Patent Publication Number: US-2022220519-A1

Title: Transaminase mutant and use thereof

Description:
TECHNICAL FELD 
     The disclosure relates to the field of biotechnologies, and in particular, to a transaminase mutant and use thereof. 
     BACKGROUND 
     Chiral amines widely exist in the natural world, and are important intermediates for synthesizing natural products and chiral drugs. Many chiral amines contain one or more chiral centers, there are significant differences in pharmacological activity, metabolic process, metabolic rate and toxicity of the different chiral drugs, usually one enantiomer is effective, but the other enantiomer is low-effective or ineffective, and even toxic. Therefore, how to efficiently and stereoselectively construct compounds containing the chiral centers is of great significance in pharmaceutical research and development. 
     The chiral amines are an important composition part for synthesizing a variety of biologically active compounds and active pharmaceutical ingredients. It is estimated that 40% of existing drugs are the chiral amines and derivatives thereof, for example, the synthesis of neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines all uses the chiral amines as intermediates (Top. Catal. 2014, 57, 284-300), this makes chiral amine compounds become an important part of a pharmaceutical industry. 
     There are many types of industrial production for the chiral amines, it mainly relies on metal-catalyzed hydrogenation of an enamide from a ketone precursor, and a process requires expensive transition metal complexes as catalysts. Because these transition metal complex resources are limited, it is difficult to achieve sustainability. At the same time, the process of asymmetric-synthesizing the chiral amine from the ketone precursor requires protection and deprotection steps of an amine, steps and waste are increased, and a yield is reduced. 
     In a method of synthesizing the chiral amine by a catalytic hydrogenation reduction, the catalyst is difficult in preparation and expensive, the device investment is large, the production cost is high, the requirements for activity of catalyst and hydrogenation performances are very high, and the catalyst is toxic, especially a sulfide in hydrogen, it easily causes a person to be poisoned. 
     Transaminase is a generic term of enzymes that use pyridoxal phosphate as a cofactor, and catalyze an amino on 1 amino donor (amino acid or amine) to be transferred to a prochiral acceptor ketone, as to obtain a chiral amine or a by-product ketone or α-keto acid thereof. Because traditional chemical methods for asymmetric-synthesizing amines have different limitations, such as low efficiency, low selectivity and serious environmental pollution, at the same time, the synthesis of the chiral amines catalyzed by the transaminase has high stereo and chemoselectivity, has safety and environmental compatibility, and is a process of green environmental protection, it is called green chemistry. At the same time, enzyme catalysis is often in place in one step, it has incomparable advantages of the chemical methods. The synthesis of the chiral compounds by the transaminase becomes a key asymmetric synthesis technology. 
     Although the progress of using the transaminase to produce the chiral amines is highly concerned, an enzymatic method has many problems in amplified production application. For example, the increase of a fermentation cost is caused by low enzyme activity and large amount of enzymes; at the same time, the large amount of the enzymes caused by the low enzyme activity seriously hinders an industrial application of an enzyme-catalyzed reaction. On the one hand, the large amount of the enzymes causes that a reaction volume is large, and a utilization rate of a catalytic container is reduced. At the same time, it is caused that a volume of post-treatment is increased, and an amount of an extraction solvent is large, so that extraction, concentration and acquisition of a product are difficult, and a product yield is low, it greatly hinders the industrial application of the enzyme catalysis. Enzymes with high activity may reduce the amount of the enzymes and the reaction volume, so that the industrial application of the enzyme catalysis is possible. Therefore, it is very important to obtain the high-activity enzymes. At the same time, a substrate spectrum of the enzymes may also be expanded, so that some enzyme-catalyzed reactions with extremely low conversion rates or even inactivity may proceed smoothly, an excellent conversion rate and an extremely high product chiral purity are achieved. 
     On the other hand, while catalyzed, the enzyme is easily affected by an organic solvent in a reaction system or easily affected to be denatured and inactivated by factors, such as high pH and high temperature of the reaction. Therefore, it is also critical that the tolerance of the enzyme to extreme conditions is increased. In the industrial production of the chiral amines, because most of existing substrates and amino donors are poorly water-soluble, in order to increase the production of chiral amine products, it is necessary to increase the content of the organic solvents in the reaction system, or use basic amino donors (such as isopropylamine), an extremely harsh reaction condition is created, so that wild transaminase is extremely easily denatured to lose activity, so the transaminase that is well tolerant to the organic solvent and high pH is required to meet the needs of the industrial production. 
     SUMMARY 
     The disclosure aims to provide a transaminase mutant and use thereof, as to improve activity of transaminase. 
     In order to achieve the above purpose, according to one aspect of the disclosure, a transaminase mutant is provided. An amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation occurred in an amino acid sequence as shown in SEQ ID NO: 1, and the mutation includes at least one of the following mutation sites: position 3, position 5, position 8, position 25, position 32, position 45, position 56, position 59, position 60, position 84, position 86, position 164, position 176, position 178, position 180, position 187, position 197, position 206, position 207, position 242, position 245, position 319, position 324, position 326, position 328, position 370, position 397, position 414, position 416, position 424, position 436, position 437 and position 442. 
     Further, the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation occurred in the amino acid sequence as shown in SEQ ID NO:1, and the mutation includes at least one of the following mutation sites: L3S, V5S, I8A, I8S, F25L, F25T, Q32N, I45W, L59V, F56M, C60F, C60Y, F84V, W86H, W86L, W86P, W86N, F164M, F164V, F176Y, F176S, A178L, I180V, S187A, T197P, L206M, K207T, V242A, T245A, T245V, R319C, R324A, R324G, E326M, V328A, V328G, L370A, L370D, L370K, T397A, P414G, Q416A, E424D, E424T, A436S, A436G, A436P, A436N, A436Y, A436Q, A436E, M437S, M437A, R442T, R442S, R442Q and R442V; or an amino acid sequence of the transaminase mutant has the mutation sites in the mutated amino acid sequence, and has more than 80% identity with the mutated amino acid sequence. 
     Further, the mutation further includes at least one of the following mutation sites: C60Y+F164V, L3S+V5S, L3S+V5S+F164V, L3S+V5S+C60Y, L3S+V5S+C60Y+F164V, I180V+L370A and L3S+V5S+L59V; preferably, the mutation further includes at least one of the following mutation site combinations: F164V+C60Y, E424D+A436G, C60Y+F164V+A436P, C60Y+F164V+A436N, W86P+F164V, F25L+L59V, F25T+F164V, C60Y+F164V+A436Y, C60Y+F164V+A436Q, C60Y+F164V+A436E, F164V+M437A, I8A+V328A, I8S+F164V, C60Y+F164V+L370A, C60Y+F164V+L370D, C60Y+F164V+L370K, I45W+F164V, C60Y+F164V+F176Y, C60Y+F176S+F164V, L3S+V5S+C60Y+F164V+L370A, C60Y+F164V+R442S, C60Y+F164V+R442Q, L3S+V5S+S187A+L370A+E424D, C60Y+F164V+R442T, L3S+V5S+E424D+L370A, L3S+V5S+F164V+C60Y+I180V+L370A, L3S+V5S+C60Y+F164V+A178L+L370A, L3S+V5S+F164V+T197P+L370A, L3S+V5S+V328A+E424D, L3S+V5S+L59V+L206M+L370A, L3S+V5S+L370A+E424D, L3S+V5S+F164V+K207T+L370A, L3S+V5S+S244A+L370A, L3S+V5S+F164V+T245A+L370A, L3S+V5S+F164V+T245V+V328A, L3S+V5S+F164V+L370A+T397A, L3S+V5S+L59V+F164V+R319C+L370A+T397A, L3S+V5S+L59V+F164V+L370A, L3S+V5S+L59V+L370A+A436G+Q416A, L3+V5S+L59V+L370A+A436G+R442Q, L3S+V5S+L59V+V328A+L370A+R442Q, L3S+V5S+L59V+L370A+R442L, L3S+V5S+L59V+C60Y+F164V+L370A+R442V, L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A, and L3S+V5S+C60Y+F164V+I180V+S187A+L370A+R442L. 
     Further, the mutation at least includes one of the following mutation sites or mutation site combinations: position 7, position 32, position 96, position 164, position 171, position 186, position 252, position 384, position 389, position 391, position 394, position 404, position 411, position 420, position 423, position 424, position 442, position 452 and position 456; preferably, the mutation at least also includes 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 at least includes one of the following mutation site combinations: 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, and L3S+V5S+F164V+C60Y+L370A+G452S+L404Q. 
     According to another aspect of the disclosure, a DNA molecule is provided. The DNA molecule encodes any one of the above transaminase mutants. 
     According to another aspect of the disclosure, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. 
     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-23a(+), pET-23b(+), pET-24a(+), 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-43a(+), 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-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. 
     According to another aspect of the disclosure, a host cell is provided. The host cell contains any one of the above recombinant plasmids. 
     Further, the host cell includes prokaryotic cell, yeast or eukaryotic cell; preferably, the prokaryotic cell is  Escherichia coli  BL21-DE3 cell or  Escherichia coli  Rosetta-DE3 cell. 
     According to another aspect of the disclosure, a method for producing a chiral amine is provided. The method includes a step of using a transaminase to catalyze a transamination reaction of a ketone compound and an amino donor, wherein the transaminase is any one of the above transaminase mutants. 
     Further, the ketone compound is 
     
       
         
         
             
             
         
       
     
     R 1  and R 2  are each independently optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted aralkyl, or optionally substituted or unsubstituted aryl; and R 1  and R 2  can form a substituted or unsubstituted ring alone or in combination;
 
preferably, R 1  and R 2  are optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted aralkyl, or optionally substituted or unsubstituted aryl having 1 to 20 carbon atoms, more preferably optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted aralkyl group, or optionally substituted or unsubstituted aryl having 1 to 10 carbon atoms;
 
preferably, the aryl includes phenyl, naphthyl, pyridyl, thienyl, oxadiazole group, imidazole group, thiazolyl, furanyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, thienyloxy, oxadiazoloxy, imidazoloxy, thiazolyloxy, furanyloxy and pyrrolyloxy;
 
preferably, the alkyl includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, t-butyl, methoxy, ethoxy, t-butoxy, methoxy carbonyl, ethoxy carbonyl, t-butoxy carbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
 
preferably, the aralkyl is a benzyl; and
 
preferably, the substitution refers to substitution with halogen atom, nitrogen atom, sulfur atom, hydroxy, nitro, cyano, methoxy, ethoxy, carboxyl, carboxymethyl, carboxyethyl or methylenedioxy.
 
