Patent Description:
Enzymes, as biocatalysts, may give full play to high efficiency and high specificity characters thereof in vivo. However, in industrial applications, there are generally problems, such as inability to adapt to industrial production conditions and low catalytic ability to non-natural substrates. Site-directed mutagenesis and saturation mutation technologies are effective means to modify enzyme molecules.

The site-directed mutagenesis (or site-specific mutagenesis) refers to a method of introducing a specific base pair at a designated site of a DNA fragment of interest. Changing an encoded amino acid sequence by changing a nucleotide sequence of a specific site of a gene is often used to study the effect of a certain amino acid residue(s) on the structure and function of a protein. In a rational design of the enzymes, a site-directed mutagenesis method may be used for screening mutant enzymes with improved catalytic activity, substrate specificity, and/or stability.

Saturation mutation is a method to obtain a mutant in which an amino acid in a target side is replaced by <NUM> other amino acids, respectively, by modifying the coding gene of the protein of interest in a short time. This method is not only a powerful tool for protein orientation modification, but also an important means for studying the structure-function relationship of proteins. The saturation mutation may often acquire a more ideal evolution than single-point mutations. These problems which the site-directed mutagenesis method may not solve are exactly the uniqueness of the saturation mutation method.

An w-transaminase (ω-TA) belongs to the class of transferase, and, like other transaminases, catalyzes a process of exchange between an amino group and a keto group. The w-transaminase uses a ketone compound as a raw material, and through stereoselective transamination, may efficiently produce chiral amines, and has attracted the attention of many researchers. Single active-site mutants are sufficient to enhance Chromobacterium violaceum transaminase activity (<NPL>).

<NUM>-aminopyrrolidine derivatives and optical isomers thereof are a chiral amine compound, and is a key intermediate for synthesizing a large number of chiral drugs or agricultural chemicals. (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine is an important <NUM>-aminopyrrolidine derivative with optical activity. It is reported by <NPL> that (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine is prepared using (R) -<NUM>-benzyl-<NUM>-pyrrolidinol as a starting raw material in a five-step reaction, an ee value thereof is <NUM>%, and the synthetic route is as follows:
<CHM>.

The starting raw material of this synthetic route is high in price, and a harmful reagent sodium azide is used in the synthetic process, requirements to operation equipment, personnel safety, three-waste treatment and the like are higher, and pollution to the environment is greater. However, there are few reports on asymmetric synthesis of chiral (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine using bioenzyme biocatalysis at present.

Although it has also been reported that the transaminase may be used as a biocatalyst to reduce a ketone substrate in one-step, to prepare the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine with high optical purity. Compared with a traditional chemical method, a biological transformation method is mild in reaction condition, and avoids using strong oxidants, strong reducing agents and dangerous reagents. The conditions are mild, and the pollution to the environment is low.

However, in applications of the biological transformation method in industrial production, there are still some problems which need to be further solved. In this method, the enzyme catalytic activity is not efficient enough, a total volume of a reaction system thereof is <NUM>-<NUM>/g substrate, and the reaction system has a large volume, which leads to an increase in production batches and production cost, and a large amount of organic solvents in a post-treatment process, the difficulty of reaction post-treatment may be increased, and a larger burden may be brought to the environment. In addition, in the prior art, D-alanine or L-alanine or a salt thereof is mostly used as an amino donor, and coupling coenzyme systems, such as glucose and GDH, ammonium formate and FDH, also need to be added to the reaction system.

Therefore, the biological transformation methods in the prior art still needs to be improved, as to improve the catalytic characteristics of the transaminase, reduce the total volume of the reaction system, reduce the production cost, and reduce the environment pollution.

The invention aims to provide a transaminase mutant and an application thereof, as to improve catalytic activity thereof.

