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CN110205310B - Transaminase mutants and uses thereof - Google Patents

️Fri Oct 29 2021

CN110205310B - Transaminase mutants and uses thereof - Google Patents

Transaminase mutants and uses thereof Download PDF

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CN110205310B

CN110205310B CN201910464742.2A CN201910464742A CN110205310B CN 110205310 B CN110205310 B CN 110205310B CN 201910464742 A CN201910464742 A CN 201910464742A CN 110205310 B CN110205310 B CN 110205310B Authority

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transaminase

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洪浩

詹姆斯·盖吉

张娜

刘芳

颜俊杰

刘冶

王祖建

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Asymchem Life Science Tianjin Co Ltd

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2019-05-30 Application filed by Asymchem Life Science Tianjin Co Ltd filed Critical Asymchem Life Science Tianjin Co Ltd

2019-05-30 Priority to CN202111205871.3A priority Critical patent/CN113817703B/en

2019-05-30 Priority to CN202111204443.9A priority patent/CN113736759B/en

2019-05-30 Priority to CN202111205863.9A priority patent/CN113817702B/en

2019-05-30 Priority to CN201910464742.2A priority patent/CN110205310B/en

2019-09-06 Publication of CN110205310A publication Critical patent/CN110205310A/en

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Abstract

The invention discloses a transaminase mutant and application thereof. The amino acid sequence of the transaminase mutant is represented by SEQ ID NO: 1, the mutation at least comprises one of the following mutation sites: 3 rd, 5 th, 8 th, 25 th, 32 th, 45 th, 56 th, 59 th, 60 th, 84 th, 86 th, 164 th, 176 th, 178 th, 180 th, 187 th, 197 th, 206 th, 207 th, 242 th, 245 th, 319 th, 324 th and the like; or the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence. The application of the mutant can improve the reaction rate, improve the enzyme stability, reduce the enzyme dosage and reduce the difficulty of post-treatment, so that the mutant can be suitable for industrial production.

Description

Transaminase mutants and uses thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a transaminase mutant and application thereof.

Background

Chiral amines are widely found in nature and are important intermediates for the synthesis of natural products and chiral drugs. Many chiral amines contain one or more chiral centers, and different chiral drugs differ significantly in pharmacological activity, metabolic processes, metabolic rates, and toxicity, usually with one enantiomer being effective and the other enantiomer being ineffective or ineffective, and even toxic. Therefore, how to efficiently and stereoselectively construct compounds containing chiral centers has important significance in medicine research and development.

Chiral amines are important components for synthesizing various bioactive compounds and active pharmaceutical ingredients, and it is estimated that at present, 40% of drugs are chiral amines and derivatives thereof, and the synthesis of drugs such as neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, vaccines and the like takes chiral amines as intermediates (top. total. 2014,57,284-300), which makes chiral amine compounds important components in the pharmaceutical industry.

There are a number of commercial processes for chiral amines, which rely primarily on the metal-catalyzed hydrogenation of enamides from ketone precursors, and which require expensive transition metal complexes as catalysts, which are difficult to achieve sustainability due to their limited resources. Meanwhile, the process of asymmetric synthesis of chiral amines from ketone precursors requires amine protection and deprotection steps, increasing steps and waste, decreasing yield.

The catalyst in the method for synthesizing chiral amine by catalytic hydrogenation reduction is difficult to prepare and expensive, the equipment investment is large, the production cost is high, the requirements on the activity and the hydrogenation conditions of the catalyst are high, and the catalyst is toxic, particularly sulfides in hydrogen are easy to generate personnel poisoning.

Transaminase is a generic term for enzymes that catalyze the transfer of amino groups from 1 amino donor (amino acid or amine) to prochiral acceptor ketones using pyridoxal phosphate as a cofactor to yield chiral amines or their by-product ketones or alpha-keto acids. Because the traditional chemical method for synthesizing the amine asymmetrically has different limitations, such as low efficiency, low selectivity, serious environmental pollution and the like, and meanwhile, the transaminase-catalyzed synthesis of the chiral amine has high stereoselectivity and chemical selectivity, has safety and environmental compatibility, is a green and environment-friendly process and is a green chemistry. Meanwhile, the enzyme catalysis is often in place by one step, and the method has incomparable advantages compared with a chemical method, and the transaminase is used for synthesizing the chiral compound and becomes a key asymmetric synthesis technology.

Although advances in the production of chiral amines using transaminases have been highly focused, enzymatic processes have presented a number of problems in scale-up production applications. For example, the low enzyme activity and the large enzyme dosage lead to the increase of the fermentation cost; meanwhile, the large enzyme dosage caused by low enzyme activity seriously hinders the industrial application of the enzyme catalysis reaction, on one hand, the large enzyme dosage causes large reaction volume, and the utilization rate of the catalysis container is reduced. Meanwhile, the volume is increased during the post-treatment, the amount of the extraction solvent is large, the extraction, concentration and obtaining of the product are difficult, the product yield is low, and the industrial application of enzyme catalysis is greatly hindered. The enzyme with high activity can reduce the enzyme dosage and the reaction volume, so that the industrial application of the enzyme catalysis becomes possible. Therefore, the method is extremely key to obtain high-activity enzyme, and can also enlarge the substrate spectrum of the enzyme, so that enzyme catalytic reactions with extremely low conversion rate and even without activity can be smoothly carried out, and extremely good conversion rate and extremely high product chiral purity can be achieved.

On the other hand, enzyme catalysis is susceptible to denaturation and inactivation by organic solvents in the reaction system or by factors such as high reaction pH and high temperature, and therefore it is also very critical to increase the tolerance of the enzyme to extreme conditions. In order to improve the production of chiral amine products, the content of organic solvents in a reaction system needs to be increased, or alkaline amino donors (such as isopropylamine) are used, so that extremely harsh reaction conditions are created, wild aminotransferases are extremely easy to denature and lose activity, and therefore, the aminotransferases which are well tolerant to both organic solvents and high pH are needed to meet the requirements of industrial production.

Disclosure of Invention

The invention aims to provide a transaminase mutant and application thereof to improve the activity of transaminase.

In order to achieve the above object, according to one aspect of the present invention, there is provided a transaminase mutant. The amino acid sequence of the transaminase mutant is represented by SEQ ID NO: 1, the mutation at least comprises one of the following mutation sites: 3 rd, 5 th, 8 th, 25 th, 32 th, 45 th, 56 th, 59 th, 60 th, 84 th, 86 th, 164 th, 176 th, 178 th, 180 th, 187 th, 197 th, 206 th, 207 th, 242 th, 245 th, 319 th, 324 th, 326 th, 328 th, 370 th, 397 th, 414 th, 416 th, 424 th, 436 th, 437 th and 442 th bits.

Further, the amino acid sequence of the transaminase mutant is represented by SEQ ID NO: 1, the mutation comprises at least one of the following mutation sites: l3, V5, I8, F25, Q32, I45, L59, F56, C60, F84, W86, F164, F176, a178, I180, S187, T197, L206, K207, V242, T245, R319, R324, E326, V328, L370, T397, P414, Q416, E424, a436, M437, R442 and R442; or an amino acid sequence of the transaminase mutant which has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence.

Further, C60Y + F164V, L3S + V5S, L3S + V5S + F164V, L3S + V5S + C60Y, L3S + V5S + C60Y + F164V, I180V + L370A, and L3S + V5S + L59V; preferably, the mutation comprises at least one of the following combinations of mutation sites: f164 + C60, E424 + A436, C60 + F164 + A436, W86 + F164, F25 + L59, F25 + F164, C60 + F164 + A436, F164 + M437, I8 + V328, I8 + F164, C60 + F164 + L370, I45 + F164, C60 + F164 + F176, C60 + F176 + F164, L3+ V5 + C60 + F164 + L370, C60 + F164 + R442, L3+ V187 + L424, C60 + F164 + R442, L164R 442, L3+ V164 + T164 + L164 + T164 + L164, L164 + T164 + L370, L3+ V + T5 + T5 + L245 + T3 + L5 + T3 + L164 + T164, L164 + T164 + L370, L164 + T164, L370, L164 + T164, L164 + T164, L164 + T164, L370, L164 + T164, L164 + T164, L370, L164 + T164, L164 + T164, L370, L164 + T70 + T164, L164 + T70, L370, L164, L370, L164 + T70 + T70, L370, L164 + T70 + T70 + T70, L164, L370, L164 + T70 + T70, L164, L370, L164 + T70 + T70 + T70 + T70, L370, L164, L370, L164, L370, l3+ V5 + L59 + F164 + L370, L3+ V5 + L59 + L370 + A436 + Q416, L + V5 + L59 + L370 + A436 + R442, L3+ V5 + L59 + V328 + L370 + R442, L3+ V5 + L59 + C60 + F164 + L370 + R442, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370, L3+ V5 + C60 + F164 + I180 + S187 + L370 + R442.