     Preferably, the ketone compound is 
     
       
         
         
             
             
         
       
     
     Further, the amino donor is isopropylamine or alanine, preferably the isopropylamine. 
     Further, in a reaction system the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor, the pH is 7 to 11, preferably 8 to 10, and more preferably 9 to 10. 
     Further, in the reaction system the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor, the temperature is 25° C. to 60° C., more preferably 30 to 55° C., and further preferably 40 to 50° C. 
     Further, a volume concentration of dimethyl sulfoxide in the reaction system the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor is 0% to 50%. 
     Further, the volume concentration of methyl tert-butyl ether in the reaction system the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor is 0% to 90%. 
     The above transaminase mutants of the disclosure are based on the transaminase shown in SEQ ID NO: 1, and are mutated through a site-directed mutation method, thereby the amino acid sequence thereof is changed, so changes in protein structure and function are achieved, and through a directed screening method, the transaminase with the above mutation sites is obtained. Therefore, these transaminase mutants have good organic solvent tolerance and high pH tolerance, and have high soluble expression characteristics and high activity characteristics, a reaction rate may be improved by use of these mutants, the enzyme stability is improved, the amount of the enzyme is reduced, and the difficulty of post-treatment is reduced, so it may be suitable for industrial production. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Drawings for constituting a part of the present disclosure are used to provide further understanding of the disclosure. Exemplary embodiments of the disclosure and descriptions thereof are used to explain the disclosure, and do not constitute improper limitation to the disclosure. In the drawings: 
         FIG. 1  shows an electrophoresis diagram of an expression condition of a protein detected by SDS-PAGE in an embodiment of the disclosure. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     It is to be noted that embodiments in the present disclosure and features in the embodiments may be combined with each other in the case without conflicting. The disclosure is described in detail below in combination with the embodiments. 
     Transaminase is a type of protein-based biocatalysts. In an industrial production process, certain organic solvent, pressure, pH and other conditions that are easy to denature a protein are often required. Therefore, the biocatalyst used needs to have high tolerance in order to meet the needs of the industrial production. However, wild transaminase often has low tolerance to industrial demand conditions, thereby its wide application is limited. 
     Transaminase ArS-ωTA derived from  Arthrobacter citreus  may specifically catalyze a ketone compound to produce an amino product, but an enzyme has low tolerance to the organic solvent, and the enzyme has low tolerance in high pH. At the same time, the enzyme is poor in soluble expression in a prokaryotic expression system, and the enzyme has low activity to a substrate, so that a usage amount of the enzyme is larger, and the ketone compound is converted less; at the same time, due to the large amount of the enzyme, the difficulty of post-treatment is also increased, a yield is low, and a process is complicated. The disclosure performs modification in allusion to the above disadvantages of the transaminase ArS-ωTA, its organic solvent tolerance is improved, pH tolerance, soluble expression characteristics and activity characteristics are improved, so that it may be applied to industrial production conditions. 
     Rational modification of the enzyme is based on a three-dimensional molecular structure of the enzyme to modify a substrate binding site, a coenzyme binding site, a surface and other parts of the enzyme, as to change catalytic properties of the enzyme and improve the characteristics, such as activity and selectivity, of the enzyme. Directed evolution of the enzyme is a non-rational design of the protein, a special evolutionary condition is artificially created, a natural evolutionary mechanism is simulated, a gene is modified in vitro, and error-prone PCR, DNA shuffling and other technologies are applied, a new enzyme with expected properties is obtained in combination with an efficient screening system. 
     A technical scheme of the disclosure rationally modifies the ArS-ωTA protein through a technology combined with rational design and random mutation, the obtained mutant uses the ketone compound for activity verification, and finally a mutant strain having good organic solvent tolerance, high pH tolerance, soluble expression, activity and selectivity is obtained. 
     The rational design may be performed by means of site-directed mutation. Herein, the site-directed mutation: means that a desired change (usually a change representing a favorable direction) is introduced into a target DNA fragment (may be a genome or a plasmid) by a polymerase chain reaction (PCR) and other methods, including base addition, deletion, point mutation and the like. The site-directed mutation may quickly and efficiently improve characters and representation of a target protein expressed by DNA, and is a very useful method in gene research. 
     The method of introducing the site-directed mutation using a whole-plasmid PCR is simple and effective, and is a more commonly used method at present. A principle is that a pair of primers (forward and reverse) containing mutation sites are annealed with a template plasmid, and “circularly extended” by a polymerase. The so-called cyclic extension means that the polymerase extends the primer according to the template, and then returns to a 5′-end of the primer to be terminated after one round, and it undergoes circulation of repeated heating and annealing extension, this reaction is different from rolling circle amplification and does not form multiple tandem copies. Extension products of the forward and reverse primers are annealed and matched to form an open-circle plasmid with a nick. The extension product of Dpn I digestion, because the original template plasmid is derived from conventional  Escherichia coli , is modified by dam methylation, and is fragmented because it is sensitive to Dpn 1, the plasmid with a mutation sequence synthesized in vitro is not digested because it is not methylated, so it may be successfully transformed in the subsequent transformation, and a clone of the mutant plasmid may be obtained. 
     The above mutant plasmid is transformed into an  Escherichia coli  cell, and overexpressed in the  Escherichia coli . Then, a crude enzyme is obtained by ultra-sonicating the cells. The optimum conditions for induction expression of the transaminase: 25° C., and 0.1 mM IPTG induction overnight. 
     Through computer simulation analysis of a three-dimensional structure of the transaminase using software, it is found that the ArS-ωTA protein is an S-type transaminase with pyridoxal 5-phosphate (PLP) as a cofactor. An amino acid near the active center of the enzyme is modified to improve its enzymatic properties, such as stabilizing a transition state, reducing free energy of a binding state between the enzyme and a reaction transition state molecule, making the substrate more easily enter the active neutrality, and reducing the steric hindrance of the substrate; and an amino acid far away from the active center is modified, chemical bond that promotes the stability, such as hydrogen bond, disulfide bond, salt bridge and hydrophobic accumulation, is added, so the stability of the protein may be improved, and a protein half-life is increased. 
     The disclosure rationally modifies the ArS-ωTA protein (SEQ ID NO: 1) and performs amino acid mutations (L3S, VSS, I8A, I8S, I45W, F25L, F25T, Q32N, L59V, F56M, C60F, C60Y, F84V, W86H, W86L, W86P, W86N, Y89F, F164M, F164V, F164Y, F176Y, F176S, A178L, I180V, S187A, T197P, L206M, K207T, T245A, T245V, R319C, V242A, V328A, V328G, T397A, P414G, E424D, E424T, L370A, L370D, L370K, R324A, R324G, E326M, Q416A, A436S, A436G, A436P, A436N, A436Y, A436Q, A436E, M437S, M437A, R442T, R442S, R442Q, R442V, R442A) and combination mutations thereof, preferably pET22b is used as an expression vector, and a plasmid containing a mutant gene is obtained, preferably BL21 (DE3) is used as an expression strain, and the mutant protein is obtained under the induction of IPTG. 
     The constructed mutant protein performs activity verification, and results are shown in Table 1: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Table 1: Strain 
               
            
           
           
               
               
               
            
               
                   
                   
                 multiple of 
               
               
                   
                 Amino acid difference 
                 activity increased 
               
               
                   
                 (compare to ArS-ωTA) 
                 (compare to ArS-ωTA) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 ArS-ωTA 
                 N/A 
                 0 
               
               
                 M1 
                 I8A 
                 0.91 
               
               
                 M2 
                 I8S 
                 2.15 
               
               
                 M3 
                 Q32N 
                 2.25 
               
               
                 M4 
                 F56M 
                 5.3 
               
               
                 M5 
                 L59V 
                 4.73 
               
               
                 M6 
                 C60F 
                 15.1 
               
               
                 M7 
                 F84V 
                 2.98 
               
               
                 M8 
                 W86H 
                 5.51 
               
               
                 M9 
                 W86L 
                 3.93 
               
               
                 M10 
                 W86P 
                 5.6 
               
               
                 M11 
                 W86N 
                 0.6 
               
               
                 M12 
                 Y89F 
                 −0.1 
               
               
                 M13 
                 F164M 
                 14.5 
               
               
                 M14 
                 F164V 
                 45.1 
               
               
                 M15 
                 V242A 
                 11.7 
               
               
                 M16 
                 V328A 
                 6.56 
               
               
                 M17 
                 V328G 
                 2.84 
               
               
                 M18 
                 P414G 
                 8.31 
               
               
                 M19 
                 E424D 
                 5.29 
               
               
                 M20 
                 E424T 
                 6.58 
               
               
                 M21 
                 A436S 
                 5.5 
               
               
                 M22 
                 A436V 
                 9.6 
               
               
                 M23 
                 R442V 
                 −1 
               
               
                 M24 
                 R442A 
                 −1 
               
               
                 M25 
                 L3S + V5S 
                 0.20 
               
               
                 M26 
                 C60Y 
                 30.17 
               
               
                 M27 
                 F164Y 
                 −0.2 
               
               
                 M28 
                 A436G 
                 3.67 
               
               
                   
               
            
           
         
       
     
     Multiple sites that may improve the catalytic activity of the transaminase mutants are obtained above through the site-directed mutation. The activity verification is performed by using 10 wt of wet weight cells at 0.02 g/ml of the substrate concentration, and the activity of the optimal mutant obtained is 4 (times greater than that of the parent ArS-ωTA. However, the activity of the original bacteria ArS-ωTA is too low, the mutant after the 45-time increase of the activity still has a large amount of the substrate that may not be converted into amino products after 16 hours of the conversation. Therefore, further modification is carried out, including introduction of beneficial site combinations and new mutation sites, the mutant after the modification is subjected to the activity verification using 5 wt˜0.5 wt of the weight cells at 0.1 g/ml of the substrate concentration, as shown in Table 2. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Multiple of 
               
               
                   
                 Amino acid difference 
                 activity increased 
               
               
                 Strain 
                 (compare to ArS-ωTA) 
                 (compare to ArS-ωTA) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 M29 
                 F164V + C60Y 
                 105.3 
               