In order to achieve the above objective, according to one aspect of the present application, a transaminase mutant is provided, the transaminase mutant having the sequence of SEQ ID NO: <NUM> with a mutation of one or more amino acids, the mutation comprises any one or more of the group consisting of:, V379L/M/T, V379L/M/T+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S, V379L/M/T+A417S, V379L/M/T+C418A/Q/S, V379L/M/T+ P243E , V379L/M/T+ K193E, V379L/M/T+ V234I, V379L/M/T+I262V, V379L/M/T+Q280K, V379L/M/T+R416A/C/H/Q/T/S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+A417S, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S, V379L/M/T+R416A/C/H/Q/T/S+K193E, V379L/M/T+R416A/C/H/Q/T/S+V234I , V379L/M/T+C418A/Q/S+F89Y/W, V379L/M/T+C418A/Q/S+P243E, V379L/M/T+C418A/Q/S+I262V, V379L/M/T+A417S+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+I262V, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+A417S and V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+I262V+ V234I∘.

In order to achieve the above objective, according to a second aspect of the present application, a DNA molecule is provided, the DNA molecule encodes any one of the transaminase mutants.

According to a third aspect of the present application, a recombinant vector is provided, the DNA molecule is effectively linked with the recombinant vector.

Further, the recombinant vector is selected from a group consisting of pET-21b (+), 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-<NUM>, pGEX-6p-<NUM>, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-<NUM>, pUC-<NUM> or pUC-<NUM>.

According to a fourth aspect of the present application, a host cell is provided. The host cell contains any one of the above recombinant vectors.

Further, the host cell is a prokaryotic cell or a eukaryocyte cell; preferably, the prokaryotic cell is a yeast cell; preferably, the host cell is a competent cell, further preferably, the competent cell is an E. coli BL21 cell or an E. coli W3110.

According to a fourth aspect of the invention, a method for synthesizing a chiral amine is provided. The method includes a step of performing a catalytic transamination reaction on a ketone compound and an amino donor with a transaminase, herein the transaminase is any one of the above transaminase mutants.

Further, the ketone compound is
<CHM>
, herein, each of R1 and R2 is independently a C1-C8 alkyl, a C5-C10 cycloalkyl, a C5-C10 aryl or a C6-C10 heteroaryl, or the R1 and the R2 jointly form a C5-C10 heterocyclyl, a C5-C10 carbocyclyl or the C5-C10 heteroaryl with carbon in a carbonyl, one or more of heteroatoms in the C5-C10 heterocyclyl and the C5-C10 heteroaryl are independently selected from at least one of nitrogen, oxygen and sulfur, and each of an aryl in the C6-C10 aryl, a heteroaryl in the C5-C10 heteroaryl, a carbocyclyl in the C5-C10 carbocyclyl or a heterocyclyl in the C5-C10 heterocyclyl is independently unsubstituted or substituted by at least one group of halogen, an alkoxy or an alkyl, preferably, the ketone compound is
<CHM>
a product of the transaminase reaction is
<CHM>
preferably, the amino donor is isopropylamine or an isopropyl amine salt.

By applying the technical scheme of the invention, the w-transaminase is improved using the site-directed mutagenesis and/or saturation mutation method, and the w-transaminase mutants with high catalytic efficiency and/or high stability is obtained. Based on partial ω-transaminase mutants obtained in the present application, in synthesis of a (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminoheterocycle compound (especially (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine and (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine), the amount e of the enzyme is reduced to <NUM>-<NUM> wt, the reaction volume is reduced to <NUM>-<NUM> V, a utilization ratio of the enzyme and a utilization ratio of a reaction kettle are greatly improved, the production batches of the enzyme solution and a usage amount of a material are reduced, and a usage amount of the organic solvent in the post-treatment is effectively reduced, so the difficulty of the post-treatment and a discharge amount of three wastes are reduced, and the labor cost is reduced. In addition, through the obtained (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine and (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine with high optical purity, the industrial production cost of the compound is greatly reduced, and the enzyme has a better application value in industrial production.

It is to be noted that embodiments in the present application and features in the embodiments may be mutually combined in the case without conflicting. The invention is described in detail below in combination with the embodiments.

Catalytic activity: refers to an amount of a raw material reactant transformed per unit volume (or mass) of a catalyst in a unit time. In the invention, a level of the catalytic activity of a transaminase is positively related to a transformation rate of the reaction raw material in the invention.