Further, the mutation at least comprises one of the following mutation sites or mutation site combinations: 7 th, 32 th, 96 th, 164 th, 171 th, 186 th, 252 th, 384 th, 389 th, 391 th, 394 th, 404 th, 411 th, 420 th, 423 th, 424 th, 442 th, 452 th and 456 th bits; preferably, the mutation further comprises at least one of the following mutation sites: K7N, Q32L, K96R, V164L, E171D, S186G, V252I, Y384F, I389M, I389F, D391E, N394D, L404Q, L404Q, G411D, Q420R, Q420K, M423K, E424R, E424K, E424Q, R442H, R442L, G452S, and K456R.

Further, the mutation includes at least one of the following combinations of mutation sites:

L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+Y384F+G452S、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+S186G+Q420R、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+M423K、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+Q420K+E424R、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+K7N+E424Q、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+D391E、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+Q32L+E171D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+I389M、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+I389F+N394D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+L404Q、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+I389F+L404Q、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+V164L+K456R、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+K96R、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+Q32L+R442H、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+R442L、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+V252I、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+E424K、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A、L3S+V5S+C60Y+F164V+Q420R+L370A、L3S+V5S+F164V+C60Y+L370A+G452S、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+E424K、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+Y384F+L404Q、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+E424K+G411D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+S186G+Q420R、L3S+V5S+C60Y+F164V+A178L+I180V+L370A+G411D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+V164L+I389F+E424Q+K96R、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+V164L+I389F+L404Q、L3S+V5S+C60Y+F164V+A178L+I180V+L370A+G411D+M423K、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+I389F+L404Q、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+V164L+E171D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+L404Q、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+V252I、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+E424Q、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+L404Q、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+E424Q、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+L404Q+E171D、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+E424Q+M423K、L3S+V5S+C60Y+F164L+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+I389F+V252I+L404Q+E171D+D391E、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+E424Q+K7N、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+E171D、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F+D391E、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D+S186G+Y384F、L3S+V5S+C60Y+F164V+I180V+L370A+G411D+R442L、C60Y+F164L+I180V+L370A+G411D+A178L+S186G+S187A+Y384F+E171D+I389F+V252I+L404Q、C60Y+F164V+I180V+L370A+G411D+A178L+S186G+S187A+Y384F+E424Q、L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A+G411D、C60Y+F164V+R442Q+G411D、L3S+V5S+F164V+C60Y+I180V+L370A+G411D、L3S+V5S+C60Y+F164V+L370A+G411D、L3S+V5S+C60Y+F164V+Q420R+L370A+G411D、C60Y+F164V+L370A+G411D、L3S+V5S+F164V+C60Y+L370A+G452S+G411D+Y384F、C60Y+F164V+R442Q+Y384F、L3S+V5S+F164V+C60Y+I180V+L370A+Y384F、L3S+V5S+C60Y+F164V+L370A+Y384F、L3S+V5S+C60Y+F164V+Q420R+L370A+Y384F、C60Y+F164V+L370A+Y384F、L3S+V5S+F164V+C60Y+L370A+G452S+Y384F、C60Y+F164V+R442Q+S186G、L3S+V5S+F164V+C60Y+I180V+L370A+S186G、L3S+V5S+C60Y+F164V+L370A+S186G、L3S+V5S+C60Y+F164V+Q420R+L370A+S186G、C60Y+F164V+L370A+S186G、L3S+V5S+F164V+C60Y+L370A+G452S+S186G、C60Y+F164V+R442Q+D391E、L3S+V5S+F164V+C60Y+I180V+L370A+D391E、L3S+V5S+C60Y+F164V+L370A+D391E、L3S+V5S+C60Y+F164V+Q420R+L370A+D391E、C60Y+F164V+L370A+D391E、L3S+V5S+F164V+C60Y+L370A+G452S+D391E、C60Y+F164V+R442Q+E171D、L3S+V5S+F164V+C60Y+I180V+L370A+E171D、L3S+V5S+C60Y+F164V+L370A+E171D、L3S+V5S+C60Y+F164V+Q420R+L370A+E171D、C60Y+F164V+L370A+E171D、L3S+V5S+F164V+C60Y+L370A+G452S+E171D、C60Y+F164V+R442Q+L404Q、L3S+V5S+F164V+C60Y+I180V+L370A+L404Q、L3S+V5S+C60Y+F164V+L370A+L404Q、L3S+V5S+C60Y+F164V+Q420R+L370A+L404Q、C60Y+F164V+L370A+L404Q、L3S+V5S+F164V+C60Y+L370A+G452S+L404Q。

according to another aspect of the present invention, there is provided a DNA molecule. The DNA molecule encodes any one of the above-described transaminase mutants.

According to still another aspect of the present invention, there is provided a recombinant plasmid. The recombinant plasmid contains the DNA molecule.

Further, the recombinant plasmid is pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), and pET-35b (+), or, pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

According to yet another aspect of the present invention, a host cell is provided. The host cell contains any of the above recombinant plasmids.

Further, host cells include prokaryotic, yeast, or eukaryotic cells; preferably, the prokaryotic cell is an Escherichia coli BL21-DE3 cell or an Escherichia coli Rosetta-DE3 cell.

According to yet another aspect of the present invention, a method of producing a chiral amine is provided. The method comprises the step of carrying out catalytic transamination reaction on ketone compounds and an amino donor by transaminase, wherein the transaminase is any one of the above transaminase mutants.

Further, the ketone compound is wherein R₁And R₂Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r₁And R₂May be used alone or in combination with each other to form a substituted or unsubstituted ring;

preferably, R₁And R₂Is an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 20 carbon atoms, more preferably an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 10 carbon atoms;

preferably, the aryl group includes phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furanyl, pyrrolyl, phenoxy, naphthoxy, pyridyloxy, thienyloxy, oxadiazolyloxy, imidazolyloxy, thiazolyloxy, furanyloxy, and pyrrolyloxy;

preferably, the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;

preferably, the aralkyl group is benzyl;

preferably, the substitution means substitution by 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.

Preferably, the ketone compound is

Further, the amino donor is isopropylamine or alanine, preferably isopropylamine.

Furthermore, in the reaction system of the transaminase for carrying out the catalytic transamination reaction on the ketone compound and the amino donor, the pH value is 7-11, preferably 8-10, and more preferably 9-10.

Further, the temperature of a reaction system in which the transaminase performs a catalytic transamination reaction on the ketone compound and the amino donor is 25 to 60 ℃, more preferably 30 to 55 ℃, and still more preferably 40 to 50 ℃.

Furthermore, the volume concentration of dimethyl sulfoxide in a reaction system for carrying out catalytic transamination reaction on ketone compounds and amino donors by using transaminase is 0-50%.

Furthermore, the volume concentration of the methyl tert-butyl ether in a reaction system for carrying out catalytic transamination reaction on the ketone compound and the amino donor by the transaminase is 0-90%.

The transaminase mutant of the invention is represented by SEQ ID NO: 1, the amino acid sequence of the transaminase is changed by a site-directed mutagenesis method, so that the structure and the function of the protein are changed, and the transaminase with the mutation sites is obtained by a directional screening method, so that the transaminase mutants have good organic solvent tolerance and high pH tolerance, high soluble expression characteristic and high activity characteristic, and the application of the mutants can improve the reaction rate, improve the enzyme stability, reduce the enzyme dosage, reduce the difficulty of post-treatment and be suitable for industrial production.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows an electrophoresis chart of SDS-PAGE detecting protein expression in the embodiment of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Transaminase is a kind of biocatalyst mainly based on protein, and in industrial production process, certain organic solvent, pressure, pH and other conditions are often needed to denature protein, so that the used biocatalyst is required to have higher tolerance so as to adapt to the needs of industrial production. Wild-type transaminases tend to be less tolerant of industrial demanding conditions, thereby limiting their widespread use.