               
                 M30 
                 E424D + A436G 
                 107.3 
               
               
                 M31 
                 C60Y + F164V + A436P 
                 116.3 
               
               
                 M32 
                 C60Y + F164V + A436N 
                 55.3 
               
               
                 M33 
                 W86P + F164V 
                 1 
               
               
                 M34 
                 F25L + L59V 
                 47.2 
               
               
                 M35 
                 F25T + F164V 
                 0.6 
               
               
                 M36 
                 C60Y + F164V + A436Y 
                 140 
               
               
                 M37 
                 C60Y + F164V + A436Q 
                 144.07 
               
               
                 M38 
                 C60Y + F164V + A436E 
                 66 
               
               
                 M39 
                 V328A + M437S 
                 −1 
               
               
                 M40 
                 F164V + M437A 
                 0.5 
               
               
                 M41 
                 I8A + V328A 
                 135.4 
               
               
                 M42 
                 I8S + F164V 
                 14.1 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 199.4 
               
               
                 M44 
                 C60Y + F164V + L370D 
                 203.4 
               
               
                 M45 
                 C60Y + F164V + L370K 
                 165.1 
               
               
                 M46 
                 I45W + F164V 
                 0.1 
               
               
                 M47 
                 C60Y + F164V + F176Y 
                 164.5 
               
               
                 M48 
                 C60Y + F176S + F164V 
                 159.3 
               
               
                 M49 
                 F164V + R324A 
                 −1 
               
               
                 M50 
                 F164V + R324G 
                 −1 
               
               
                 M51 
                 E326M + E424T 
                 0 
               
               
                 M52 
                 L3S + V5S + C60Y + F164V + 
                 238.9 
               
               
                   
                 L370A 
               
               
                 M53 
                 C60Y + F164V + R442S 
                 1004.1 
               
               
                 M54 
                 C60Y + F164V + R442Q 
                 2526.6 
               
               
                 M55 
                 L3S + V5S + S187A + L370A + 
                 1274.6 
               
               
                   
                 E424D 
               
               
                 M56 
                 C60Y + F164V + R442T 
                 567.7 
               
               
                 M57 
                 L3S + V5S + E424D + L370A 
                 858.6 
               
               
                 M58 
                 L3S + V5S + F164V + C60Y + 
                 2655.4 
               
               
                   
                 I180V + L370A 
               
               
                 M59 
                 L3S + V5S + C60Y + F164V + 
                 2467.4 
               
               
                   
                 A178L + L370A 
               
               
                 M60 
                 L3S + V5S + F164V + T197P + 
                 16.5 
               
               
                   
                 L370A 
               
               
                 M61 
                 L3S + V5S + V328A + E424D 
                 1089.9 
               
               
                 M62 
                 L3S + V5S + L59V + L206M + 
                 1357.9 
               
               
                   
                 L370A 
               
               
                 M63 
                 L3S + V5S + L370A + E424D 
                 630.6 
               
               
                 M64 
                 L3S + V5S + F164V + K207T + 
                 807 
               
               
                   
                 L370A 
               
               
                 M65 
                 L3S + V5S + S244A + L370A 
                 1189.2 
               
               
                 M66 
                 L3S + V5S + F164V + T245A + 
                 13.9 
               
               
                   
                 L370A 
               
               
                 M67 
                 L3S + V5S + F164V + T245V + 
                 23.4 
               
               
                   
                 V328A 
               
               
                 M68 
                 L3S + V5S + F164V + L370A + 
                 281.2 
               
               
                   
                 T397A 
               
               
                 M69 
                 L3S + V5S + L59V + F164V + 
                 20.8 
               
               
                   
                 R319C + L370A + T397A 
               
               
                 M70 
                 L3S + V5S + L59V + F164V + 
                 5.2 
               
               
                   
                 L370A 
               
               
                 M71 
                 L3S + V5S + L59V + L370A + 
                 1725.9 
               
               
                   
                 A436G + Q416A 
               
               
                 M72 
                 L3 + V5S + L59V + L370A + 
                 620.8 
               
               
                   
                 A436G + R442Q 
               
               
                 M73 
                 L3S + V5S + L59V + V328A + 
                 1671 
               
               
                   
                 L370A + R442Q 
               
               
                 M74 
                 L3S + V5S + L59V + L370A + 
                 2343.4 
               
               
                   
                 R442L 
               
               
                 M75 
                 L3S + V5S + L59V + C60Y + 
                 1121.9 
               
               
                   
                 F164V + L370A + R442V 
               
               
                 M76 
                 L3S + V5S + C60Y + F164V + 
                 3455.4 
               
               
                   
                 A178L + S187A + I180V + L370A 
               
               
                 M77 
                 L3S + V5S + C60Y + F164V + 
                 3200.8 
               
               
                   
                 I180V + S187A + L370A + R442L 
               
               
                   
               
            
           
         
       
     
     After the above modification, multiple sites that may improve the catalytic activity of the transaminase mutant are obtained, and a beneficial mutation combination is carried out at the same time, and multiple mutation site combinations with the improved activity are obtained. The activity of the best strain is 3455 times greater than that of the ArS-ωTA. The optimal mutant strain is subjected to the activity verification using 0.5 wt of wet weight cells at 0.1 g/ml of the substrate concentration in a reaction system (a volume is very small, 10 V). After 16 hours of the conversation, a conversation rate reaches more than 95%. 
     It may be seen that the mutant strain is excellently improved in catalytic activity, and improved from the basic non-catalytic activity of the wild bacteria (10 wt of the wet weight cells, 0.02 g/ml of the substrate concentration, and 0.1% of the conversion rate), to the excellent catalytic activity: a very small amount of the enzyme (0.5 wt) and a very small reaction volume (10 V) are used to achieve 95% of a conversion effect. 
     The disclosure simultaneously performs directed evolution on the above mutant protein obtained by the rational modification, and a mutant protein that greatly improves (improvement of quality) the activity of the ketone substrate is obtained. This mutant protein is used as an original strain, error-prone PCR is used as a technical means to process random mutations, as to further improve its activity and characteristics such as organic solvent tolerance and high pH tolerance. At the same time, combined with a site-directed mutation technology and a staggered extension PCR random recombination technology, the beneficial mutations obtained by the error-prone PCR are continuously accumulated. The obtained mutants construct a mutant library containing the random mutations, the organic solvent concentration is continuously increased, the pH of the screening and reaction system is continuously increased, and a screening pressure is set, to obtain a target property mutant. The mutant uses pET22b as an expression vector and uses BL21 (DE3) as an expression strain, and the mutant protein is obtained under the induction of IPTG. The induced mutant strains containing the target protein are lysed by an ultrasonication mode to release the target protein, and an expression condition of the target protein is detected by SDS-PAGE. 
     Herein, the error-prone PCR is to change a mutation frequency in an amplification process by adjusting reaction conditions while a DNA polymerase is used to amplify a target gene, the tendency of an inherent mutation sequence of the polymerase is reduced, and the diversity of a mutation spectrum is increased, so that wrong bases are randomly incorporated into the amplified gene at a certain frequency, thereby a randomly mutated DNA group is obtained. 
     In the disclosure, a variety of the mutations are obtained by the above method of random mutation and directed screening, and through the activity verification, the mutant improves the tolerance to the organic solvent, the tolerance to the pH, the activity to the substrate, and the solubility of the target protein. 
     Tolerance verification: a tolerance improvement degree of the mutation site obtained by the error-prone PCR in 35% of dimethyl sulfoxide 
     Tolerance verification results of a beneficial mutation site obtained using M76 (L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A) as the original strain in 35% of the dimethyl sulfoxide are shown in Table 3. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                   
                   
                 Tolerance 
               
               
                   
                   
                 Amino acid difference 
                 improvement 
               
               
                   
                 Strain 
                 (compare to M76) 
                 degree 
               
               
                   
                   
               
             
            
               
                   
                 M76 
                 N/A 
                 Null 
               
               
                   
                 M78 
                 G411D 
                 225.83% 
               
               
                   
                 M79 
                 Y384F + G452S 
                 218.95% 
               
               
                   
                 M80 
                 S186G + Q420R 
                 554.20% 
               
               
                   
                 M81 
                 M423K 
                 150.10% 
               
               
                   
                 M82 
                 Q420K + E424R 
                 183.15% 
               
               
                   
                 M83 
                 K7N + E424Q 
                 83.33% 
               
               
                   
                 M84 
                 D391E 
                 401.29% 
               
               
                   
                 M85 
                 Q32L + E171D 
                 594.07% 
               
               
                   
                 M86 
                 I389M 
                 335.16% 
               
               
                   
                 M87 
                 I389F + N394D 
                 37.76% 
               
               
                   
                 M88 
                 L404Q 
                 12.06% 
               
               
                   
                 M89 
                 I389F + L404Q 
                 137.91% 
               
               
                   
                 M90 
                 V164L + K456R 
                 351.86% 
               
               
                   
                 M91 
                 K96R 
                 23.48% 
               
               
                   
                 M92 
                 Q32L + R442H 
                 131.25% 
               
               
                   
                 M93 
                 R442L 
                 157.16% 
               
               
                   
                 M94 
                 V252I 
                 131.58% 
               
               
                   
                 M95 
                 E424K 
                 59.79% 
               
               
                   
                   
               
            
           
         
       
     
     Tolerance versification: a tolerance improvement degree of the mutation site obtained by the error-prone PCR in 50% of methyl tert-butyl ether. 
     Tolerance verification results of a beneficial mutation site obtained using M76 (L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A) as the original strain in 50% of the methyl tert-butyl ether are shown in Table 4. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                   
                 Tolerance 
               
               
                   
                   
                 Amino acid difference 
                 improvement 
               
               
                   
                 Strain 
                 (compare to M76) 
                 degree 
               
               
                   
                   
               
             
            
               
                   
                 M76 
                 N/A 
                 Null 
               
               
                   
                 M78 
                 G411D 
                 26.22% 
               
               
                   
                 M79 
                 Y384F + G452S 
                 27.56% 
               
               
                   
                 M80 
                 S186G + Q420R 
                 63.70% 
               
               
                   
                 M81 
                 M423K 
                 350.13% 
               
               
                   
                 M82 
                 Q420K + E424R 
                 443.27% 
               
               
                   
                 M83 
                 K7N + E424Q 
                 17.87% 
               
               
                   
                 M84 
                 D391E 
                 245.61% 
               
               
                   
                 M85 
                 Q32L + E171D 
                 117.28% 
               
               
                   
                 M86 
                 I389M 
                 116.47% 
               
               
                   
                 M87 
                 I389F + N394D 
                 251.31% 
               
               
                   
