using the transaminase mutant according to any one of claims 1 to 4. 13.根据权利要求12所述的方法,其特征在于,所述酮类化合物为
Figure FDA0003752745150000031
其中R1和R2各自独立地为选取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基;或者
13. The method according to claim 12, characterized in that the ketone compound is
Figure FDA0003752745150000031
wherein R1 and R2 are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon group; or
R1和R2可单独或两者互相结合形成取代或未被取代的环, R1 and R2 may be combined alone or together to form a substituted or unsubstituted ring. 所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。The substitution refers to substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.
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