The transaminase ArS-omega TA derived from Arthrobacter citreus can specifically catalyze ketone compounds to generate amino products, but the tolerance of the enzyme to organic solvents is low, the tolerance of the enzyme in high pH is low, meanwhile, the soluble expression of the enzyme in a prokaryotic expression system is poor, and the activity of the enzyme to substrates is low, so that the use amount of the enzyme is large, and the conversion to the ketone compounds is less; meanwhile, the enzyme dosage is large, so that the difficulty of post-treatment is increased, the yield is low, and the steps are complex. The invention improves the above deficiency of the transaminase ArS-omega TA, improves the tolerance of organic solvent, and improves the pH tolerance, the soluble expression characteristic and the activity characteristic, so that the transaminase ArS-omega TA can be applied to industrial production conditions.

The rational modification of the enzyme is to modify a substrate binding site, a coenzyme binding site, a surface and other sites of the enzyme based on the three-dimensional molecular structure of the enzyme so as to change the catalytic property of the enzyme and improve the properties of the enzyme such as activity, selectivity and the like. The directed evolution of the enzyme is a non-rational design of protein, artificially creates special evolution conditions, simulates a natural evolution mechanism, transforms genes in vitro, applies error-prone PCR, DNA shuffling (DNA shuffling) and other technologies, and combines a high-efficiency screening system to obtain a new enzyme with expected characteristics.

According to the technical scheme, the ArS-omega TA protein is rationally transformed by a technology combining rational design and random mutation, and the obtained mutant is subjected to activity verification by using a ketone compound, so that the mutant strain with better organic solvent tolerance, high pH tolerance, soluble expression, activity and selectivity is finally obtained.

Rational design can be carried out by means of site-directed mutagenesis. Wherein, site-directed mutagenesis: it is intended to introduce a desired change (usually, a change indicating a favorable direction) including addition, deletion, point mutation or the like of a base into a DNA fragment of interest (which may be a genome or a plasmid) by a method such as Polymerase Chain Reaction (PCR). The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work.

The method for introducing site-directed mutation by utilizing whole plasmid PCR is simple and effective, and is a means which is used more at present. The principle is that a pair of primers (positive and negative) containing mutation sites and a template plasmid are annealed and then are circularly extended by polymerase, wherein the circular extension refers to a cycle that the polymerase extends the primers according to the template, returns to the 5' end of the primers after one circle to terminate, and is repeatedly heated, annealed and extended, and the reaction is different from rolling circle amplification and cannot form a plurality of tandem copies. The extension products of the forward and reverse primers are annealed and then paired to form nicked open-loop plasmids. The original template plasmid is derived from conventional Escherichia coli, is subjected to dam methylation modification, is sensitive to Dpn I and is cut up, and the plasmid with a mutant sequence synthesized in vitro is not cut up due to no methylation, so that the plasmid is successfully transformed in subsequent transformation, and clone of the mutant plasmid can be obtained.

The mutant plasmid is transformed into Escherichia coli cells and is over-expressed in Escherichia coli. Then the crude enzyme is obtained by a method of disrupting cells by ultrasonication. Optimal conditions for transaminase-induced expression: induction was carried out overnight at 25 ℃ with 0.1mM IPTG.

Computer simulation analysis is carried out on the three-dimensional structure of the transaminase by adopting software, and the results show that the ArS-omega TA protein is S-type transaminase taking 5-pyridoxal phosphate (PLP) as a cofactor, and the amino acid near the neutral enzymatic activity is modified, so that the enzymological properties of the transaminase can be improved, such as a stable transition state, the free energy of the binding state of the enzyme and a reaction transition state molecule is reduced, a substrate can enter the neutral activity more easily, and the steric hindrance of the substrate is reduced; the amino acid far away from active neutrality is modified, and chemical bonds such as hydrogen bonds, disulfide bonds, salt bridges and hydrophobic accumulation are added to promote stability of the protein, so that the half-life period of the protein is prolonged.

The invention rationally modifies ArS- ω TA protein (SEQ ID NO: 1), makes amino acid mutations (L3, V5, I8, I8, I45, F25, F25, Q32, L59, F56, C60, C60, F84, W86, W86, W86, W86, Y89, F164, F164, F176, F176, A178, I180, S187, T197, L206, K207, T245, T245, R319, V242, V328, V328, T397, P414, E424, E424, L370, L370, R324, R324, E326, Q416, A436, A436, A436, A436, A436, A436, M437, R442, R442, R442) and their combined mutations, preferably obtains pET22 as a gene expression vector, preferably obtains mutant of gene expression vector (DE G) for inducing expression of protein under the strain with preferably IPTG.

The constructed mutant protein is subjected to activity verification, and the result is shown in table 1:

TABLE 1

The site capable of improving the catalytic activity of the transaminase mutant is obtained through site-directed mutagenesis, 10wt wet-weight cells are used, the substrate concentration of 0.02g/ml is used for activity verification, the activity of the obtained optimal mutant is improved by 45 times compared with that of the parent ArS-omega TA, however, due to the fact that the activity of the outgrowth ArS-omega TA is too low, a large amount of substrate which is converted for 16 hours is still remained and can not be converted into amino products after the 45 times of the outgrowth ArS-omega TA is converted, further modification is carried out, the modification comprises the introduction of beneficial site combination and new mutation sites, and the activity of the modified mutant is verified by 5wt to 0.5wt wet-weight cells and the substrate concentration of 0.1g/ml, and the activity is verified, and the table 2 shows.

TABLE 2

Through the modification, a plurality of sites capable of improving the catalytic activity of the transaminase mutant are obtained, beneficial mutation combination is carried out, a plurality of mutation site combinations with improved activity are obtained, and the activity of the strain is improved by 3455 times compared with that of ArS-omega TA. The optimum mutant strain is subjected to activity verification in a reaction system (with an extremely small volume of 10V) with a substrate concentration of 0.1g/ml by the weight of 0.5wt wet cells, and after 16h of conversion, the conversion rate reaches over 95%.

It can be seen that the mutant strains obtained excellent improvement in catalytic activity, from essentially non-catalytic activity of the wild species (10wt wet cell weight, 0.02g/ml substrate concentration, 0.1% conversion) to excellent catalytic activity: a conversion of 95% was achieved with very little enzyme (0.5wt) and very little reaction volume (10V).

The mutant protein obtained by rational modification is subjected to directed evolution to obtain the mutant protein with greatly improved activity (improved quality) on the ketone substrate. The mutant protein is used as an original strain, error-prone PCR is used as a technical means to carry out random mutation, and the activity, the organic solvent tolerance, the high pH tolerance and the like of the mutant protein are further improved. Meanwhile, by combining the site-directed mutagenesis technology and the staggered extension PCR random recombination technology, beneficial mutations obtained by error-prone PCR are continuously accumulated. Constructing a mutant library containing random mutation by the obtained mutant, continuously increasing the concentration of an organic solvent, continuously increasing the pH value of a screening and reaction system, and setting screening pressure to obtain the target characteristic mutant. The mutant takes pET22b as an expression vector and BL21(DE3) as an expression strain, and obtains mutant protein under the induction of IPTG. Cell lysis is carried out on induced mutant thalli containing the target protein in an ultrasonic disruption mode to release the target protein, and the expression condition of the target protein is detected by SDS-PAGE.

Wherein, the error-prone PCR is that when DNA polymerase is used for target gene amplification, the mutation frequency in the amplification process is changed by adjusting reaction conditions, the inherent mutation sequence tendency of the polymerase is reduced, the diversity of the mutation spectrum is improved, and the error base is randomly doped into the amplified gene at a certain frequency, so that a randomly mutated DNA population is obtained.

According to the invention, through activity verification, multiple mutations are obtained by the method of directional screening through random mutation, so that the tolerance of the mutant to an organic solvent is improved, the tolerance to pH is improved, the activity of a substrate is improved, and the solubility of a target protein is improved.

And (3) verifying tolerance: tolerance improvement degree of mutation sites obtained by error-prone PCR in 35% dimethyl sulfoxide

The beneficial mutation sites obtained by taking M76(L3S + V5S + C60Y + F164V + A178L + S187A + I180V + L370A) as the starting strain are subjected to tolerance verification in 35% dimethyl sulfoxide, and the results are shown in Table 3.

TABLE 3

And (3) verifying tolerance: the error-prone PCR-derived mutation site was more tolerant in 50% MTBE.

The results of verifying the tolerance of the beneficial mutation sites obtained by taking M76(L3S + V5S + C60Y + F164V + A178L + S187A + I180V + L370A) as the starting strain in 50% methyl tert-butyl ether are shown in Table 4.

TABLE 4

The obtained beneficial mutation uses an iteration-error-prone PCR-directional screening method and simultaneously uses a site-directed mutagenesis technology to continuously improve the activity of the mutant strain and the tolerance of the mutant strain to an organic solvent.