                 M88 
                 L404Q 
                 149.03% 
               
               
                   
                 M89 
                 I389F + L404Q 
                 157.31% 
               
               
                   
                 M90 
                 V164L + K456R 
                 74.62% 
               
               
                   
                 M91 
                 K96R 
                 10.78% 
               
               
                   
                 M92 
                 Q32L + R442H 
                 14.82% 
               
               
                   
                 M93 
                 R442L 
                 46.52% 
               
               
                   
                 M94 
                 V252I 
                 75.41% 
               
               
                   
                 M95 
                 E424K 
                 48.17% 
               
               
                   
                   
               
            
           
         
       
     
     The obtained beneficial mutation uses an iterative—error-prone PCR—directed screening method, and use the site-directed mutation technology to continuously improve the activity of the mutant strain and the tolerance to the organic solvent. 
     Different mutant strains are used as original strains for the tolerance site verification: 
     The plasmids of the different mutant strains are extracted, and the mutant strains are constructed by the site-directed mutation technology. The obtained mutant strains are subjected to the verification of tolerance sites in 35% of the dimethyl sulfoxide, and results are shown in Table 5: 
     
       
         
           
               
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Original strain (amino acid difference 
                 Mutation site 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 compare to ArS-ωTA) 
                 G411D 
                 Y384F 
                 S186G 
                 D391E 
                 E171D 
                 L404Q 
               
               
                   
               
               
                 C60Y + F164V + R442Q 
                 ++ 
                 ++ 
                 +++ 
                 ++ 
                 ++ 
                 + 
               
               
                 L3S + V5S + F164V + C60Y + I180V + L370A 
                 ++ 
                 ++ 
                 +++ 
                 +++ 
                 +++ 
                 + 
               
               
                 L3S + V5S + C60Y + F164V + L370A 
                 ++ 
                 ++ 
                 +++ 
                 ++ 
                 ++ 
                 + 
               
               
                 L3S + V5S + C60Y + F164V + Q420R + L370A 
                 ++ 
                 ++ 
                 +++ 
                 +++ 
                 +++ 
                 + 
               
               
                 C60Y + F164V + L370A 
                 ++ 
                 ++ 
                 +++ 
                 ++ 
                 ++ 
                 + 
               
               
                 L3S + V5S + F164V + C60Y + L370A + G452S 
                 ++ 
                 ++ 
                 ++ 
                 ++ 
                 ++ 
                 + 
               
               
                   
               
               
                 + means that the enzyme tolerance improvement degree in 35% of the dimethyl sulfoxide is 1%~100%, 
               
               
                 ++ means that the enzyme tolerance improvement degree in 35% of the dimethyl sulfoxide is 100%~300%, 
               
               
                 +++ means that the enzyme tolerance improvement degree in 35% of the dimethyl sulfoxide is 400%~600%. 
               
            
           
         
       
     
     It may be seen that the mutation sites obtained by random mutation+directed screening have a significant effect on increasing the tolerance of the strain in the organic solvent (dimethyl sulfoxide). 
     Tolerance verification: a tolerance improvement degree of the mutation site obtained by the error-prone PCR in 40% of the dimethyl sulfoxide 
     Tolerance verification results of a beneficial mutation site obtained using M76(L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A) as the original strain in 45% of the dimethyl sulfoxide are shown in Table 6. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                 Tolerance 
               
               
                   
                 Amino acid difference 
                 improvement 
               
               
                 Strain 
                 (compare to M76) 
                 degree 
               
               
                   
               
             
            
               
                 M76 
                 N/A 
                 Null 
               
               
                 M96 
                 G411D + E424K 
                 59.47% 
               
               
                 M97 
                 Y384F + L404Q 
                 56.18% 
               
               
                 M98 
                 E424K + G411D 
                 82.19% 
               
               
                 M99 
                 G411D + S186G 
                 406.83% 
               
               
                 M100 
                 S186G + Q420R 
                 757.07% 
               
               
                 M101 
                 G411D + A187S 
                 1269.27% 
               
               
                 M102 
                 G411D + V164L + I389F + E424Q + K96R 
                 1220.24% 
               
               
                 M103 
                 V164L + I389F + L404Q 
                 891.46% 
               
               
                 M104 
                 G411D + M423K + A187S 
                 1311.22% 
               
               
                 M105 
                 G411D + S186G + I389F + L404Q 
                 1441.71% 
               
               
                 M106 
                 G411D + S186G + Y384F + V164L 
                 1027.07% 
               
               
                 M107 
                 G411D + S186G + Y384F + V164L + E171D 
                 1659.27% 
               
               
                 M108 
                 G411D + S186G + Y384F + I389F + L404Q 
                 2086.59% 
               
               
                 M109 
                 G411D + S186G + Y384F + V164L + V252I 
                 1060.00% 
               
               
                 M110 
                 G411D + S186G + Y384F + V164L + I389F + 
                 1978.78% 
               
               
                   
                 V252I 
               
               
                 M111 
                 G411D + S186G + Y384F + V164L + E424Q 
                 2036.34% 
               
               
                 M112 
                 G411D + S186G + Y384F + I389F + V164L 
                 1915.61% 
               
               
                 M113 
                 G411D + S186G + Y384F + V164L + I389F + 
                 2085.85% 
               
               
                   
                 V252I + L404Q 
               
               
                 M114 
                 G411D + S186G + Y384F + V164L + I389F + 
                 2225.85% 
               
               
                   
                 V252I + E424Q 
               
               
                 M115 
                 G411D + S186G + Y384F + V164L + I389F + 
                 2281.71% 
               
               
                   
                 V252I + L404Q + E171D 
               
               
                 M116 
                 G411D + S186G + Y384F + V164L + I389F + 
                 2228.05% 
               
               
                   
                 V252I + E424Q + M423K 
               
               
                 M117 
                 G411D + S186G + Y384F + V164L + I389F + 
                 2147.32% 
               
               
                   
                 V252I + L404Q + E171D + D391E 
               
               
                 M118 
                 G411D + S186G + Y384F + E424Q + K7N 
                 1237.04% 
               
               
                 M119 
                 G411D + S186G + Y384F + E171D 
                 395.41% 
               
               
                 M120 
                 G411D + S186G + Y384F + D391E 
                 297.45% 
               
               
                 M121 
                 G411D + S186G + Y384F 
                 273.21% 
               
               
                 M122 
                 G411D + R442L + L178A + A187S 
                 137.54% 
               
               
                   
               
            
           
         
       
     
     Tolerance verification: a tolerance improvement degree of the mutation site obtained by the error-prone PCR in 70% of methyl tert-butyl ether. 
     Tolerance verification results of a beneficial mutation site obtained using M76 (L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A) as the original strain in 70% of the methyl tert-butyl ether are shown in Table 7. 
     
       
         
           
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Tolerance 
               
               
                   
                 Amino acid difference 
                 improvement 
               
               
                 Strain 
                 (compare to M76) 
                 degree 
               
               
                   
               
             
            
               
                 M76 
                 N/A 
                 Null 
               
               
                 M96 
                 G411D + E424K 
                 37.79% 
               
               
                 M97 
                 Y384F + L404Q 
                 57.63% 
               
               
                 M98 
                 E424K + G411D 
                 107.45% 
               
               
                 M99 
                 G411D + S186G 
                 294.79% 
               
               
                 M100 
                 S186G + Q420R 
                 353.59% 
               
               
                 M101 
                 G411D + A187S 
                 373.16% 
               
               
                 M102 
                 G411D + V164L + I389F + E424Q + K96R 
                 462.54% 
               
               
                 M103 
                 V164L + I389F + L404Q 
                 314.26% 
               
               
                 M104 
                 G411D + M423K + A178S 
                 543.56% 
               
               
                 M105 
                 G411D + S186G + I389F + L404Q 
                 561.26% 
               
               
                 M106 
                 G411D + S186G + Y384F + V164L 
                 455.26% 
               
               
                 M107 
                 G411D + S186G + Y384F + V164L + E171D 
                 600.39% 
               
               
                 M108 
                 G411D + S186G + Y384F + I389F + L404Q 
                 685.05% 
               
               
                 M109 
                 G411D + S186G + Y384F + V164L + V252I 
                 486.73% 
               
               
                 M110 
                 G411D + S186G + Y384F + V164L + I389F + 
                 698.92% 
               
               
                   
                 V252I 
               
               
                 M111 
                 G411D + S186G + Y384F + V164L + E424Q 
                 806.69% 
               
               
                 M112 
                 G411D + S186G + Y384F + I389F + V164L 
                 820.06% 
               
               
                 M113 
                 G411D + S186G + Y384F + V164L + I389F + 
                 835.50% 
               
               
                   
                 V252I + L404Q 
               
               
                 M114 
                 G411D + S186G + Y384F + V164L + I389F + 
                 843.56% 
               
               
                   
                 V252I + E424Q 
               
               
                 M115 
                 G411D + S186G + Y384F + V164L + I389F + 
                 850.34% 
               
               
                   
                 V252I + L404Q + E171D 
               
               
                 M116 
                 G411D + S186G + Y384F + V164L + I389F + 
                 840.12% 
               
               
                   
                 V252I + E424Q + M423K 
               
               
                 M117 
                 G411D + S186G + Y384F + V164L + I389F + 
                 811.60% 
               
               
                   
                 V252I + L404Q + E171D + D391E 
               
               
                 M118 
                 G411D + S186G + Y384F + E424Q + K7N 
                 457.64% 
               
               
                 M119 
                 G411D + S186G + Y384F + E171D 
                 274.31% 
               
               
                 M120 
                 G411D + S186G + Y384F + D391E 
                 324.87% 
               
               
                 M121 
                 G411D + S186G + Y384F 
                 398.67% 
               
               
                 M122 
                 G411D + R442L + L178A + A187S 
                 357.69% 
               
               
                   
               
            
           
         
       
     
     The tolerance of the modified mutant protein in the solvents dimethyl sulfoxide and methyl tertiary ether is greatly improved, and the activity of the mutant at high pH is also verified. 
     High pH tolerance verification: the obtained mutants are verified for the activity in 40% of the dimethyl sulfoxide and 70% of the methyl tert-butyl ether under a condition of pH=10.0, after 16 hours the mutant substrate is basically completely conversation, and results are shown in Table 8. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 8 
               
             
            
               
                   
                   
               
               
                   
                   
                 pH = 10′ 
                 pH = 10, 
               
               
                   