Evolution: means of mutation or recombination and the like are used to create molecular diversity, and then screen the diversity, as to obtain a gene or DNA with a new function. In the invention, a wild-type transaminase is modified by means of mutation or recombination and the like, as to obtain the transaminase with improved performance.

Wild-type: refers to those obtained from nature, without artificial mutagenesis or modified. In the invention, the wild-type ω-transaminase refers to a natural transaminase encoded by a gene sequence which is not artificially modified and obtained by screening from Genebank.

Immobilized enzyme: refers to an enzyme whose catalytic function can be used repeatedly and continuously in a certain space range. Generally, an enzyme-catalyzed reaction is performed in an aqueous solution, and the immobilized enzyme is a water-soluble enzyme which is physically or chemically treated to make it insoluble in water, but still has enzyme activity. After the enzyme is immobilized, the stability is increased generally, it is easy to separate from a reaction system and easy to control, it may be used for multiple times, it is convenient for transporting and storing, and beneficial to automatic production, but the activity is reduced, and the usage range is reduced.

Immobilized cell: is a method used to obtain enzymes and metabolites of a cell, and developed on the basis of the immobilized enzyme. The immobilized cell refers to a cell which is immobilized on an insoluble carrier and performs life activity in a certain space range. Because the immobilized cell may perform normal growth, reproduction and metabolism, the immobilized cell is also called an immobilized viable cell or an immobilized proliferative cell.

In the invention, the related <NUM> wt refers to <NUM> of transaminase mutant recombinant wet cells required to transform <NUM> of a main raw material.

In the invention, the related <NUM> V is equal to a volume of a reaction system/a mass of a substrate.

The site-directed mutagenesis and/or saturation mutation method is used for transforming the transaminase, as to improve the catalytic activity thereof, substrate specificity and/or stability, it is helpful to solve problems existing in an prior art, such as a dosage of enzyme solution is larger, a reaction system is large, and production cost is high. A main objective of the invention is to improve an w-transaminase by using an enzyme molecule transformation method to obtain an ω-transaminase mutant with high catalytic efficiency and/or high stability, as to solve the deficiency in the prior art, and improve an application value of industrial production thereof.

In the invention, a wild-type ω-transaminase gene derived from Chromobacterium violaceum is used as a starting gene, because the transaminase (having the amino acid sequence as shown in SEQ ID NO: <NUM>) encoded by the wild-type gene has relatively higher activity itself, <NUM> wt of wild-type transaminase bacterial sludge is used, and a reaction may be basically transformed and completed. Based on the wild-type transaminase with relatively higher catalytic activity, it is difficult to obtain a transaminase with further improved catalytic activity through evolution transformation of an enzyme molecule. Therefore, the invention performs the site-directed mutagenesis in <NUM> sites, and <NUM> mutant strains are screened by the saturation mutation, so that the following w-transaminase mutants with improved catalytic activity and/or stability are obtained.

An amino acid site of a mutation capable of improving the catalytic activity and/or the stability of the w-transaminase mutant is selected from one or more of F89, K193, P243, V234, I262, Q280, V379, R416, A417 and C418. Herein, a site capable of improving the catalytic activity is selected from: F89, K193, P243, V234, I262, Q280, V379, R416, A417 and C418, these sites are positioned near an enzyme catalytic center, and may be related to substrate entry or binding.

The above transaminase is obtained, through selecting SEQ ID NO: <NUM> as a basic sequence, and genetically engineered to obtain a mutation containing one or more amino acid residues changes, and the catalytic activity and/or stability thereof is remarkably improved.

On the basis of mutations at the above sites, the inventor found that when these sites are mutated into different amino acids and tested for changes in the transaminase activity thereof, and found that when these amino acid sites are mutated into arbitrary one or a combination of the following, the activity and/or the stability of the transaminase is further improved The mutation includes arbitrary one or more of the followings: P243E, F89Y/W, K193E, V234I, I262V, Q280K, V379L/M/T, R416A/C/H/Q/T/S, A417S and C418A/Q/S, herein, "/" means "or".

