阅读说明：本技术 ω-转氨酶突变体及其在制备西他列汀中间体中的应用 (Omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate ) 是由 柳志强 程峰 陈秀玲 张晓健 郑裕国 何人宝 金逸中 林娇华 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种ω-转氨酶突变体及其在制备西他列汀中间体中的应用,该ω-转氨酶突变体由SEQ ID No.2所示氨基酸序列的第56位或第134位进行单突变获得；第56位的苯丙氨酸替换成缬氨酸、组氨酸或酪氨酸；第134位的苏氨酸替换成甘氨酸。本发明通过分子对接和同源建模等方式获得一些可能影响其催化活性的位点,对其进行了定点突变,最终得到的ω-转氨酶突变体不仅具有较高的酶活,酶活均高于300U/g,至少是野生型的2.7倍,而且能够高效催化西他列汀中间体前体酮1-哌啶-4-(2,4,5-三氟苯基)-1,3-二丁酮合成西他列汀中间体(R)-3-氨基-1-哌啶-4-(2,4,5-三氟苯基)-1-丁酮,转化率高达85％。(The invention discloses a omega-transaminase mutant and application thereof in preparing sitagliptin intermediates, wherein the omega-transaminase mutant is obtained by performing single mutation on the 56 th site or the 134 th site of an amino acid sequence shown in SEQ ID No. 2; a substitution of phenylalanine at position 56 with valine, histidine or tyrosine; threonine at position 134 is replaced with glycine. According to the invention, some sites which may influence the catalytic activity of the mutant are obtained through molecular docking, homologous modeling and other modes, site-directed mutation is carried out on the mutant, and the finally obtained omega-transaminase mutant not only has higher enzyme activity which is higher than 300U/g and at least 2.7 times of that of a wild type, but also can efficiently catalyze sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrup to synthesize the sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone, and the conversion rate is as high as 85%.)

1. A ω -transaminase mutant, characterized in that it is obtained by single mutation at position 56 or 134 of the amino acid sequence shown in SEQ ID No. 2;

Wherein the phenylalanine at position 56 is mutated to valine, histidine or tyrosine; threonine at position 134 is mutated to glycine.

2. A gene encoding the ω -transaminase mutant of claim 1.

3. A recombinant vector comprising the encoding gene of claim 2.

4. A genetically engineered bacterium comprising the coding gene of claim 2.

5. The use of the ω -transaminase mutant according to claim 1 in biocatalytic synthesis of sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone from sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutbutanone.

6. The use of the genetically engineered bacterium of claim 4 in catalyzing sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrrone to synthesize sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone.

7. A method for synthesizing a sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone by biocatalyzing a sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutbutanone is characterized by comprising the following steps: the method comprises the steps of carrying out a biocatalytic synthesis reaction in a buffer solution by using 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutanone as a substrate, a omega-transaminase mutant as claimed in claim 1 or a genetically engineered bacterium as claimed in claim 4 as a biocatalyst, isopropylamine as an amino donor and pyridoxal phosphate as a coenzyme to obtain the (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone.

8. The method as claimed in claim 7, wherein the reaction temperature is 40-45 ℃, the stirring speed is 500-600rpm, and the reaction time is 40-50 h.

9. the method of claim 7, wherein the molar ratio of substrate to amino donor is from 1:7 to 12; the molar ratio of the substrate to the biocatalyst is 1: 1-3.

10. The method of claim 9, wherein the final concentration of the substrate is 2 to 50g/L, the final concentration of the amino donor is 25 to 95g/L, the final concentration of the coenzyme is 0.3 to 0.5g/L, and the amount of the biocatalyst added is 10 to 50g/L, based on the total volume of the buffer solution in the reaction system.

Technical Field

The invention relates to the technical field of biochemical engineering, in particular to an omega-transaminase mutant and application thereof in preparation of a sitagliptin intermediate, and especially relates to application in preparation of a sitagliptin intermediate 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyryl ethyl ketone.