                   
                 40% DMSO, 
                 70% MTBE, 
               
               
                   
                   
                 0.5 wt 
                 0.5 wt 
               
               
                   
                 Site 
                 enzyme 
                 enzyme 
               
            
           
           
               
               
               
               
               
               
            
               
                 Strain 
                 (amino acid difference compare to ArS-ωTA) 
                 5 h 
                 16 h 
                 5 h 
                 16 h 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 M76 
                 L3S + V5S + C60Y + F164V + A178L + S187A + I180V + L370A 
                 ND 
                 &lt;0.1% 
                 ND 
                 &lt;0.1% 
               
               
                 M110 
                 L3S + V5S + C60Y + F164L + A178L + S187A + I180V + L370A + G411D + 
                 45.50% 
                 90.55% 
                 75.10% 
                 91.78% 
               
               
                   
                 S186G + Y384F + I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164L + A178L + S187A + I180V +L370A + G411D + 
                 95.12% 
                 96.94% 
                 91.56% 
                 94.66% 
               
               
                   
                 S186G + Y384F + I389F + V252I + L404Q 
               
               
                 M114 
                 L3S + V5S + C60Y + F164L + I180V + L370A + G411D + A178L + S186G + 
                 76.61% 
                 93.41% 
                 91.86% 
                 95.06% 
               
               
                   
                 S187A + Y384F + V252I + I389F + E424Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164L + I180V + L370A + G411D + A178L + S186G + 
                 94.29% 
                 95.80% 
                 93.33% 
                 96.24% 
               
               
                   
                 S187A + Y384F + E171D + I389F + V252I + L404Q 
               
               
                 M116 
                 3S + V5S + C60Y + F164L + I180V + L370A + G411D + A178L + S186G + 
                 68.30% 
                 92.40% 
                 92.70% 
                 95.46% 
               
               
                   
                 S187A + Y384F + I389F + V252I + E424Q + M423K 
               
               
                 M117 
                 L3S + V5S + C60Y + F164L + A178L + S187A + I180V +L370A + G411D + 
                 73.04% 
                 95.15% 
                 84.75% 
                 95.13% 
               
               
                   
                 S186G + Y384F + I389F + V252I + L404Q + E171D + D391E 
               
               
                 M123 
                 C60Y + F164L + I180V + L370A + G411D + A178L + S186G + S187A + 
                 95.12% 
                 93.17% 
                 90.67% 
                 95.49% 
               
               
                   
                 Y384F + E171D + I389F + V252I + L404Q 
               
               
                 M124 
                 C60Y + F164V + I180V + L370A + G411D + A178L + S186G + S187A + 
                 89.64% 
                 94.87% 
                 89.17% 
                 95.46% 
               
               
                   
                 Y384F + E424Q 
               
               
                   
               
            
           
         
       
     
     The obtained mutants are broken by ultrasonication, supernatant enzyme solution and precipitated enzyme solution are obtained by centrifuging, and SDS-PAGE is performed to detect the expression condition of the protein. Generally, the soluble expressed protein exists in the supernatant in a dissolved state, and the abnormally folded protein exists in the form of an inclusion body, namely a precipitated protein. After SDS-PAGE detection, results are shown in  FIG. 1  (Note: Lane 1: Marker, Lane 2: ArS-Ωta soluble protein, Lane 3: ArS-Ωta protein inclusion body, Lane 4: Marker, Lane 5: mutant M115 soluble protein, and Lane 6: mutant M115 protein inclusion body), and the soluble expression of the optimal mutant protein is 4 times greater than that of the original bacteria. 
     According to a typical implementation mode of the disclosure, a transaminase mutant is provided. 
     An amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation occurred in an amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 1: MGLTVQKINW EQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLITPGGTRLLDFFNQLCCVNLGQKNQKVNAAI KEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLW TREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSCSSAVLMAPSSNTFQDSNGNYL KDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGVGSTMPPYEYVPQIRKMTKELGVLWISDEVL TGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVVSKEIAAFMDKHRWESVSTYAGHPV AMAAVCANLEVMMEENLVEQAKNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYV KLDRNFRHGMNPNQIPTQIIMEKALEKGVLIGGAMPNTMRIGASLNVSRGDIDKAMDALDYALDYL ESGEWQQS), and the mutation includes at least one of the following mutation sites: position 3, position 5, position 8, position 25, position 32, position 45, position 56, position 59, position 60, position 84, position 86, position 164, position 176, position 178, position 180, position 187, position 197, position 206, position 207, position 242, position 245, position 319, position 324, position 326, posit ion 328, position 370, position 397, position 414, position 416, position 424, position 436, position 437 and position 442. Preferably, the mutation includes at least one of the following mutation sites: L3S, VSS, I8A, I8S, F25L, F25T, Q32N, I45W, L59V, F56M, C60F, C60Y, F84V, W86H, W86L, W86P, W86N, F164M, F164V, F176Y, F176S, A178L, I180V, S187A, T197P, L206M, K207T, V242A, T245A, T245V, R319C, R324A, R324G, E326M, V328A, V328G, L370A, L370D, L370K, T397A, P414G, Q416A, E424D, E424T, A436S, A436G, A436P, A436N, A436Y, A436Q, A436E, M437S, M437A, R442T, R442S, R442Q and R442V; or an amino acid sequence of the transaminase mutant has the mutation sites in the mutated amino acid sequence, and has more than 80% identity with the mutated amino acid sequence. 
     The above transaminase mutant of the disclosure is based on the transaminase shown in SEQ ID NO: 1, and is mutated through a site-directed mutation method, thereby the amino acid sequence thereof is changed, so changes in protein structure and function are achieved, the transaminase mutant has high solubility expression characteristics and high activity characteristics, a reaction rate may be improved by the application of this mutant, the enzyme stability is improved, the amount of the enzyme is reduced, and the difficulty of post-treatment is reduced, so it may be suitable for industrial production. 
     A term “identity” used herein has the meaning generally known in the field, and those skilled in the art are also familiar with rules and standards for measuring the identity between different sequences. The sequences defined by the disclosure with different degrees of the identity must also have the improved tolerance of the transaminase to the organic solvent. In the above implementation mode, preferably the amino acid sequence of the transaminase mutant has the above identity and has or encodes the amino acid sequence with the improved tolerance to the organic solvent. Those skilled in the art may obtain such mutant sequences under the teaching of the disclosed content of the present disclosure. 
     Preferably, the mutation includes at least one of the following mutation site combinations: C60Y+F164V, L3S+V5S, L3S+V5S+F164V, L3S+V5S+C60Y, L3S+V5S+C60Y+F164V, I180V+L370A and L3S+V5S+L59V; more preferably, the mutation includes at least one of the following mutation site combinations: F164V+C60Y  E424D+A436G  C60Y+F164V+A436P  C60Y+F164V+A436N  W86P+F164V  F25L+L59V  F25T+F164V  C60Y+F164V+A436Y  C60Y+F164V+A436Q  C60Y+F164V+A436E  F164V+M437A  I8A+V328A  I8S+F164V  C60Y+F164V+L370A  C60Y+F164V+L370D  C60Y+F164V+L370K  I45W+F164V  C60Y+F164V+F176Y  C60Y+F176S+F164V  L3S+V5S+C60Y+F164V+L370A  C60Y+F164V+R442S  C60Y+F164V+R442Q  L3S+V5S+S187A+L370A+E424D  C60Y+F164V+R442T  L3S+V5S+E424D+L370A  L3S+V5S+F164V+C60Y+I180V+L370A  L3S+V5S+C60Y+F164V+A178L+L370A  L3S+V5S+F164V+T197P+L370A  L3S+V5S+V328A+E424D  L3S+V5S+L59V+L206M+L370A  L3S+V5S+L370A+E424D  L3S+V5S+F164V+K207T+L370A  L3S+V5S+S244A+L370A  L3S+V5S+F164V+T245A+L370A  L3S+V5S+F164V+T245V+V328A  L3S+V5S+F164V+L370A+T397A  L3S+V5S+L59V+F164V+R319C+L370A+T397A  L3S+V5S+L59V+F164V+L370A  L3S+V5S+L59V+L370A+A436G+Q416A  L3+V5S+L59V+L370A+A436G+R442Q  L3S+V5S+L59V+V328A+L370A+R442Q  L3S+V5S+L59V+L370A+R442L  L3S+V5S+L59V+C60Y+F164V+L370A+R442V  L3S+V5S+C60Y+F164V+A178L+S187A+I180V+L370A  L3S+V5S+C60Y+F164V+I180V+S187A+L370A+R442L. 
     According to a typical implementation mode of the disclosure, the mutation further includes at least one of the following mutation sites or mutation site combinations: position 7, position 32, position 96, position 164, position 171, position 186, position 252, position 384, position 389, position 391, position 394, position 404, position 411, position 420, position 423, position 424, position 442, position 452 and position 456; preferably, the mutation also includes 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 mutation is performed through the site-directed mutation method, thereby its amino acid sequence is changed, and changes in protein structure and function are achieved. Through the directed screening method, the transaminases with the above mutation sites are obtained. Therefore, these transaminase mutants have good organic solvent tolerance and high pH tolerance, and have high soluble expression characteristics and high activity characteristics, a reaction rate may be improved by use of these mutants, the enzyme stability is improved, the amount of the enzyme is reduced, and the difficulty of post-treatment is reduced, so it may be suitable for industrial production. 
     More preferably, the mutation includes at least one of the following mutation site combinations: 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 includes at least one of the following mutation site combinations: 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, and L3S+V5S+F164V+C60Y+L370A+G452S+L404Q. Through the directed screening method, the transaminases with the above mutation sites are obtained. Therefore, these transaminase mutants have good organic solvent tolerance and high pH tolerance, and have high soluble expression characteristics and high activity characteristics, a reaction rate may be improved by use of these mutants, the enzyme stability is improved, the amount of the enzyme is reduced, and the difficulty of post-treatment is reduced, so it may be suitable for industrial production. 
     According to a typical implementation mode of the disclosure, a DNA molecule is provided. The DNA molecule encodes the above transaminase mutant tolerant to an organic solvent. The above transaminase mutant encoded by the DNA molecule has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristics and high activity characteristics. 
     The above DNA molecule of the disclosure may also exist in the form of an “expression cassette”. The “expression cassette” refers to a linear or circular nucleic acid molecule, includes DNA and RNA sequences that may direct the expression of a specific nucleotide sequence in an appropriate host cell. Generally speaking, it includes a promoter operatively linked with a target nucleotide, and it is optionally operatively linked with a termination signal and/or other regulatory elements. The expression cassette may also include a sequence required for proper translation of the nucleotide sequence. 
     A coding region usually encodes the target protein, but also encodes a target functional RNA in sense or antisense direction, such as an antisense RNA or an untranslated RNA. The expression cassette containing the target polynucleotide sequence may be chimeric, it means that at least one of its components is heterologous to at least one of the other components thereof. The expression cassette may also be naturally existent, but obtained by efficient recombination for heterologous expression. 
     According to a typical implementation mode of the disclosure, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecule in the above recombinant plasmid is inserted in an appropriate position of the recombinant plasmid, so that the above DNA molecule may be replicated, transcribed or expressed correctly and smoothly. 
     Although a qualifier used in the disclosure to define the above DNA molecule is “containing”, it does not mean that other sequences that are not related to its function may be arbitrarily added to both ends of the DNA sequence. It is known by those skilled in the art that in order to meet requirements of a recombination operation, it is necessary to add appropriate digestion sites of a restriction endonuclease at both ends of the DNA sequence, or additionally add a start codon, a stop codon and the like. Therefore, if closed-type expression is used to limit, these situations may not be truly covered. 
     A term “plasmid” used in the disclosure includes any plasmids, cosmids, bacteriophages or agrobacterium binary nucleic acid molecules in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, or a prokaryotic expression plasmid or a eukaryotic expression plasmid, but preferably the prokaryotic expression plasmid. In some implementation schemes, the recombinant plasmid is selected from 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-23a(+), pET-23b(+), pET-24a(+), 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-43a(+), 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-1, pGEX-6p-2, 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 implementation mode of the disclosure, a host cell is provided, and the host cell contains any one of the above recombinant plasmids. The host cell suitable for the disclosure includes but is not limited to a prokaryotic cell, yeast or a eukaryotic cell. Preferably, the prokaryotic cell is eubacteria, such as gram-negative bacteria or gram-positive bacteria. More preferably, the prokaryotic cell is an  Escherichia coli  BL21 cell or an  Escherichia coli  DH5α competent cell. 
     According to a typical implementation mode of the disclosure, a method for producing a chiral amine is provided. The method includes a step of performing a catalytic transamination reaction on a ketone compound and an amino donor by transaminase, and the transaminase is any one of the above transaminase mutants tolerant to an organic solvent. Because the above transaminase mutant of the disclosure has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristics and high activity characteristics, the reaction rate may be improved by using the chiral amine prepared by the transaminase mutant of the disclosure, the enzyme stability is improved, the amount of the enzyme is reduced, and the difficulty of post-treatment is reduced. Further, the ketone compound is 
     