And (3) carrying out tolerance site verification by taking different mutant strains as starting strains:

different mutant strain plasmids are extracted, mutant strains are constructed by a site-directed mutagenesis technology, and the obtained mutant strains are subjected to the verification of tolerance sites in 35% dimethyl sulfoxide, and the results are shown in a table 5:

TABLE 5

+ represents 1-100% improvement of enzyme tolerance in 35% dimethyl sulfoxide, + + represents 100-300% improvement of enzyme tolerance in 35% dimethyl sulfoxide, and +++ represents 400-600% improvement of tolerance in 35% dimethyl sulfoxide

It can be seen that the mutation site obtained by random mutation and directional screening has obvious effect on increasing the tolerance of the strain in an organic solvent (dimethyl sulfoxide).

And (3) verifying tolerance: tolerance improvement degree of mutation sites obtained by error-prone PCR in 40% dimethyl sulfoxide

The results of verifying the tolerance in 45% dimethyl sulfoxide of the beneficial mutation sites obtained by taking M76(L3S + V5S + C60Y + F164V + A178L + S187A + I180V + L370A) as the starting strain are shown in Table 6.

TABLE 6

And (3) verifying tolerance: the error-prone PCR-derived mutation site was more tolerant in 70% MTBE.

The results of verifying the tolerance of the beneficial mutation sites obtained by taking M76(L3S + V5S + C60Y + F164V + A178L + S187A + I180V + L370A) as the starting strain in 70% methyl tert-butyl ether are shown in Table 7.

TABLE 7

The tolerance of the modified mutant protein in dimethyl sulfoxide and methyl tertiary ether as solvents is greatly improved, and the activity of the mutant at high pH is verified.

High pH tolerance verification: the obtained mutants were tested for activity in 40% dmso and 70% mtbe at pH 10.0 and after 16 hours the obtained mutant substrates were essentially completely converted, the results are shown in table 8.

TABLE 8

The obtained mutant is crushed by ultrasonic crushing, centrifuged to obtain supernatant enzyme liquid and precipitated enzyme liquid, and SDS-PAGE is carried out to detect the expression condition of the protein, wherein the soluble expressed protein exists in the supernatant in a dissolved state, and the abnormally folded protein exists in an inclusion body form, namely the precipitated protein. The results of SDS-PAGE are shown in FIG. 1 (lane 1: Marker, lane 2: ArS-omega ta soluble protein, lane 3: inclusion body of ArS-omega ta protein, lane 4: Marker, lane 5: soluble protein of mutant M115, lane 6: inclusion body of mutant M115), and the optimal mutant protein has a 4-fold higher soluble expression than the starting strain.

According to an exemplary embodiment of the present invention, a transaminase mutant is provided. The amino acid sequence of the transaminase mutant is represented by SEQ ID NO: 1(SEQ ID NO: 1: MGLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLITPGGTRLLDFFNQLCCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSCSSAVLMAPSSNTFQDSNGNYLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGVGSTMPPYEYVPQIRKMTKELGVLWISDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVVVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENLVEQAKNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFRHGMNPNQIPTQIIMEKALEKGVLIGGAMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQQS), wherein the mutation at least comprises one of the following mutation sites: 3 rd, 5 th, 8 th, 25 th, 32 th, 45 th, 56 th, 59 th, 60 th, 84 th, 86 th, 164 th, 176 th, 178 th, 180 th, 187 th, 197 th, 206 th, 207 th, 242 th, 245 th, 319 th, 324 th, 326 th, 328 th, 370 th, 397 th, 414 th, 416 th, 424 th, 436 th, 437 th and 442 th bits. Preferably, the mutation comprises at least one of the following mutation sites: l3, V5, I8, F25, Q32, I45, L59, F56, C60, F84, W86, F164, F176, a178, I180, S187, T197, L206, K207, V242, T245, R319, R324, E326, V328, L370, T397, P414, Q416, E424, a436, M437, R442 and R442; 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 80% or more homology with the mutated amino acid sequence.

The transaminase mutant of the invention is represented by SEQ ID NO: 1, the transaminase ArS-omega TA is mutated by a site-directed mutagenesis method, so that the amino acid sequence of the transaminase is changed, the change of the protein structure and function is realized, the transaminase ArS-omega TA has high solubility expression characteristic and high activity characteristic, and the application of the transaminase ArS-omega TA can improve the reaction rate, improve the stability of the transaminase, reduce the dosage of the transaminase and the difficulty of aftertreatment, so that the transaminase ArS-omega TA can be suitable for industrial production.

The term "homology" as used herein has the meaning generally known in the art and rules, standards for determining homology between different sequences are well known to those skilled in the art. The sequences defined by different degrees of homology according to the invention must also simultaneously have improved tolerance of the transaminase to organic solvents. In the above embodiments, it is preferable that the amino acid sequence of the transaminase mutant has the above homology and has or encodes an amino acid sequence having improved tolerance to organic solvents. One skilled in the art can obtain such variant sequences under the teachings of the present disclosure.

Preferably, the mutation comprises at least one of the following combinations of mutation sites: C60Y + F164V, L3S + V5S, L3S + V5S + F164V, L3S + V5S + C60Y, L3S + V5S + C60Y + F164V, I180V + L370A and L3S + V5S + L59V; more preferably, the mutation comprises at least one of the following combinations of mutation sites: f164 + C60, E424 + A436, C60 + F164 + A436, W86 + F164, F25 + L59, F25 + F164, C60 + F164 + A436, F164 + M437, I8 + V328, I8 + F164, C60 + F164 + L370, I45 + F164, C60 + F164 + F176, C60 + F176 + F164, L3+ V5 + C60 + F164 + L370, C60 + F164 + R442, L3+ V187 + L424, C60 + F164 + R442, L164R 442, L3+ V164 + T164 + L164 + T164 + L164, L164 + T164 + L370, L3+ V + T5 + T5 + L245 + T3 + L5 + T3 + L164 + T164, L164 + T164 + L370, L164 + T164, L370, L164 + T164, L164 + T164, L164 + T164, L370, L164 + T164, L164 + T164, L370, L164 + T164, L164 + T164, L370, L164 + T70 + T164, L164 + T70, L370, L164, L370, L164 + T70 + T70, L370, L164 + T70 + T70 + T70, L164, L370, L164 + T70 + T70, L164, L370, L164 + T70 + T70 + T70 + T70, L370, L164, L370, L164, L370, l3+ V5 + L59 + F164 + L370, L3+ V5 + L59 + L370 + A436 + Q416, L + V5 + L59 + L370 + A436 + R442, L3+ V5 + L59 + V328 + L370 + R442, L3+ V5 + L59 + C60 + F164 + L370 + R442, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370, L3+ V5 + C60 + F164 + I180 + S187 + L370 + R442.

According to a typical embodiment of the invention, the mutation further comprises at least one of the following mutation sites or combinations of mutation sites: 7 th, 32 th, 96 th, 164 th, 171 th, 186 th, 252 th, 384 th, 389 th, 391 th, 394 th, 404 th, 411 th, 420 th, 423 th, 424 th, 442 th, 452 th and 456 th bits; preferably, the mutation further comprises at least one of the following mutation sites: K7N, Q32L, K96R, V164L, E171D, S186G, V252I, Y384F, I389M, I389F, D391E, N394D, L404Q, L404Q, G411D, Q420R, Q420K, M423K, E424R, E424K, E424Q, R442H, R442L, G452S, and K456R. The transaminase mutants have good organic solvent tolerance and high pH tolerance, and have high soluble expression characteristic and high activity characteristic, so that the application of the mutants can improve the reaction rate, improve the enzyme stability, reduce the enzyme dosage, reduce the difficulty of post-treatment and ensure that the mutants can be suitable for industrial production.