Background

Sitagliptin (sitagliptin), which was developed and developed by Merck corporation and Codexis corporation in the united states, was the first active ingredient to obtain a dipeptidyl peptidase-4 (DPP-4) inhibitor approved by FDA for the treatment of type II diabetes (2016 (10 months), and also an oral therapeutic drug for type II diabetes, linagliptin (sitagliptin hydrochloride tablet, JANUVIA). Sitagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor, the activity of sitagliptin is inhibited by combining an amide part and a DPP-4 active part, the half-life period of the Incretin (Incretin) is prolonged, the GLP-1 and GIP activity in plasma is improved, the content of the Incretin is only slightly increased, side effects caused by overhigh GLP-1 content are avoided, and the sitagliptin has better safety and tolerance.

In the aspect of combined medication, the combined medication of the metformin hydrochloride and the pioglitazone is better than the independent medication of the metformin hydrochloride and the pioglitazone. Compared with similar medicines, the application of the sitagliptin in the clinic has outstanding medication advantages, and the sitagliptin has better research value based on good market prospect. Jiranavir (janivia), currently approved for use in over 70 countries around the world, has been sold around $ 40 billion in recent years ($ 61 billion in total in 2016 in combination with metformin), and is a top-20-strength drug worldwide.

The preparation of the optically pure sitagliptin and the intermediate thereof mainly comprises a chemical synthesis method, a chiral resolution method and an asymmetric synthesis method. The chemical synthesis method has various process steps, the price of the needed synthetic reagent is high, the cost investment is high, and part of the reagent has certain toxic action on the environment and human body, so the current green and environment-friendly requirements cannot be met. Chiral reagents are used in the process of preparing the optically pure sitagliptin and the intermediate thereof by chiral resolution, the resolution yield is only 50% at most, and the chiral resolution process is complex. The asymmetric synthesis method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easy separation and purification and the like, and is suitable for large-scale preparation of sitagliptin and intermediates thereof. The key to the asymmetric synthesis is to obtain an omega-transaminase which catalyzes the asymmetric transamination reaction to obtain an optically pure sitagliptin intermediate.

international patent application W02004087650 discloses the synthesis route of sitagliptin intermediates by merck company, using asymmetric hydrogenation of ketones with chiral ruthenium catalysts to build chiral alcohols, which are then converted to chiral amines. In the synthesis method, asymmetric hydrogenation catalyzed by ruthenium is needed, the catalyst is expensive, the total yield is only 52%, high-pressure hydrogen is used in the process, and the stereoselectivity is not high.

International patent application W02007050485 discloses a synthesis method of sitagliptin intermediates by merck corporation, which adopts asymmetric hydrogenation of enamine by chiral germanium catalyst to synthesize chiral amine, the yield reaches 84%, the ee value is 94%, but the method needs expensive germanium chiral catalyst, and the removal and recovery are difficult.

chinese patent application document CN102838511A discloses a production method of sitagliptin intermediate in Zhejiang Haixiang pharmaceutical industry, which adopts Grignard reagent to carry out nucleophilic substitution on chiral epichlorohydrin, and then uses cyanide to carry out substitution hydrolysis to synthesize beta-hydroxy acid, wherein the total yield of the method is only 40%, and the method adopts highly toxic cyanide and has limited application.

chinese patent application CN102485718A discloses a route for sitagliptin intermediate synthesis in the pharmaceutical industry in the zhejiang sea, by using methionine as chiral source, but with a yield of only 14%.

Chinese patent application CN103014081A discloses transamination of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -methyl butyrate to (R) -3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate using transaminase by suzhou han enzyme company, but does not disclose the sequence and cloning method of the specific transaminase.

In recent years, asymmetric synthesis methods have the advantages of high selectivity and environmental friendliness, and gradually become the first choice for synthesizing chiral medicinal chemicals and intermediates thereof. The omega-aminotransferase is a key enzyme for producing sitagliptin, and a plurality of omega-aminotransferase genes are expressed in different hosts (escherichia coli, pichia pastoris and the like), so that the genetic engineering bacteria with high enzyme activity and selectivity are obtained. Nevertheless, there are some problems that ω -transaminase has low enzymatic activity for a specific substrate and low conversion rate, especially when the substrate contains large groups such as benzene ring.