The inventor performed a multi-point combination mutation on the above sites which have a positive effect to the catalytic activity and/or the stability, and obtained an w-transaminase mutant with further improved catalytic properties through a directed screening method, compared with the wild-type w-transaminase, the catalytic activity and/or the stability of the mutant is remarkably improved.

In a more preferable embodiment, the above mutation includes arbitrary one of the following combinations: V379L/M/T+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S, V379L/M/T+A417S, V379L/M/T+C418A/Q/S, V379L/M/T+ P243E , V379L/M/T+ K193E, V379L/M/T+ V234I, V379L/M/T+I262V, V379L/M/T+Q280K, R416A/C/H/Q/T/S+A417S, R416A/C/H/Q/T/S+C418A/Q/S, R416A/C/H/Q/T/S+ F89Y/W, R416A/C/H/Q/T/S+A417S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+A417S, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S, V379L/M/T+R416A/C/H/Q/T/S+ K193E, V379L/M/T+R416A/C/H/Q/T/S+ V234I , V379L/M/T+C418A/Q/S+ F89Y/W, V379L/M/T+C418A/Q/S+ P243E, V379L/M/T+C418A/Q/S+ I262V, V379L/M/T+A417S+ Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+ F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+ Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+ Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+ Q280K+I262V, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+ Q280K+ A417S and V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+ Q280K+I262V+ V234I, but not limited to this.

A specific screening process of the above mutant and the combinations thereof is as follows:
<NUM> pairs of site-directed mutagenesis primers are used for performing the site-directed mutagenesis on <NUM> sites (F22V, F22A, F22L, L59V, L59A, W60F, C61S, C61A, F88V, F89W, F89Y, Y153F, Y153M, Y153V, A231G, R416K, R416C, R416A, A417H, C418Q, F320V, P94E, S101K, P243E, Q280K, Q346S, P354A, F397A, W60L, T87A, V234M, V234I, I262V, T321A, V379L, V379M). <NUM> saturation mutation primers are used for performing the saturation mutation on <NUM> sites (W60, T321, V379, F89, Y153, A231, Y322, R416, A417 and C418), herein the site-directed mutagenesis primers uses primer sequences obtained by a Quick Change Primer Design website design for this experiment, sequences of the saturation mutation primers are as shown in Table <NUM> below. A complete linear fragment is obtained by a full vector PCR. The PCR product is digested with Dpn I so as to remove a female parent template of a starting gene, and then transformed into escherichia coli BL21 (DE3), spread in an LB culture dish containing 50µg/ml of ampicillin, and cultured overnight in <NUM> DEG C. The site-directed mutagenesis uses gene sequencing to determine the mutation sites, and the saturation mutation is screened by high throughput screening, and then determines the mutation sites by the gene sequencing.

According to the system as shown in the above Table <NUM>, other components except the enzyme solution are uniformly mixed in the <NUM>-shallow-well plate, background detection is performed in <NUM>, and 96µL of the prepared mutant enzyme solution is respectively added to each well, and the mixed system is immediately placed in <NUM> DEG C, a shaker reaction is performed in <NUM> rpm, after <NUM>-<NUM>, an absorbance change of OD<NUM> is detected with the microplate reader.

Through comparing with the enzyme activity of the wild-type transaminase, a mutant strain with higher activity is screened, and rescreening and gene sequencing are performed.

The above preliminary-screened mutant of which the enzyme activity is higher than that of the wild-type transaminase is inoculated in <NUM> of the LB liquid culture medium containing 100µg/ml of the ampicillin, and shake-cultured at <NUM> DEG C until OD<NUM>=<NUM>, the IPTG is added until the final concentration is <NUM>, the induced expression is performed at <NUM> DEG C. After induction is performed for <NUM>, cells are collected by centrifuging at <NUM> for <NUM>. The cells are disrupted with an ultrasonic disrupter (JY92-2D, Ningbo Xinzhi Biotechnology Co. ), and supernatant is obtained by centrifuging in <NUM> DEG C and <NUM> for <NUM>, and used for activity detection.