More preferably, the mutation comprises at least one of the following combinations of mutation sites: G411D + S186G, G411D + S186G + Y384F, G411D + S186G + Y384F + V164L, G411D + S186G + Y384F + V164L + I389F and G411D + S186G + Y384F + V164L + I389F + V252I; further preferably, the mutation comprises at least one of the following combinations of mutation sites: l3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Y384 + G452, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Q186, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + M423, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Q420 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + K7 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + D391, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Q370 + L20 + L178 + L187 + F187 + L180 + L178 + L180 + L178 + L164 + L178 + L180 + L187 + F187 + K180 + L187 + K180 + L178 + L180 + L178 + L2 + L178 + L180, L187 + L178 + K180 + L178 + L187 + K180 + K20 + K187 + K180 + L187 + K180 + K187 + L178C 178 + K180 + L178 + K180, L187 + L178 + K20 + L178 + K20 + L187 + L178C 80 + K20 + L178 + K20 + L187 + K20 + L178C 80 + L187 + L178C 80 + L178C 80, L178 + L187 + K20 + L187 + L178C 178 + K20 + L178 + L187 + L178 + K20 + L178C 80, L178C 80, L178 + K20 + K178C 80, L178C 80 + K20 + L187 + K20 + K187 + K20 + K187 + K20 + K187 + K20 + K187 + K20, L178C 180, L178C 80, L178C 80, L180, L178C 80 + L178C 80, L187 + K20 + K187 + L178C 180, L180 + K187 + L187 + K, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + R442, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + V252, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370, L3+ V5 + C60 + F164 + Q164 + L370, L3+ V5 + F164 + C60 + L370 + G452, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + L411 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + L411 + Y5 + L404, L3+ V5 + V164 + L164 + K180 + L178 + L187 + K180 + L187 + K180 + L187 + K178 + L180 + L178 + L180 + L178 + L164 + L180 + L178 + L164 + K180 + L187 + K180 + L187 + L178 + K + L180 + K + L180 + L178 + K + L180 + K + L187 + L178 + L180 + K + L180 + L187 + L180 + L178 + K + L + K + L178 + K + L + K + L + K + L + K + L180, L3+ V5 + C60 + F164 + A178 + I180 + L370 + G411 + M423, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + I389 + L404, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L187 + G384 + L178 + S186 + Y384 + E171, L3+ V5 + C60 + F164 + A178 + S187 + I187 + L370 + G411 + S187 + I187 + G187 + Y187 + I389 + L404, L3+ V5 + V384 + S187 + V187 + L178 + L180 + L187 + L178 + V180 + L187 + S187 + L178 + V180 + L187 + L178 + L187 + V180 + L187 + L178 + V2 + L180 + L187 + L178 + L2 + E411 + L187 + L178 + V2 + L187 + L180 + G411 + L187 + L178 + Y180 + E411, L187 + L178 + L187 + L178 + L180 + E171, L187 + L178 + L187 + L180 + L187 + E171, L187 + L178 + L180 + L187 + D, L178, L187 + L178, L187 + L180 + L178, L187 + L180 + E171, L187 + L178, L187 + L, L180 + L187 + L, L187 + L, L180, L187 + L180, L187 + L187, L180, and the same methods, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + I389 + V252 + L404 + E171, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + I389 + V252 + E424 + M423, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + I384 + 389 + V252 + L404 + E171 + D384, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + E424 + K7, L3+ V5 + F164 + A178 + S187 + I180 + L180 + G411 + S164 + L164 + E164 + G187 + L187 + I.G 180 + L187 + I178 + I180 + L187 + G411 + L178 + E411 + L178 + E411 + D180 + L178 + L180 + L187 + E411, L187 + L178 + L180 + L187 + E, C60 + F164 + R442 + G411, L3+ V5 + F164 + C60 + I180 + L370 + G411, L3+ V5 + C60 + F164 + Q420 + L370 + G411, C60 + F164 + L370 + G411, L3+ V5 + F164 + C60 + L370 + G452 + Y384, C60 + F164 + R442 + Y384, L3+ V5 + F164 + C60 + I180 + L370 + Y384, L3+ V5 + C60 + F164 + L370 + Y384, L3+ V5 + F164 + Q420 + L370 + Y384, C60 + F164 + L370 + Y384, L70 + V70 + L70 + F + V70 + F + R70 + V + R70 + L70 + V + R + V70 + L70 + R + V + R + L70 + V + R + L70 + R + V + R + L70 + G411, L3+ V + R + V + R + F + G +, C60Y + F164V + L370A + D391E, L3S + V5S + F164V + C60Y + L370A + G452S + D391E, C60Y + F164V + R442Q + E171D, L3S + V5S + F164V + C60Y + I180Y + L370Y + E171Y, L3Y + V5Y + C60Y + F164 + L370Y + E171Y + E Y, L3Y + V5Y + C60Y + F Y + L Y + L Y + F Y + L Y + F Y + 36. The transaminase with the mutation sites is obtained by a directional screening method, and the transaminase mutants have good organic solvent tolerance, high pH tolerance, high soluble expression characteristic and high activity characteristic, so that the application of the mutants can improve the reaction rate, improve the enzyme stability, reduce the enzyme dosage, reduce the difficulty of post-treatment and be suitable for industrial production.

According to an exemplary embodiment of the present invention, a DNA molecule is provided. The DNA molecule encodes the above-described organic solvent-resistant transaminase mutant. The transaminase mutant coded by the DNA molecule has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristic and high activity characteristic.

The above-described DNA molecules of the invention may also be present in the form of "expression cassettes". An "expression cassette" refers to a nucleic acid molecule, linear or circular, encompassing DNA and RNA sequences capable of directing the expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter is included that is operably linked to a nucleotide of interest, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest in the sense or antisense orientation, e.g., an antisense RNA or an untranslated RNA. An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component. The expression cassette may also be naturally occurring but obtained with efficient recombinant formation for heterologous expression.

According to an exemplary embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the above DNA molecules. The DNA molecule in the recombinant plasmid is placed in a proper position of the recombinant plasmid, so that the DNA molecule can be correctly and smoothly replicated, transcribed or expressed.

Although the term "comprising" is used in the present invention when defining the above DNA molecule, it does not mean that other sequences unrelated to their functions may be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to satisfy the requirements of recombinant operation, it is necessary to add suitable restriction sites for restriction enzymes at both ends of a DNA sequence, or additionally add initiation codons, termination codons, etc., and thus, if defined by closed expressions, these cases cannot be truly covered.

The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, either prokaryotic or eukaryotic, but preferably prokaryotic, selected from the group consisting of pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), and, pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, QEP 9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pR A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6-p-1, pGEX-6-P-2-pGEX-2 b (+), pET-39b (+), pET-40b (+) pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. More preferably, the above recombinant plasmid is pET-22b (+).

According to a typical embodiment of the present invention, there is provided a host cell containing any one of the above recombinant plasmids. Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells, yeast, or eukaryotic cells. Preferably the prokaryotic cell is a eubacterium, such as a gram-negative or gram-positive bacterium. More preferably, the prokaryotic cell is an E.coli BL21 cell or an E.coli DH5 alpha competent cell.

According to an exemplary embodiment of the present invention, a method for producing chiral amines is provided. The method comprises the step of carrying out catalytic transamination reaction on ketone compounds and an amino donor by transaminase, wherein the transaminase is any one of the above transaminase mutants resistant to organic solvents. Because the transaminase mutant has good organic solvent tolerance, high pH tolerance, high soluble expression characteristic and high activity characteristic, the chiral amine prepared by the transaminase mutant can improve the reaction rate, improve the enzyme stability, reduce the enzyme dosage and reduce the difficulty of post-treatment.

Further, the ketone compound is

Wherein R is₁And R₂Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r₁And R₂May be used alone or in combination with each other to form a substituted or unsubstituted ring;

preferably, the aralkyl group is benzyl;

Preferably, the ketone compound is

According to a typical embodiment of the invention, the ketone compound is

The transamination reaction product is

Reaction formula is

In a typical embodiment of the invention, the amino donor is isopropylamine or alanine, preferably isopropylamine.

In a reaction system for carrying out a catalytic transamination reaction on a ketone compound and an amino donor by using the transaminase of the invention, the pH is 7-11, preferably 8-10, more preferably 9-10, that is, the value of the pH can be selected from 7-11, such as 7, 7.5, 8, 8.6, 9, 10, 10.5, etc. The temperature of the reaction system in which the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor is 25 to 60 ℃, more preferably 30 to 55 ℃, and still more preferably 40 to 50 ℃, that is, the temperature can be selected from 25 to 60 ℃, for example, 30, 31, 32, 35, 37, 38, 39, 40, 42, 45, 48, 50, 51, 52, 55, and the like. The volume concentration of dimethyl sulfoxide in the reaction system of the transaminase for carrying out the catalytic transamination reaction on the ketone compound and the amino donor is 0-50%, for example, 10%, 15%, 18%, 20%, 30%, 35%, 38%, 40%, 42%, 48%, 49%, etc. The volume concentration of methyl tert-butyl ether in the reaction system for the catalytic transamination reaction of the ketone compound and the amino donor by the transaminase is 0-90%, for example, 10%, 16%, 18%, 20%, 30%, 35%, 38%, 40%, 42%, 48%, 49%, 55%, 60%, 70%, 80%, 90%, etc.