       
         
         
             
             
         
       
     
     herein R1 and R2 each independently represent an optionally substituted or unsubstituted alkyl, an optionally substituted or unsubstituted aralkyl, or an optionally substituted or unsubstituted aryl; R 1  and R 2  may be singly or combined with each other to form a substituted or unsubstituted ring;
 
preferably, R 1  and R 2  are optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted aralkyl, or optionally substituted or unsubstituted aryl having 1 to 20 carbon atoms, more preferably optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted aralkyl group, or optionally substituted or unsubstituted aryl having 1 to 10 carbon atoms;
 
preferably, the aryl includes phenyl, naphthyl, pyridyl, thienyl, oxadiazole group, imidazole group, thiazolyl, furanyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, thienyloxy, oxadiazoloxy, imidazoloxy, thiazolyloxy, furanyloxy and pyrrolyloxy;
 
preferably, the alkyl includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, t-butyl, methoxy, ethoxy, t-butoxy, methoxy carbonyl, ethoxy carbonyl, t-butoxy carbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
 
preferably, the aralkyl is benzyl; and
 
preferably, the substitution refers to substitution with halogen atom, nitrogen atom, sulfur atom, hydroxy, nitro, cyano, methoxy, ethoxy, carboxyl, carboxymethyl, carboxyethyl or methylenedioxy.
 
     Preferably, the ketone compound is 
     
       
         
         
             
             
         
       
     
     According to a typical implementation mode of the disclosure, the ketone compound is 
     
       
         
         
             
             
         
       
     
     a transamination reaction product is 
     
       
         
         
             
             
         
       
     
     and a reaction formula is 
     
       
         
         
             
             
         
       
     
     In a typical implementation mode of the disclosure, the amino donor is isopropylamine or alanine, preferably the isopropylamine. 
     In a reaction system the transaminase of the disclosure is applied to perform a catalytic transamination reaction on the ketone compound and the amino donor, a pH is 7-11, preferably 8-10, more preferably 9-10, in other words, the pH may be a value optionally selected from 7 to 11, such as 7, 7.5, 8, 8, 8.6, 9, 10, and 10.5. A temperature of the reaction system in which the transaminase is used to perform the catalytic transamination reaction on the ketone compound and the amino donor is 25-60° C., more preferably 30-55° C., further preferably 40-50° C., in other words, the temperature may be a value optionally selected from 25° C. to 60° C., such as 30, 31, 32, 35, 37, 38, 39, 40, 42, 45, 48, 50, 51, 52, and 55. The volume concentration of the dimethyl sulfoxide in the reaction system in which the transaminase is used to perform 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%, and 49%. The volume concentration of the methyl tert-butyl ether in the reaction system in which the transaminase is used to perform the catalytic transamination reaction on the ketone compound and the amino donor is 0% to 90%, for example, 10%, 16%, 18%, 20%, 30%, 35%, 38%, 40%, 42%, 48%, 49%, 55%, 60%, 70%, 80%, and 90%. 
     It is known by those skilled in the art that many modifications may be made to the disclosure without departing from spirit of the disclosure, and such modifications also fall within a scope of the disclosure. In addition, the following experimental methods are conventional methods unless otherwise specified, and experimental materials used may be easily obtained from commercial companies unless otherwise specified. 
     Embodiment 1 
     Catalytic activity of ArS-ωTA mutant and wild enzyme on substrate 1 in organic solvent-free system: 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg a raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, 250 μL crude enzyme solution of ArS-ωTA mutant or wild enzyme (0.05 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8.5) is added, and 0.41 mL 100 mM PB8.5 is added, so that a final volume of the system is 1 mL, it is stirred at 30° C. for 16h, the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. Mutant information and results are shown in Table 9. 
     
       
         
           
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                 Conversion 
               
               
                 Strain 
                 Sequence 
                 rate (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 ArS- 
                 ArS-ωTA 
                 &lt;0.1% 
               
               
                 ωTA 
               
               
                 M76 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 95.93% 
               
               
                   
                 I180V + L370A 
               
               
                 M110 
                 L3S + V5S + C60Y + F164L + A178L + S187A + 
                 98.01% 
               
               
                   
                 I180V + L370A + G411D + S186G + Y384F + 
               
               
                   
                 I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164L + A178L + S187A + 
                 98.05% 
               
               
                   
                 I180V + L370A + G411D + S186G + Y384F + 
               
               
                   
                 I389F + V252I + L404Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 98.61% 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 E171D + I389F + V252I + L404Q 
               
               
                   
               
            
           
         
       
     
     It may be seen from the results in Table 9 that the catalytic activity of the ArS-ωTA mutant on the substrate 1 is greatly improved compared with the wild bacteria. After the modification of the disclosure, the catalytic activity of the ArS-ωTA mutant is greatly improved, the catalytic activity on an original non-catalyzed substrate is obtained, and a substrate spectrum is enlarged. At the same time, it may be seen that the conversion is carried out in a very small reaction volume, a utilization rate of a reactor is improved. 
     Embodiment 2 
     Catalytic activity of ArS-ωTA wild enzyme and mutant on substrate 1 in organic solvent system (40%) DMSO: 
     In a 10 mL reaction flask, 100 mg raw material (same as Embodiment 1) is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, 250 μL crude enzyme solution of ArS-ωTA mutant or wild enzyme (0.05 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8.5) is added, 0.01 mL 100 mM PB8.5 is added, and 0.4 mL dimethyl sulfoxide is added, so that a final volume of the system is 1 mL, it is stirred at 30° C. for 16h, the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. Mutant information and results are shown in Table 10. 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Conversion 
               
               
                 Strain 
                 Sequence 
                 rate (%) 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                 ND 
               
               
                 ωTA 
               
               
                 M76 
                 L3S + V5S + C60Y + F164V + A178L + 
                 4.75% 
               
               
                   
                 S187A + I180V + L370A 
               
               
                 M110 
                 L3S + V5S + C60Y + F164L + A178L + 
                 80.51% 
               
               
                   
                 S187A + I180V + L370A + G411D + 
               
               
                   
                 S186G + Y384F + I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164L + A178L + 
                 97.46% 
               
               
                   
                 S187A + I180V + L370A + G411D + 
               
               
                   
                 S186G + Y384F + I389F + V252I + L404Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164L + I180V + 
                 98.20% 
               
               
                   
                 L370A + G411D + A178L + S186G + 
               
               
                   
                 S187A + Y384F + E171D + I389F + 
               
               
                   
                 V252I + L404Q 
               
               
                   
               
               
                 ND: No product generation detected 
               
            
           
         
       
     
     Embodiment 3 
     Chiral amine generated by catalyzing substrate through organic solvent-tolerant ArS-ωTA mutant and wild enzyme in 70% methyl tert-butyl ether solvent system: 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg raw material (same as Embodiment 1) is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, 250 μL crude enzyme solution of ArS-ωTA mutant or wild enzyme (0.05 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8.5) is added, 1.4 mL methyl tert-butyl ether is added, so that a final volume of the system is 2 mL, it is stirred at 35° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, the methyl tert-butyl ether reagent is blown dry with nitrogen. 100 μL sample is taken from the remaining, and 2 ml acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate, the conversion rate of the ArS-ωTA mutant is 95%, and the ArS-ωTA wild enzyme may not detect production of a product. Mutant information and results are shown in Table 11. 
     