Partial detection results are as shown in Table <NUM>.

It may be observed from Table <NUM> that a reaction speed of the transaminase is rapider, the transformation effects in <NUM> and overnight <NUM> are basically the same, and a difference between the transformation rates does not exceed <NUM>%, however, in order to facilitate sampling operation, an overnight reaction is used in the subsequent experiments. According to some ω-transaminase mutants in the invention, in synthesis of a (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminoheterocycle compound (especially (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine), the amount of enzyme is further reduced to <NUM>-<NUM> wt, the reaction volume is <NUM> V, and ee value of the obtained (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine is greater than <NUM>%, so an utilization rate of the enzyme is greatly improved, and industrial production cost of the compound is greatly reduced.

In addition, reaction conditions used for synthesizing (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine and partial reaction results are shown in Table <NUM>.

It may be observed from Table <NUM> that according to some ω-transaminase mutants in the invention, in synthesis of the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine, the amount of the enzyme is reduced to <NUM> wt, the reaction volume is reduced to <NUM> V, the transformation rate is improved by <NUM>%-<NUM>%, the ee value is greater than <NUM>%. A utilization ratio of the enzyme and a reaction kettle is greatly improved, production batches of enzyme solution and a usage amount of a material are effectively reduced, and the production cost is reduced.

(<NUM>) The N-Cbz-<NUM>-pyrrolidone is used as a substrate for detecting tolerance of the transaminase mutants. The following system is used:
<NUM> of the N-Cbz-<NUM>-pyrrolidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> of <NUM> of isopropylamine hydrochloride, <NUM> of <NUM>/ml of PLP, and <NUM>-<NUM> wt of the recombinant crude enzyme which is treated by <NUM>% of DMSO at <NUM> DEG C and pH <NUM> for <NUM> are added, the reaction system is supplemented to <NUM>-<NUM> V with <NUM> of NaHCOs, the pH is adjusted to <NUM>, reaction is performed at <NUM> rpm in a constant temperature shaker at <NUM> DEG C. 200µL of the system is taken at <NUM>, 400µL of the methanol is added and mixed uniformly, the centrifugation is performed at <NUM> rpm for <NUM> minutes, 200µL of the supernatant is taken and 800µL of the methanol is added and mixed uniformly, it is sent to the HPLC for detecting the transformation rate. The mutant with the improved stability is determined. The partial results are as shown in Table <NUM>.

The enzyme may lose the activity under extreme conditions, such as a high temperature, a strong acid, a strong alkali and an organic solvent, for a long time. Residual enzyme activity is total activity of the enzyme which still maintains the activity under the environments of the high temperature, the strong acid, the strong alkali or the organic solvent and the like. Relative residual activity refers to a percentage of the measured enzyme activity of the enzyme solution treated under the extreme conditions of the high temperature, the alkalinity, the organic solvent and the like, and the enzyme activity of the enzyme solution in the optimal conditions without being treated under the extreme environments. In a same treatment condition, the relative residual activity is high, it is indicated that the enzyme is more stable in the condition.

It may be observed from Table <NUM> that in the same extreme conditions, the relative residual activity of the mutant is two to three times higher than that of the wild-type transaminase. Therefore, the stability of the w-transaminase mutant obtained in the invention is greatly improved, which creates better preconditions for subsequent immobilization and continuous flow reaction.

In conclusion, the catalytic activity and/or the stability of the w-transaminase mutant obtained by the invention through the directed screening method is greatly improved, so the enzyme amount thereof in the transamination reaction is reduced, and the reaction system is reduced, especially the cheap isopropylamine is used as the amino donor for catalyzing a reaction of synthesizing the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine and the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine.

In a second typical implementation mode of the invention, a DNA molecule is provided, the DNA molecule encodes any one of the above w-transaminase mutants. The coded transaminase mutant has an advantage of remarkably improved catalytic activity and/or stability.

In a third typical implementation mode of the invention, a recombinant vector is provided, the recombinant vector is linked with the above DNA molecule.