It will be apparent to those skilled in the art that many modifications can be made to the present invention without departing from the spirit thereof, and such modifications are intended to be within the scope of the invention. The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.

Example 1

Catalytic activity of ArS- ω TA mutant and wild enzyme on substrate 1 in an organic solvent-free system:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 250. mu.L of a crude enzyme solution of ArS-. omega.TA mutant or wild enzyme (20% crude enzyme solution obtained by ultrasonication of 0.05g of wet mutant cells, pH 8.5) was added, 100mM PB8.50.41mL was added to make the final volume of the system 1mL, the mixture was stirred at 30 ℃ for 16 hours under constant temperature, the system was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the sample was dissolved in 2mL of acetonitrile, and the product conversion rate was measured by HPLC after centrifugation at 12000rpm for 5 minutes, and the information on the mutant and the results are shown in Table 9.

TABLE 9

The results in Table 9 show that the catalytic activity of the ArS-omega TA mutant on the substrate 1 is greatly improved compared with that of the wild strain. After the improvement of the invention, the catalytic activity of the ArS-omega TA mutant is greatly improved, the catalytic activity of the original basically un-catalyzed substrate is obtained, the substrate spectrum is expanded, and meanwhile, the conversion can be carried out in a very small reaction volume, so that the utilization rate of a reactor is improved.

Example 2

Catalytic activity of ArS- ω TA wild-type enzyme and mutant on substrate 1 in organic solvent system (40%) DMSO:

in a 10mL reaction flask, 100mg of the starting material (same as example 1) was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 250. mu.L of a crude enzyme solution of an ArS-. omega.TA mutant or wild enzyme (20% crude enzyme solution pH 8.5 was obtained by ultrasonication of 0.05g of wet cells of the mutant), 100mM PB8.50.01mL of the crude enzyme solution and 0.4mL of dimethyl sulfoxide were added to make the final volume of the system 1mL, the mixture was stirred at a constant temperature of 35 ℃ for 16 hours, after the system was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the crude enzyme solution was sampled and dissolved in 2mL of acetonitrile, and after centrifugation at 12000rpm for 5 minutes, HPLC was carried out to examine the conversion rate of the product, and the information and the results of the mutant are shown in Table 10.

Watch 10

ND: no product formation detected

Example 3

The ArS-omega TA mutant and the wild enzyme which are resistant to organic solvent catalyze the substrate to generate chiral amine in a 70% methyl tertiary ether solvent system:

in a 10mL reaction flask, 100mg of the starting material (same as example 1) was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 250. mu.L of crude enzyme solution of ArS-. omega.TA mutant or wild enzyme (20% crude enzyme solution pH 8.5 was obtained by ultrasonication of 0.05g of wet cells of the mutant) was added, 1.4mL of methyl-tert-ether was added to make the final volume of the system 2mL, the system was stirred at a constant temperature of 35 ℃ for 16 hours, after centrifuging the system at 12000rpm for 5 minutes, the methyl-tert-ether reagent was blown dry with nitrogen, 100. mu.L of the remaining sample was taken, 2mL of acetonitrile was added and dissolved, after centrifuging at 12000rpm for 5 minutes, HPLC was performed to determine the conversion rate of the product, the conversion rate of the ArS-. omega.TA mutant was 95%, no product was produced by detection of the wild enzyme of ArS-. omega.TA, and the mutant information and results are shown in Table 11.

TABLE 11

Although the mutant M76 obtained catalytic activity for the substrate 1 (example three), most of the proteins lose activity due to low tolerance to organic solvents in 40% of dimethyl sulfoxide, the catalytic activity is greatly reduced, and the mutants M113 and M115 still maintain high catalytic activity, which indicates that the tolerance to organic solvents dimethyl sulfoxide is greatly improved on the basis of obtaining high catalytic activity by the evolved mutants.

Example 4

Catalytic activity verification of ArS-omega TA mutant and wild enzyme under different temperature and pH conditions

In a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 500. mu.L of ArS-. omega.TA mutant M52 or M115 (20% crude enzyme solution, pH 11, obtained by ultrasonication of 0.1g of wet mutant cells) or crude enzyme solution of wild enzyme (20% crude enzyme solution, pH 11, obtained by ultrasonication of 1g of wet mutant cells) was added, 100mM phosphate buffer, pH 11, was added to make the final volume 5.5mL, the mixture was stirred at a constant temperature of 35 ℃ for 16 hours, the mixture was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the mixture was sampled, 2mL of acetonitrile was added to dissolve the mixture, and the mixture was centrifuged at 12000rpm for 5 minutes and then subjected to HPLC to determine the product conversion rate.

The results show that under the extreme pH condition, although ArS-omega TA uses a large amount of enzyme (1g of wet cells), no product generation is detected after catalysis, mutant M52 does not detect the product generation, and mutant M115 uses less enzyme (0.1g) to detect the product generation of more than 80%, which indicates that the mutant is modified to obtain excellent high pH tolerance, so that the catalytic space of the enzyme is improved, and the high activity and high pH tolerance have the dual excellent characteristics of suitability for industrial production.

In a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 500. mu.L of ArS-. omega.TA mutant M52 or M115 (20% crude enzyme solution, pH 8, obtained by ultrasonication of 0.1g of wet mutant cells) or crude enzyme solution of wild enzyme (20% crude enzyme solution, pH 8, obtained by ultrasonication of 1g of wet mutant cells) was added, 100mM phosphate buffer, pH 8, was added to make the final volume 5.5mL, the mixture was stirred at a constant temperature of 60 ℃ for 16 hours, the mixture was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the mixture was sampled, 2mL of acetonitrile was added to dissolve the mixture, and the mixture was centrifuged at 12000rpm for 5 minutes and then subjected to HPLC to determine the product conversion rate.

The results show that under the extreme temperature conditions, although ArS-omega TA uses a large amount of enzyme (1g of wet cells), no product generation is detected after catalysis, mutant M52 does not detect the product generation, and mutant M115 uses less enzyme (0.1g) to detect the product generation of more than 80%, which indicates that the mutant is modified to obtain excellent high-temperature tolerance, so that the catalytic space of the enzyme is improved, and the high-activity and high-temperature tolerance have the dual excellent characteristics of suitability for industrial production.

Example 5

The ArS-omega TA mutant and the wild enzyme catalyze the substrate to generate chiral amine:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 5mL to 250 μ L of an ArS- ω TA mutant (20% crude enzyme solution, pH7.0 and pH 10.5, obtained by ultrasonication of 1g to 0.05g of wet cells of the mutant), or crude enzyme solution of wild enzyme (20% crude enzyme solution, pH7.0 and pH 10.5, obtained by ultrasonication of 1g of wet cells of ArS- ω TA), 0.41mL of 100mM pH7.0 phosphate buffer (or pH 10.5 phosphate buffer) was added to make the final volume of the system 3mL, the mixture was stirred at a constant temperature of 30 ℃ for 16 hours, the system was centrifuged at 12000rpm for 5min, 200 μ L of the sample was taken, 2mL of acetonitrile was added and dissolved, and the product conversion rate was measured by HPLC after centrifugation at 12000rpm for 5 min.

The mutant information and results are shown in Table 12. The results show that the wild ArS-omega TA enzyme has no product formation under the two pH systems of 7.0 and 10.5, while the product formation is detected under the two pH systems by a plurality of mutants, and the activity of partial mutants is excellent, such as that the mutant M115 uses extremely small enzyme amount (0.05g under the condition of pH 10.5) and the conversion rate is more than 95%, and the chiral purity of the product is extremely high and more than 99%. The mutant obtained by transforming ArS-omega TA obtains excellent catalytic activity, can catalyze the generation of chiral amine under high pH, and has good catalytic effect, which shows that the mutant obtains qualitative breakthrough in catalytic activity and tolerance.

TABLE 12

"ND" means that no product formation was detected using 1g of wet cell catalysis, "-" means that < 5% of product formation was detected at pH7, "+" means that 5% to 20% of product formation was detected at pH7, "+++" means that 20% to 50% of product formation was detected at pH7, "+++++++" means that 50% to 80% of product formation was detected at pH7, "++++" means that 80% to 90% of product formation was detected at pH7, "++++++++++++++" means that 80% to 95% of product formation was detected at pH7, and at the same time 90% to 100% of product formation was detected using 0.05g of wet cell catalysis at pH 10.5.