       
         
           
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                   
                   
                 Conversion 
               
               
                 Strain 
                 Sequence 
                 rate (%) 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                    0% 
               
               
                 ωTA 
               
               
                 M76 
                 L3S + V5S + C60Y + F164V + A178L + 
                 12.01% 
               
               
                   
                 S187A + I180V + L370A 
               
               
                 M110 
                 L3S + V5S + C60Y + F164L + A178L + 
                 95.58% 
               
               
                   
                 S187A + I180V + L370A + G411D + 
               
               
                   
                 S186G + Y384F + I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164L + A178L + 
                 97.61% 
               
               
                   
                 S187A + I180V + L370A + G411D + 
               
               
                   
                 S186G + Y384F + I389F + V252I + L404Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164L + I180V + 
                 97.57% 
               
               
                   
                 L370A + G411D + A178L + S186G + 
               
               
                   
                 S187A + Y384F + E171D + I389F + 
               
               
                   
                 V252I + L404Q 
               
               
                   
               
            
           
         
       
     
     Although the mutant M76 obtains the catalytic activity to the substrate 1 (Embodiment 3), in 40% of dimethyl sulfoxide, due to the low tolerance thereof to the organic solvent, most of the proteins are denatured to lose the activity, and the catalytic activity is greatly reduced. However, the mutants M113 and M115 still maintain the higher catalytic activity, it is indicated that the evolved mutant has greatly improved tolerance to the organic solvent dimethyl sulfoxide on the basis of acquiring the higher catalytic activity. 
     Embodiment 4 
     Catalytic activity verification of ArS-ωTA mutant and wild enzyme under different temperature and pH conditions 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, and 500 μL crude enzyme solution of ArS-ωTA mutant M52 or M115 (0.1 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=11), or wild enzyme (1 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=11) is added, 100 mM phosphate buffer (pH=11) is added, so that a final volume of the system is 5.5 mL, it is stirred at 35° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Results show that even though the catalytic system of ArS-ωTA uses a lot of the enzyme (1 g wet cells) under an extreme pH condition, no product is detected after catalysis, and no product is detected in the catalytic system of mutant M52, while the catalytic system of mutant M115 uses the less enzyme (0.1 g), &gt;80% of the product is detected, it is indicated that the mutant obtains the excellent high pH tolerance after modification, so that a catalytic space of the enzyme may be improved, and the double excellent characteristics of high activity and high pH tolerance make it suitable for the needs of the industrial production. 
     In a10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5′-phosphate is added, 2 mM isopropylamine hydrochloride is added, and 500 μL crude enzyme solution of ArS-ωTA mutant M52 or M115 (0.1 g of mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8), or wild enzyme (1 g mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8) is added, 100 mM phosphate buffer (pH=8) is added, so that a final volume of the system is 5.5 mL, it is stirred at 60° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Results show that even though catalytic system of ArS-ωTA uses a lot of the enzyme (1 g wet cells) under an extreme pH condition, no product is detected after catalysis, and no product is detected in the catalytic system of mutant M52, while the catalytic system of mutant M115 uses the less enzyme (0.1 g), &gt;80% of the product is detected, it is indicated that the mutant obtains the excellent high temperature tolerance after modification, so that a catalytic space of the enzyme may be improved, and the double excellent characteristics of high activity and high temperature tolerance make it suitable for the needs of the industrial production. 
     Embodiment 5 
     Chiral amine generated by catalyzing substrate through ArS-ωTA mutant and wild enzyme: 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg raw material is weighed, 1 mg pyridoxal 5′-phosphate is added, 2 mM isopropylamine hydrochloride is added, and 5 mL-250 μL crude enzyme solution of ArS-ωTA mutant (1 g-0.05 g-mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=7.0 and pH=10.5), or wild enzyme (1 g-ArS-ωTA female parent wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=7.0 and pH=10.5) is added, 0.41 mL 100 mM phosphate buffer (pH=7.0) or phosphate buffer (pH=10.5) is added, so that a final volume of the system is 3 mL, it is stirred at 30° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 NL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Mutant information and results are shown in Table 12. The results show that the ArS-ωTA wild enzyme does not produce a product in the two pH systems of 7.0 and 10.5, while the product is detected in multiple mutants in the two pH systems, and some mutants show good activity, for example, the catalytic system of mutant M115 uses a very small amount of the enzyme (0.05 g, under a condition of pH=10.5), the conversion rate is &gt;95%, and a chiral purity of the product is extremely high (&gt;99%). The mutant obtained after modification of ArS-ωTA obtains the good catalytic activity, and at the same time, it may catalyze the production of the chiral amine at high pH, and the catalytic effect is good, it is indicated that the mutant achieves the qualitative breakthrough on catalytic activity and tolerance. 
     
       
         
           
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Number 
                 Amino acid difference (compare to ArS-ωTA) 
                 Activity 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                 ND 
               
               
                 ωTA 
               
               
                 M29 
                 C60Y + F164V 
                 + 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 + 
               
               
                 M26 
                 C60Y 
                 ND 
               
               
                 M14 
                 F164V 
                 − 
               
               
                 M52 
                 L3S + V5S + C60Y + F164V + L370A 
                 +++ 
               
               
                 M56 
                 C60Y + F164V + R442T 
                 ++ 
               
               
                 M6-2 
                 L3S + V5S + C60Y + F164V + L370A + Y384F + 
                 +++ 
               
               
                   
                 G452S 
               
               
                 M104 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + M423K 
               
               
                 M108 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + L404Q 
               
               
                 M119 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 E171D 
               
               
                 M120 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 D391E 
               
               
                 M101 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L 
               
               
                 M78 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S187A 
               
               
                 M99 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A 
               
               
                 M121 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F 
               
               
                 M106 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L 
               
               
                 M109 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + V252I 
               
               
                 M112 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F 
               
               
                 M111 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + E424Q 
               
               
                 M110 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q 
               
               
                 M114 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I++E424Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q + E171D 
               
               
                 M116 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I++E424Q + M423K 
               
               
                 M117 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q + E171D + 
               
               
                   
                 D391E 
               
               
                   
               
               
                 “ND” means that no product is detected by using 1 g wet cells for catalysis, 
               
               
                 “−” means that &lt;5% of the product is detected under a condition of pH = 7, 
               
               
                 “+” means that 5%-20% of the product is detected under the condition of pH = 7, 
               
               
                 “++” means that 20%-50% of the product is detected under the condition of pH = 7, 
               
               
                 “+++” means that 50%-80% of the product is detected under the condition of pH = 7, 
               
               
                 “++++” means that 80%-90% of the product is detected under the condition of pH = 7, 
               
               
                 “+++++” means that 80%-95% of the product is detected under the condition of pH = 7, and at the same time, 90-100% of the product is detected by using 0.05 g wet cells for catalysis under a condition of pH = 10.5. 
               
            
           
         
       
     
     Embodiment 6 
     Chiral amine generated by catalyzing substrate through ArS-ωTA mutant and wild enzyme: 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, and 100 μL crude enzyme solution of ArS-ωTA mutant (0.02 g mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8.5), or 1000 μL crude enzyme solution of wild enzyme (0.2 g ArS-ωTA wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8.5) is added, 0.41 mL 100 mM PB8.5 is added, so that a final volume of the system is 3 mL, it is stirred at 30° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Mutant information and results are shown in Table 13. The results show that the catalytic system of ArS-ωTA wild enzyme uses 10 times of the amount of the enzyme of the mutant, but only a small amount of the product is produced (&lt;20%). The catalytic system of mutants, such as M118 and M115, use very small amount of enzyme to obtain the conversion rates which are all &gt;90%, and the chiral purity of the product is extremely high (&gt;99%). At the same time, the activity of multiple mutants is greatly improved compared with that of the ArS-ωTA female parent, so the excellent catalytic effect is obtained. 
     
       
         
           
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 Number 
                 Amino acid difference (compare to ArS-ωTA) 
                 Activity 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                 − 
               
               
                 ωTA 
               
               
                 M26 
                 C60Y 
                 − 
               
               
                 M14 
                 F164V 
                 − 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 + 
               
               
                 M45 
                 C60Y + F164V + L370K 
                 + 
               
               
                 M48 
                 C60Y + F164V + F176S 
                 + 
               
               
                 M52 
                 L3S + V5S + C60Y + F164V + L370A 
                 +++ 
               
               
                 M54 
                 C60Y + F164V + R442Q 
                 ++ 
               
               
                 M36 
                 C60Y + F164V + A436Y 
                 ++ 
               
               
                 M3-2 
                 L3S + V5S + C60Y + F164V + L370A + Y384F 
                 +++ 
               
               
                 M3-6 
                 L3S + V5S + C60Y + F164V + L370A + L404Q 
                 +++ 
               
               
                 M6-2 
                 L3S + V5S + C60Y + F164V + L370A + Y384F + 
                 ++ 
               
               
                   
                 G452S 
               
               
                 M3-1 
                 L3S + V5S + C60Y + F164V + L370A + G411D 
                 +++ 
               
               
                 M89 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 +++ 
               
               
                   
                 I180V + L370A + I389F + L404Q 
               
               
                 M94 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 +++ 
               
               
                   
                 I180V + L370A + V252I 
               
               
                 M88 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 +++ 
               
               
                   
                 I180V + L370A + I389F + L404Q 
               
               
                 M4-3 
                 L3S + V5S + C60Y + F164V + L370A + S186G + 
                 +++ 
               
               
                   
                 Q420R 
               
               
                 M104 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + M423K 
               
               
                 M105 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 +++ 
               
               
                   
                 I180V + L370A + G411D + S186G + I389F + 
               
               
                   
                 L404Q 
               
               
                 M82 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 ++++ 
               
               
                   
                 I180V + L370A + Q420K + E42R 
               
               
                 M92 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 ++++ 
               
               
                   
                 I180V + L370A + Q32L + R442H 
               
               
                 M101 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L 
               
               
                 M122 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + R442L 
               
               
                 M78 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S187A 
               
               
                 M109 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V252I 
               
               
                 M112 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F 
               
               
                 M111 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 E424Q 
               
               
                 M110 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I 
               
               
                 M108 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + L404Q 
               
               
                 M118 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 K7N + E424Q 
               
               
                 M119 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 E171D 
               
               
                 M120 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 D391E 
               
               
                 M113 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I + L404Q 
               
               
                 M114 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I ++ E424Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I + L404Q + E171D 
               
               
                 M116 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I ++ E424Q + M423K 
               
               
                 M117 
                 L3S + V5S + C60Y + F164L + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + V252I + L404Q + E171D + D391E 
               
               
                   
               
               
                 “−” means that the conversion rate obtained by the original strain using 10 times of the amount of the enzyme (0.2 g) of the mutant is less than 20%, or the conversion rate obtained by the mutant using the same amount of the enzyme (0.2 g) as the original strain is reduced or not increased, 
               
               
                 “+” means that the mutant uses a very small amount of the enzyme (0.02 g) to increase the conversion rate by 0.2-1 time, 
               
               
                 “++” means that the mutant uses a very small amount of the enzyme (0.02 g) to increase the conversion rate by 1-2 times, 
               
               
                 “+++” means that the mutant uses a very small amount of the enzyme (0.02 g) to increase the conversion rate by 2-4 times, and 
               
               
                 “++++” means that the mutant uses a very small amount of the enzyme (0.02 g) to increase the conversion rate by more than 4 times. 
               