In the above recombinant vector, any recombinant vector capable of expressing a DNA molecule of the transaminase mutant is suitable for use in the present invention. In the preferable embodiments of the invention, the recombinant vector is selected from the followings: pET-21b(+), 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-<NUM>, pGEX-6p-<NUM>, pGEX-6p-<NUM>, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-<NUM>, pUC-<NUM> or pUC-<NUM>.

In a fourth typical implementation mode of the invention, a host cell is provided. The host cell contains any one of the above recombinant vectors. The specific host cell is a prokaryotic cell or a eukaryocyte cell; preferably, preferably the prokaryotic cell is a yeast cell. More preferably, the above host cell is a competent cell, further preferably, the competent cell is an escherichia coli BL21 cell or an escherichia coli W3110.

In a fifth typical implementation mode of the invention, a method for producing a chiral amine is provided. The method includes a step of performing a catalytic transamination reaction on a ketones compound and an amino donor with a transaminase, herein the transaminase is any one of the above transaminase mutants.

The ketones compound is
<CHM>
, herein, each of R<NUM> and R<NUM> is independently a C1-C8 alkyl, a C5-C10 cycloalkyl, a C6-C10 aryl or a C5-C10 heteroaryl, or the R<NUM> and the R<NUM> jointly form a C5-C10 heterocyclyl, a C5-C10 carbocyclyl or the C5-C10 heteroaryl with carbon in a carbonyl, one or more of heteroatoms in the C5-C10 heterocyclyl and the C5-C10 heteroaryl are independently selected from at least one of nitrogen, oxygen and sulfur, and an aryl in the C6-C10 aryl, a heteroaryl in the C5-C10 heteroaryl, a carbocyclyl in the C5-C10 carbocyclyl or a heterocyclyl in the C5-C10 heterocyclyl is independently unsubstituted or substituted by at least one group of halogen, an alkoxy or an alkyl, preferably, the ketonecompound is
<CHM>
and a product of the transamination reaction is
<CHM>
preferably, the amino donor is isopropylamine or an isopropyl amine salt.

These transaminase mutants with remarkably improved catalytic activity and/or stability may be used for greatly reducing the amount and the reaction volume of the enzyme in the synthesis of the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminoheterocycle compound, and greatly reducing the production batches and the production cost, so the enzyme has the better application value in the industrial production. The invention uses the cheap amino donor (such as the isopropylamine and the hydrochloride thereof) to react, and a coupling coenzyme system may not be required in the reaction. There are few types of reaction materials, and the operation is simpler.

In a process of preparing the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminoheterocycle compound, such as the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopyrrolidine or the (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine, with the above transaminase mutant of the invention as a catalyst, because the catalytic activity and/or the stability of the enzyme is remarkably improved, the amount of the enzyme is apparently reduced, the reaction volume is reduced to <NUM>-<NUM>/g the substrate, and it is apparently smaller than a reaction volume in an prior art. On this basis, the above reaction temperature, time and pH value may be rationally adjusted and optimized on the basis of the present reaction conditions. When the preferable reaction conditions of the present application are used, the reaction efficiency is higher.

Unless otherwise specified, the following experimental methods are conventional methods, and experimental materials used may be easily obtained from commercial companies unless otherwise specified.

<NUM> pairs of site-directed mutagenesis primers designed with a Quick Change Primer Design website and <NUM> saturation mutation primers as shown in Table <NUM> are used, a complete linear fragment is obtained by a full vector PCR, after a product of the above PCR is digested with Dpn I so as to remove a female parent template of a starting gene, it is transformed into escherichia coli BL21 (DE3), spread in an LB culture dish containing 50µg/ml of ampicillin, and cultured overnight in <NUM> DEG C. Saturation mutation is screened by high throughput screening. Specifically, the above mutants are screened with the following method to perform the high throughput screening:.