Disclosure of Invention

The invention provides an omega-transaminase mutant and application thereof in preparation of a sitagliptin intermediate, and particularly relates to application of the mutant in preparation of a sitagliptin intermediate 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrrone, wherein the mutant not only has high enzyme activity, but also can efficiently catalyze a sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrrone to synthesize a sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone, and the conversion rate is as high as 85%.

The specific technical scheme is as follows:

A ω -transaminase mutant obtained by single mutation at position 56 or 134 of the amino acid sequence shown in SEQ ID No. 2;

Wherein the phenylalanine at position 56 is mutated to valine, histidine or tyrosine; threonine at position 134 is mutated to glycine.

The amino acid sequence shown in SEQ ID No.2 is the amino acid sequence of wild type omega-transaminase from Arthrobacter calmette guensis (Arthrobacter cumminsii), and the nucleotide sequence is shown in SEQ ID No. 1.

According to the invention, some sites which can influence the catalytic activity of the mutant are obtained through modes such as molecular docking, homologous modeling and the like, and the sites are distributed near an active pocket, in a PLP binding region and on some sites far away from an active center.

The invention also provides a coding gene of the omega-transaminase mutant.

The omega-transaminase mutant is obtained by mutating phenylalanine (codon TTC) at the 56 th site of an amino acid sequence shown in SEQ ID No.2 into valine (codon GTC), histidine (codon CAC) and tyrosine (codon GAC); alternatively, the mutation from threonine (codon ACT) at position 134 to glycine (codon GGT).

The invention also provides a recombinant vector containing the coding gene.

The invention also provides a genetic engineering bacterium containing the coding gene.

The invention also provides application of the omega-aminotransferase mutant in biocatalysis of sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrne to synthesis of sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone.

The invention also provides application of the genetic engineering bacteria in catalyzing sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrrone to synthesize sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone.

The invention also provides a method for synthesizing a sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone by biocatalyzing a sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutbutanone, which comprises the following steps: the method comprises the steps of carrying out a biocatalytic synthesis reaction in a buffer solution by using 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutanone as a substrate, a omega-transaminase mutant as claimed in claim 1 or a genetically engineered bacterium as claimed in claim 4 as a biocatalyst, isopropylamine as an amino donor and pyridoxal phosphate as a coenzyme to obtain the (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone.

Furthermore, in the reaction system, the reaction temperature is 40-45 ℃, the stirring speed is 500-600rpm, and the reaction time is 40-50 h.

Further, based on the total volume of buffer solution in the reaction system, the final concentration of the substrate is 2-50g/L, the final concentration of the amino donor is 25-95g/L, the final concentration of the coenzyme is 0.3-0.5g/L, and the addition amount of the biocatalyst is 10-50 g/L.

Compared with the prior art, the invention has the following beneficial effects:

According to the invention, through modes such as molecular docking, homologous modeling and the like, a large number of sites which can influence the catalytic activity of 336 amino acids (among 1008 DNA bases) are obtained through calculation and analysis, site-directed mutation is carried out on the sites, and finally the obtained omega-transaminase mutant not only has high enzymatic activity (the enzymatic activity is higher than 245U/g and at least 2 times of that of a wild type), but also can efficiently catalyze sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrup to synthesize sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone: the conversion rate of the mutant T134G can reach 80% after 24h reaction, and the conversion rate reaches 85% after 36h reaction; compared with wild enzyme, the reaction time is shortened by half, the conversion rate can reach 80%, and the total conversion rate is greatly improved.

Drawings

FIG. 1 is a schematic diagram of a reaction equation for synthesizing a sitagliptin intermediate (R) -3-amino-1-piperidine-4- (2,4, 5-trifluorophenyl) -1-butanone by catalyzing sitagliptin intermediate precursor ketone 1-piperidine-4- (2,4, 5-trifluorophenyl) -1, 3-dibutyrrone with omega-transaminase according to the invention.

FIG. 2 is a SDS-PAGE pattern after purification of the transaminase in example 2;

Wherein, lane 1 is protein molecular weight Marker, and lane 2 is purified transaminase AbTA.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.