            
           
         
       
     
     Embodiment 7 
     Chiral amine generated by catalyzing substrate through ArS-ωTA mutant and wild enzyme: 
     
       
         
         
             
             
         
       
     
     In a 10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, 5 mL ArS-ωTA female parent and mutant crude enzyme solution (1 g mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8), 100 mM phosphate buffer (pH=8) is added, so that a final volume of the system is 5.5 mL, it is stirred at 35° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Mutant information and results are shown in Table 14. It may be seen that the activity of the transformed mutant is significantly improved compared with the original strain ArS-ωTA, and the chiral purity of the product is extremely high (&gt;99%). 
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 Number 
                 Amino acid difference (compare to ArS-ωTA) 
                 Activity 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                 + 
               
               
                 ωTA 
               
               
                 M99 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A 
               
               
                 M6-2 
                 L3S + V5S + C60Y + F164V + L370A + Y384F + 
                 +++ 
               
               
                   
                 G452S 
               
               
                 M56 
                 C60Y + F164V + R442T 
                 +++ 
               
               
                 M54 
                 C60Y + F164V + R442Q 
                 +++ 
               
               
                 M45 
                 C60Y + F164V + L370K 
                 ++++ 
               
               
                 M4-3 
                 L3S + V5S + C60Y + F164V + L370A ++ 
                 +++ 
               
               
                   
                 S186G + Q420R 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 ++++ 
               
               
                 M36 
                 C60Y + F164V + A436Y 
                 +++ 
               
               
                 M3-1 
                 L3S + V5S + C60Y + F164V + L370A + G411D 
                 +++ 
               
               
                 M2-1 
                 L3S + V5S + C60Y + F164V + L370A + I180V + 
                 +++ 
               
               
                   
                 G411D 
               
               
                 M178 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S187A 
               
               
                 M122 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + R442L 
               
               
                 M121 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F 
               
               
                 M113 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q 
               
               
                 M109 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + V252I 
               
               
                 M106 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L 
               
               
                 M101 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L 
               
               
                   
               
               
                 “+” means that the conversion rate is less than 20%, 
               
               
                 “++” means that the conversion rate is 20%-30%, 
               
               
                 “+++” means that the conversion rate is 30%-40%, and 
               
               
                 “+++” means that the conversion rate is 40%-50% 
               
            
           
         
       
     
     Embodiment 8 
     Chiral amine generated by catalyzing substrate through ArS-ωTA mutant and wild enzyme: 
     
       
         
         
             
             
         
       
     
     In a10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM isopropylamine hydrochloride is added, 5 mL or 500 μL of crude enzyme solution (g ArS-Ωta or mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8), 100 mM phosphate buffer (pH=8) is added, so that a final volume of the system is 5.5 mL, it is stirred at 35° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, a pH is adjusted to 10 with NaOH, and 2 mL MTBE is added to extract. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect a product conversion rate. 
     Mutant information and results are shown in Table 15. It may be seen that the activity of the transformed mutant is significantly improved compared with the original strain ArS-ωTA. Multiple mutants may convert all the substrates into the product within 16 h, and the chiral purity of the product is extremely high (&gt;99%). 
     
       
         
           
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Number 
                 Amino acid difference (compare to ArS-ωTA) 
                 Activity 
               
               
                   
               
             
            
               
                 ArS- 
                 ArS-ωTA 
                 ++ 
               
               
                 ωTA 
               
               
                 M29 
                 C60Y + F164V 
                 +++ 
               
               
                 M36 
                 C60Y + F164V + A436Y 
                 ++++ 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 +++ 
               
               
                 M48 
                 C60Y + F164V + F176S 
                 ++++ 
               
               
                 M52 
                 L3S + V5S + C60Y + F164V + L370A 
                 +++ 
               
               
                 M54 
                 C60Y + F164V + R442Q 
                 ++++ 
               
               
                 M99 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A 
               
               
                 M112 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F 
               
               
                 M113 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q 
               
               
                 M114 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + E424Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q + E171D 
               
               
                 M119 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 E171D 
               
               
                 M78 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S187A 
               
               
                   
               
               
                 “++” means that the conversion rate obtained by using 1 g wet cells containing the target transaminase is less than 50%, 
               
               
                 “+++” means that the conversion rate obtained by using 1 g wet cells containing the target transaminase is 70%-90%, and 
               
               
                 “+++” means that the conversion rate obtained by using 0.1 g of wet cells containing the target transaminase is 90%-100%. 
               
            
           
         
       
     
     Embodiment 9 
     Chiral amine generated by catalyzing substrate through ArS-ωTA mutants (M52, M118 and M111) and wild enzyme: 
     
       
         
         
             
             
         
       
     
     In a10 mL reaction flask, 100 mg raw material is added, 1 mg pyridoxal 5-phosphate is added, 2 mM of isopropylamine hydrochloride is added, 5 mL crude enzyme solution (1 g ArS-Ωta or mutant wet cells are ultrasonicated to prepare 20% of the crude enzyme solution, pH=8), 100 mM phosphate buffer (pH=8) is added, so that a final volume of the system is 5.5 mL, it is stirred at 35° C. for 16h. After the system is centrifuged at 12000 rpm for 5 min, 200 μL sample is taken, and 2 mL acetonitrile is added to dissolve. After being centrifuged at 12000 rpm for 5 min, the sample is sent for HPLC to detect product. After being detected, it is found that the product may not be detected in the catalytic system of the starting bacteria ArS-Ωta, and the product is detected in all of the catalytic systems of M52, M118 and M111 mutants. It may be seen that the mutant obtains the catalytic activity to the substrate after the modification. 
     Embodiment 10 
     A single microbial colony of  Escherichia coli  containing each plasmid encoding the target transaminase is inoculated into 50 μg/Ml of an ampicillin-containing 50 mL Luria Bertani medium. Cells are cultured and grown overnight (about 16 h) in 37° C. shaker at 200 rpm. 5 mL culture is inoculated into a 2 L flask 50 mL Luria Bertani medium with 50 μg/Ml ampicillin, and cultured in 37° C. of the constant temperature shaker at 200 rpm until OD is 0.6 to 0.8, through adding isopropyl s D-sulfur Galactoside (IPTG) to a final concentration of 0.06 mM, the expression of a transaminase gene is induced, and then culture solution is continuously cultured in 25° C. of a constant temperature shaker at 200 rpm for about 16 h. The cells are collected by centrifugation (6000 rpm, 15 min, and 4° C.), and supernatant is discarded. The cells are resuspended in 100 mM phosphate buffer with a pH 7.0, the cells are lysed by ultrasonication to obtain a crude enzyme, and the crude enzyme is centrifuged (12000 rpm, 3 me, and 4° C.) to separate the supernatant and precipitate (including an inclusion body protein). The obtained precipitate is resuspended in an equal volume of 100 mM phosphate buffer with pH 7.0. An SOS-PAGE mode is used to detect expression conditions of soluble protein and inclusion body protein in the supernatant and precipitate. 
     Expression results of ArS-Ωta and each mutant are shown in Table 16, it is indicated that the introduction of mutation sites gradually makes the soluble expression condition of the mutant proteins better and better. The original bacteria ArS-ωTA only has a small amount of the protein expressed in the supernatant, and a large amount of the protein is expressed in the precipitate. The mutant M52 doubles the expression of the supernatant protein, but the final mutant M115 and the like make almost all the proteins expressed in the supernatant, and a very little protein is expressed in the precipitate. The expression condition of the mutant is greatly improved. 
     
       
         
           
               
               
             
               
                 TABLE 16 
               
               
                   
               
               
                 Number 
                 Amino acid difference (compare to ArS-ωTA) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 ArS- 
                 ArS-ωTA 
                 − 
               
               
                 ωTA 
               
               
                 M26 
                 C60Y 
                 − 
               
               
                 M14 
                 F164V 
                 − 
               
               
                 M29 
                 C60Y + F164V 
                 − 
               
               
                 M43 
                 C60Y + F164V + L370A 
                 − 
               
               
                 M52 
                 L3S + V5S + C60Y + F164V + L370A 
                 + 
               
               
                 M76 
                 L3S + V5S + C60Y + F164V + A178L + S187A + 
                 + 
               
               
                   
                 I180V + L370A 
               
               
                 M121 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 + 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F 
               
               
                 M108 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 I389F + L404Q 
               
               
                 M118 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 K7N + E424Q 
               
               
                 M109 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + V252I 
               
               
                 M112 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F 
               
               
                 M110 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I 
               
               
                 M113 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q 
               
               
                 M114 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + E424Q 
               
               
                 M115 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 ++++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q + E171D 
               
               
                 M116 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + E424Q + M423K 
               
               
                 M117 
                 L3S + V5S + C60Y + F164V + I180V + L370A + 
                 +++ 
               
               
                   
                 G411D + A178L + S186G + S187A + Y384F + 
               
               
                   
                 V164L + I389F + V252I + L404Q + E171D + D391E 
               
               
                   
               
               
                 “−” means a soluble expression level of the female parent ArS-ωTA: only a small amount is expressed in the supernatant, and the most is expressed in the precipitate; 
               
               
                 “+” means that the soluble expression is increased by 1 time; 
               
               
                 “++” means that the soluble expression is increased by 2 times; 
               
               
                 “+++” means that the soluble expression is increased by more than 3 times; and 
               
               
                 “++++” means that the soluble expression is increased by more than 4 times. 
               
            
           
         
       
     
     It may be seen from the above descriptions that the above embodiments of the disclosure achieve the following technical effects: the above transaminase mutant of the disclosure has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristics and high activity characteristics. 
     The above are only preferred embodiments of the disclosure, and are not used to limit the disclosure. Various modifications and changes may be made to the disclosure by those skilled in the art. Any modifications, equivalent replacements, improvements and the like made within the spirit and principle of the disclosure shall be included in the scope of protection of the disclosure.