The above mutant of which enzyme activity is higher than that of a wild-type transaminase is inoculated in <NUM> of the LB liquid culture medium containing 100µg/ml of ampicillin, and shake-cultured at <NUM> DEG C until OD<NUM>=<NUM>, IPTG is added until final concentration of <NUM>, induced expression is performed at <NUM> DEG C. After induction is performed for <NUM>, cells are collected by centrifuging at <NUM> for <NUM>. The cells are disrupted with an ultrasonic disrupter (JY92-2D, Ningbo Xinzhi Biotechnology Co. ), and supernatant is obtained by centrifuging in <NUM> DEG C and <NUM> for <NUM>, and used for activity detection.

<NUM> of a N-Cbz-<NUM>-pyrrolidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> of <NUM> of an isopropylamine hydrochloride, <NUM> of <NUM>/ml of a PLP and <NUM>-<NUM> wt of a recombinant crude enzyme are added, a reaction system is supplemented to <NUM>-<NUM> V with <NUM> of phosphate buffer solution with pH <NUM>, the pH is adjusted to <NUM>, and reaction is performed overnight at <NUM> rpm in a constant temperature shaker at <NUM> DEG C. 200µL of the system is taken at <NUM>, 400µL of methanol is added for uniformly mixing, centrifugation is performed at <NUM> rpm for <NUM> minutes, supernatant is sent to a HPLC for detecting a transformation rate. The mutant with improved catalytic activity is determined.

Results of the mutant with improved catalytic activity are as shown in Table <NUM>.

"<NUM>" means <NUM> wt of a ketoreductase crude enzyme, while a reaction system is <NUM> V, a transformation rate in <NUM> is greater than <NUM>%; "+" means <NUM> wt of the ketoreductase crude enzyme, while the reaction system is <NUM> V, the transformation rate in <NUM> is greater than <NUM>%; "++" means <NUM> wt of the ketoreductase crude enzyme, while the reaction system is <NUM> V, the transformation rate in <NUM> is greater than <NUM>%; "+++" means <NUM> wt of the ketoreductase crude enzyme, while the reaction system is <NUM> V, the transformation rate in <NUM> is greater than <NUM>%; and "++++" means <NUM>-<NUM> wt of the ketoreductase crude enzyme, while the reaction system is <NUM> V, the transformation rate in <NUM> is <NUM>%-<NUM>%.

<NUM> induction-expressed transaminases mutants numbered <NUM> to <NUM>, <NUM> to <NUM> and <NUM> are used for screening synthesis reactions of (S)-<NUM>-benzyloxycarbonyl-<NUM>-aminopiperidine, and the following reaction system is used: <NUM> of a N-BOC-<NUM>-piperidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> ul of <NUM> of isopropylamine hydrochloride, <NUM> of PLP, and <NUM>-<NUM> wt of a recombinant crude enzyme are added, a reaction system is supplemented to <NUM>-<NUM> V with <NUM> of phosphate buffer solution in pH <NUM>, thepH is adjusted to <NUM>, and reaction is performed overnight at <NUM> rpm, <NUM> DEG C. Through reaction screening, reaction results of strains with relatively better activity are as shown in Table <NUM>. Catalytic activity of <NUM> residual transaminase mutants except those shown in Table <NUM> is not increased compared to the activity of a wild-type transaminase.

<NUM> wt of a transaminase crude enzyme, after treated by <NUM>% of DMSO in <NUM> DEG C and pH <NUM> for <NUM>, is used for the following reaction: <NUM> of the N-Cbz-<NUM>-piperidone substrate is dissolved in <NUM> of the DMSO and mixed uniformly, <NUM> ul of <NUM> of isopropylamine hydrochloride, and <NUM> of <NUM>/ml of PLP are added, a reaction system is supplemented to <NUM> V with <NUM> of NaHCOs, the pH is adjusted to <NUM>, reaction is performed overnight at <NUM> rpm on a constant temperature shaker at <NUM> DEG C. 200µL of the system is taken at <NUM>, 400µL of the methanol is added and mixed uniformly, centrifugation is performed in <NUM> rpm for <NUM> minutes, supernatant is sent to a HPLC for detecting a transformation rate. Specific results are as shown in Table <NUM>.