Example 6

The ArS-omega TA mutant and the wild enzyme catalyze the substrate to generate chiral amine:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 100. mu.L of an ArS-. omega.TA mutant (0.02g of wet cells of the mutant was sonicated to obtain a 20% crude enzyme solution, pH 8.5) was added, or 1000. mu.L of a crude enzyme solution of a wild enzyme (0.2g of wet cells of ArS-. omega.TA was sonicated to obtain a 20% crude enzyme solution, pH 8.5) was added, 100mM PB8.50.41mL was added to make the final volume of the system 3mL, the mixture was stirred at 30 ℃ for 16 hours under constant temperature, centrifuged at 12000rpm for 5 minutes, 200. mu.L of the sample was dissolved in 2mL of acetonitrile, and centrifuged at 12000rpm for 5 minutes, followed by HPLC to determine the product conversion.

The mutant information and results are shown in Table 13. The results show that the wild ArS-omega TA enzyme is only produced in a small amount (< 20%) by using 10 times of the enzyme amount of the mutant, the mutants such as M118, M115 and the like obtain the conversion rate of > 90% by using very few enzymes, and the chiral purity of the product is very high of > 99%. Meanwhile, compared with an ArS-omega TA female parent, the activity of a plurality of mutants is greatly improved, and an excellent catalytic effect is obtained.

Watch 13

"-" indicates that the conversion rate obtained by catalyzing the starting strain by using 10 times of enzyme amount (0.2g) of the mutant is lower than 20%, or indicates that the conversion rate obtained by catalyzing the mutant by using the enzyme amount (0.2g) equal to that of the starting strain is reduced or not increased, "+" indicates that the conversion rate is improved by 0.2-1 time by using a very small amount of enzyme (0.02g) of the mutant, "+++" indicates that the conversion rate is improved by 2-4 times by using a very small amount of enzyme (0.02g) of the mutant, and "++++" indicates that the conversion rate is improved by more than 4 times by using a very small amount of enzyme (0.02g) of the mutant.

Example 7

The ArS-omega TA mutant and the wild enzyme catalyze the substrate to generate chiral amine:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 5mL of ArS-. omega.TA parent and mutant crude enzyme solution (20% crude enzyme solution was prepared by ultrasonication of 1g of wet mutant cells, pH 8) were added, 100mM phosphate buffer pH 8 was added to adjust the final volume of the system to 5.5mL, the mixture was stirred at 35 ℃ for 16 hours under constant temperature, the system was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the sample was dissolved in 2mL of acetonitrile, and the product conversion was measured by HPLC after centrifugation at 12000rpm for 5 minutes.

The mutant information and results are shown in table 14, and it can be seen that the activity of the modified mutant is significantly improved compared with that of the original strain ArS-omega TA, and the chiral purity of the product is extremely high and is more than 99%.

TABLE 14

"+" indicates a conversion rate of less than 20%, "+ +" indicates a conversion rate of 20% to 30%, "+++" indicates a conversion rate of 30% to 40%, "+++" indicates a conversion rate of 40% to 50%

Example 8

The ArS-omega TA mutant and the wild enzyme catalyze the substrate to generate chiral amine:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 5mL or 500uL of the crude enzyme solution (20% crude enzyme solution obtained by ultrasonication of 1g of ArS- Ω ta or wet cells of the mutant, pH 8) was added, 100mM phosphate buffer, pH 8 was added to give a final volume of 5.5mL, the mixture was stirred at 35 ℃ for 16 hours, the mixture was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the sample was taken, NaOH was adjusted to pH 10, 2mL of MTBE was added and the product was extracted, and the product conversion was checked by HPLC after centrifugation at 12000rpm for 5 minutes.

The mutant information and results are shown in table 15, it can be seen that the activity of the modified mutant is significantly improved compared with that of the original strain ArS-omega TA, a plurality of mutants can completely convert substrates into products within 16h, and the chiral purity of the products is extremely high and is more than 99%.

Watch 15

"+ + +" indicates less than 50% conversion using 1g of wet cells containing the transaminase of interest, "+ + + + + +" indicates 70% to 90% conversion using 1g of wet cells containing the transaminase of interest, and "+ + + + +" indicates 90% to 100% conversion using 0.1g of wet cells containing the transaminase of interest.

Example 9

The ArS- ω TA mutant (M52, M118 and M111) and the wild enzyme catalyze the substrate to generate chiral amine:

in a 10mL reaction flask, 100mg of the starting material was weighed, 1mg of pyridoxal 5' -phosphate was added, 2mM isopropylamine hydrochloride was added, 5mL of the crude enzyme solution (20% crude enzyme solution obtained by ultrasonication of 1g of ArS-Omegata or wet cells of the mutant, pH 8) was added, 100mM phosphate buffer pH 8 was added to adjust the final volume of the system to 5.5mL, the mixture was stirred at a constant temperature of 35 ℃ for 16 hours, the system was centrifuged at 12000rpm for 5 minutes, 200. mu.L of the sample was dissolved in 2mL of acetonitrile, and the resultant product was centrifuged at 12000rpm for 5 minutes and then subjected to HPLC for product formation. The detection shows that the generation of the product cannot be detected in a catalytic system of the development ArS-omega ta, and the generation of the product is detected by M52 mutants, M118 mutants and M111 mutants. It can be seen that the mutant obtained catalytic activity on the substrate after modification.

Example 10

A single colony of E.coli comprising each plasmid encoding the transaminase of interest was inoculated into 50Ml Luria Bertani medium containing 50ug/Ml of ampicillin. Cells were grown overnight (approximately 16h) in a constant temperature shaker at 37 ℃ at 200 rpm. 5mL of the culture was inoculated into 500mL of Luria Bertani medium containing 50ug/mL of ampicillin in a 2-liter flask, cultured in a 200rpm, 37 ℃ incubator until the OD was 0.6 to 0.8, expression of the transaminase gene was induced by adding isopropyl β D-thiogalactoside (IPTG) to a final concentration of 0.06mM, and then the culture was incubated in a 200rpm, 25 ℃ incubator for about 16 hours. The cells were collected by centrifugation (6000rpm, 15min, 4 ℃) and the supernatant was discarded. The cells were resuspended in 100mM phosphate buffer pH7.0, crude enzyme was obtained by disrupting the cells by sonication, the supernatant was separated from the pellet (containing the inclusion body proteins) by centrifugation (12000rpm, 3min, 4 ℃) and the pellet was resuspended in an equal volume of 100mM phosphate buffer pH 7.0. The expression of soluble protein and inclusion body protein in the supernatant and the precipitate was detected by SDS-PAGE.

The expression results of ArS-omega TA and each mutant are shown in the following table 16, which shows that the introduction of mutation sites gradually makes the soluble expression condition of mutant protein better and better, only a small amount of protein of initial ArS-omega TA is expressed in the supernatant, a large amount of protein is expressed in the precipitate, the expression quantity of the supernatant protein is doubled by mutant M52, and the final mutant M115 and the like make the protein basically and completely expressed in the supernatant, and the precipitate expression is very little. The expression of the mutant is improved remarkably.

TABLE 16

"-" indicates the soluble expression level of the parent ArS-. omega.TA: only a small amount of expression in the supernatant, most in the pellet; "+" indicates a 1-fold increase in soluble expression; "+ +" indicates a 2-fold increase in soluble expression; "+ + + +" indicates a greater than 3-fold increase in soluble expression; "+ +++" indicates a greater than 4-fold increase in soluble expression.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the transaminase mutant has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristic and high activity characteristic.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Kai Lai En Life sciences technology (Tianjin) Co., Ltd

<120> transaminase mutants and uses thereof

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Met Gly Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp

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Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln

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Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Thr Pro Gly

35 40 45

Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Cys Cys Val Asn Leu

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Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp

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Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys

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Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro

100 105 110

Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala

115 120 125

Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg

130 135 140

Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg

145 150 155 160

Leu Arg Ser Phe Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe

165 170 175

Ser Ala Gln Ile Pro Gly Ser Ser Cys Ser Ser Ala Val Leu Met Ala

180 185 190

Pro Ser Ser Asn Thr Phe Gln Asp Ser Asn Gly Asn Tyr Leu Lys Asp

195 200 205

Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu

210 215 220

Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln

225 230 235 240

Gly Val Gly Ser Thr Met Pro Pro Tyr Glu Tyr Val Pro Gln Ile Arg

245 250 255

Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Ser Asp Glu Val Leu

260 265 270

Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly

275 280 285

Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser

290 295 300

Leu Pro Ala Gly Ala Val Val Val Ser Lys Glu Ile Ala Ala Phe Met

305 310 315 320

Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val

325 330 335

Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn

340 345 350

Leu Val Glu Gln Ala Lys Asn Ser Gly Glu Tyr Ile Arg Ser Lys Leu

355 360 365

Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr

370 375 380

Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro

385 390 395 400

Tyr Val Lys Leu Asp Arg Asn Phe Arg His Gly Met Asn Pro Asn Gln

405 410 415

Ile Pro Thr Gln Ile Ile Met Glu Lys Ala Leu Glu Lys Gly Val Leu

420 425 430

Ile Gly Gly Ala Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn

435 440 445

Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala

450 455 460

Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser

465 470 475

Claims (18)

1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant consists of SEQ ID NO: 1, the mutation is an amino acid sequence obtained by mutating an amino acid sequence shown in the formula (1): l3+ V5 + F164, L3+ V5 + C60 + F164 + L370, L3+ V5 + F164 + C60 + I180 + L370, L3+ V5 + C60 + F164 + A178 + L370, L3+ V5 + F164 + T197 + L370, L3+ V5 + F164 + K207 + L370, L3+ V5 + F164 + T245 + V328, L3+ V5 + F164 + T397, L3+ V5 + L370 + T397, L3+ V5 + L59 + F59 + R442, L3+ V5 + R319 + L370 + T397, L3+ V5 + L59 + F164 + L370 + R442, L3+ V5 + V187 + L187 + V5 + L59 + L164 + F164 + R442, L3+ K164 + L164 + R442 + K187 + L180 + L178 + L + K187 + K20 + K178 + K180 + K178 + K180 + K178 + K178 + K20 + K178 + K20 + K180 + K178 + K20 + K + 178, L178 + K20 + K20 + K20 + K + 178 + K + 178 + K + 178, L178 + K + 178 + K + 178, L178 + K + 178, L178 + K + 178, L164 + K + 180, L164 + K + 180, L + K + 180, L + K + 180, L178 + K + 180, L164 + K + 180, L + K + 180, L + K + 180, L + K + 180, L + K +, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + D391, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Q32 + E171, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + I389, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + N394, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + L404, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L187 + L404, L3+ V5 + C60 + F164 + L178 + L180 + L178 + L164 + K456, L3+ V5 + L187 + L60 + L187 + L178 + L180 + L178 + L164 + K, L164 + L178 + L180 + L178 + L180 + L187 + L178 + L180 + L187 + K164 + L178 + L180 + L178 + L187 + K164 + L180 + L178 + L180 + K164, L180 + L187 + K164, L178 + L187 + K164 + L178 + L180 + K164 + L187 + L178 + L187 + K164, L180 + K, L187 + K164 + L178 + K164 + L180 + L178 + L187 + L180 + L187 + K180 + L187 + K164, L178C 180 + L187 + L178 + K164, L178 + K, L164, L187 + K180 + L187 + K180 + K180, L187 + K180, L187 + K180 + K180, L187 + K180, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + Y384 + L404, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + E424 + G411, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + S186 + Q420, L3+ V5 + C60 + F164 + A178 + I180 + L370 + G411, L3+ V5 + F60 + F164 + G178 + L187 + I180 + L1 + G411 + G164 + L164 + K187 + L65 + G411, L3+ V5 + L60 + F164 + F178 + I178 + K187 + K180 + L187 + L180 + L178 + L180 + L164G 411 + E + L164 + D164, L164 + K389 + K, L164 + K, L187 + K, L180 + K + L187 + K, L180 + K, L180 + L187 + K, L180 + K, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + V252, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + I389 + V252, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + E424, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S411 + L384 + G411 + S186 + Y384 + I389, L3+ V5 + C60 + F164 + A178 + S164 + F164 + A178 + S187 + I180 + L370 + S411 + Y384 + I187 + V180 + L187 + V187 + S411 + S187 + S186 + Y384 + I187 + V20 + V411 + V2 + L178 + L20 + I187 + V20 + I187 + I20 + I187 + I20 + I187 + L20 + I187 + L20 + I187 + L20 + I187 + L187 + I187 + L187 + I187 + L20 + L187 + I187 + L178 + L187 + L20 + L187 + L20 + L187 + I187 + L187 + I187 + L187 + I187 + L20 + I187 + L20 + L187 + I187 + L20 + I187 + L20 + I187 + L20 + L187 + I187 + L20, L20 + L187 + L20 + L187 + L20, L187 + L20 + L187 + L20 + L187 + L20 + L187 + L20, L20 + L187 + L20 + L187, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + E171, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384 + D391, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411 + S186 + Y384, L3+ V5 + C60 + F164 + I180 + L370 + G411 + R442, L3+ V5 + C60 + F164 + A178 + S187 + I180 + L370 + G411, L3+ V5 + F164 + C60 + I180 + L370 + G411, L3+ V5 + F384 + L370 + G411, L3+ V5 + L164 + L70 + L164 + Q164 + L5 + V + F + V + L5 + L70 + L370 + L + G + L60 + V + F + L5 + L + F + R411, L3+ V + F + 5 + L + E + L70 + L + R < 5 + R < SP > 411, L + K, L3S + V5S + F164V + C60Y + I180V + L370A + D391E, L3S + V5S + C60Y + F164V + L370A + D391E, L3S + V5S + C60Y + F164V + Q420R + L370A + D391A, L3A + V5A + F164A + C60A + L370 + G452 + D391A, L3A + V5A + F A + C A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + L A + A.

2. A DNA molecule encoding the transaminase mutant of claim 1.

3. A recombinant plasmid comprising the DNA molecule of claim 2.

4. The recombinant plasmid according to claim 3, wherein the recombinant plasmid is pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), or, pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc 53999, pTwin1, pZZ 685 18, pK 232-18, pK-18-19 or pK-19.

5. A host cell comprising the recombinant plasmid of claim 3 or 4.

6. The host cell of claim 5, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell.

7. The host cell of claim 6, wherein the prokaryotic cell is an E.coli BL21-DE3 cell or an E.coli Rosetta-DE3 cell and the eukaryotic cell is yeast.

8. A method for producing a chiral amine, comprising the step of subjecting a ketone compound and an amino donor to a catalytic transamination reaction by a transaminase, wherein the transaminase is the transaminase mutant of claim 1.

9. The method of claim 8, wherein the ketone compound is

The transamination reaction product is

Wherein R is₁And R₂Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r₁And R₂Can be used aloneOr both may be bonded to each other to form a substituted or unsubstituted ring.

10. The method of claim 9, wherein R is₁And R₂Is an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.

11. The method of claim 10, wherein R is₁And R₂Is an optionally substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.

12. The method of claim 11, wherein the aryl group comprises phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furyl, pyrrolyl, phenoxy, naphthoxy, pyridyloxy, thienyloxy, oxadiazoyloxy, imidazolyloxy, thiazolyloxy, furanyloxy, and pyrrolyloxy;

the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;

the aralkyl group is a benzyl group;

the substitution means substitution by 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.

13. The method of claim 12, wherein the ketone compound is

14. The method of claim 8, wherein the amino donor is isopropylamine or alanine.

15. The method of claim 14, wherein the amino donor is isopropylamine.

16. The method of claim 8, wherein the reaction system in which the transaminase catalyzes the transamination of the ketone compound and the amino donor has a pH of 7 to 11; the temperature of a reaction system for the transaminase to carry out catalytic transaminase reaction on the ketone compound and the amino donor is 25-60 ℃; the volume concentration of dimethyl sulfoxide in a reaction system for carrying out catalytic transamination reaction on ketone compounds and an amino donor by using transaminase is 0-50%; the volume concentration of methyl tert-butyl ether in a reaction system for carrying out catalytic transamination reaction on ketone compounds and amino donors by using transaminase is 0-90%.

17. The method of claim 16, wherein the transaminase is used to catalyze the transamination of ketones and amino donors at a pH of 8 to 10; the temperature of a reaction system for the transaminase to carry out the catalytic transaminase reaction on the ketone compound and the amino donor is 30-55 ℃.

18. The method of claim 17, wherein the transaminase is used to catalyze the transamination of ketones and amino donors at a pH of 9 to 10; the temperature of a reaction system for the transaminase to perform the catalytic transaminase reaction on the ketone compound and the amino donor is 40-50 ℃.

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