It may be observed from Table <NUM> that in a same treatment condition, stability of some transaminase mutants is higher compared to the wild-type transaminase.

<NUM> transaminase crude enzymes in <NUM> wt, after treated by <NUM>%-<NUM>% of DMSO in <NUM> DEG C and pH <NUM> for <NUM>, are used for the following reaction: <NUM> of a N-Cbz-<NUM>-piperidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> ul of <NUM> of isopropylamine hydrochloride, and <NUM> of <NUM>/ml of PLP are added, a reaction system is supplemented to <NUM> with <NUM> of NaHCOs, the pH is adjusted to <NUM>, and reaction is performed overnight at <NUM> rpm on a constant temperature shaker at <NUM> DEG C. 200µL of the system is taken at <NUM>, 400µL of the methanol is added and mixed uniformly, centrifugation is performed in <NUM> rpm for <NUM> minutes, supernatant is sent to a HPLC for detecting a transformation rate. Results are as shown in Table <NUM>.

A reaction equation thereof is as follows:
<CHM>.

A reaction system is as follows: <NUM> of a N-Cbz-<NUM>-pyrrolidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> of <NUM> of isopropylamine hydrochloride, <NUM> of <NUM>/ml of PLP (pyridoxal phosphate), and <NUM>-<NUM> wt of a V379T+R416A+C418A+F89Y recombinant crude enzyme are added, the reaction system is supplemented to <NUM> V with <NUM> of phosphate buffer solution in pH <NUM>, the pH is adjusted to <NUM>, and reaction is performed overnight at <NUM> rpm on a constant temperature shaker at <NUM> DEG C. Detection is performed by HPLC, a transformation rate in <NUM> is <NUM>%, after the reaction is finished, the system is adjusted to be alkalinity, methyl tert-ether is added for extracting for <NUM> times, after extracted organic phases are merged, a magnesium sulfate is added for drying, rotary evaporation is performed until it is dried, a yield is <NUM>-<NUM>%, and a ee value is greater than <NUM>%.

A reaction system is as follows: <NUM> of a N-BOC-<NUM>-piperidone substrate is dissolved in <NUM> of DMSO and mixed uniformly, <NUM> of <NUM> of isopropylamine hydrochloride, <NUM> of PLP (pyridoxal phosphate), and <NUM> wt of a V379T+R416A+C418S+F89Y recombinant crude enzyme are added, the reaction system is supplemented to <NUM> V with <NUM> of phosphate buffer solution in pH <NUM>, the pH is adjusted to <NUM>, and reaction is performed overnight at <NUM> rpm on constant temperature shaker at <NUM> DEG C. Detection is performed by HPLC, a transformation rate in <NUM> is <NUM>%, after the reaction is finished, the system is adjusted to be alkalinity, methyl tert-ether is added for extracting for <NUM> times, after extracted organic phases are merged, a magnesium sulfate is added for drying, rotary evaporation is performed until it is dried, a yield is <NUM>%, and a ee value is greater than <NUM>%.

Claim 1:
A transaminase mutant, wherein the transaminase mutant has the sequence of SEQ ID NO: <NUM> with a mutation of one or more amino acids, wherein the mutation comprises any one or more of the group consisting of: V379L/M/T, V379L/M/T+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S, V379L/M/T+A417S, V379L/M/T+C418A/Q/S, V379L/M/T+ P243E , V379L/M/T+ K193E, V379L/M/T+ V234I, V379L/M/T+I262V, V379L/M/T+Q280K, V379L/M/T+R416A/C/H/Q/T/S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+A417S, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S, V379L/M/T+R416A/C/H/Q/T/S+K193E, V379L/M/T+R416A/C/H/Q/T/S+V234I, V379L/M/T+C418A/Q/S+F89Y/W, V379L/M/T+C418A/Q/S+P243E, V379L/M/T+C418A/Q/S+I262V, V379L/M/T+A417S+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+I262V, V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+A417S and V379L/M/T+R416A/C/H/Q/T/S+C418A/Q/S+F89Y/W+Q280K+I262V+V234I.