CN108048419A - Transaminase mutant and its application - Google Patents

Transaminase mutant and its application Download PDF

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CN108048419A
CN108048419A CN201711131103.1A CN201711131103A CN108048419A CN 108048419 A CN108048419 A CN 108048419A CN 201711131103 A CN201711131103 A CN 201711131103A CN 108048419 A CN108048419 A CN 108048419A
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transaminase
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amino
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CN108048419B (en
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洪浩
詹姆斯·盖吉
卢江平
徐幸福
于文燕
黄鑫
马玉磊
程逸冰
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Asymchem Life Science Tianjin Co Ltd
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Abstract

The invention discloses a kind of transaminase mutant and its applications.The amino acid sequence of the transaminase mutant is SEQ ID NO：The amino acid sequence that amino acid sequence shown in 1 is undergone mutation, the amino acid sites of mutation are selected from one or more of F89, K193, P243, V234, I262, Q280, V379, R416, A417 and C418.Transaminase mutant with above-mentioned at least one mutational site, enzymatic activity and/or stability greatly improve.

Description

Transaminase mutant and its application
Technical field
The present invention relates to enzyme engineering field, in particular to a kind of transaminase mutant and its application.
Background technology
Enzyme can give full play to it efficiently and the characteristics of high specific as biocatalyst in vivo.But in work In industry application, but generally existing can not adapt to industrial process conditions and to the problems such as the catalytic capability of non-natural substrates is low.It is fixed Point mutation and saturation mutation technology are the effective means that enzyme molecule is transformed.
Rite-directed mutagenesis (site-directed mutagenesis or site-specific mutagenesis) refers to The method that specific base-pair is introduced on the specified site of target DNA fragment.By changing gene specific site nucleotide sequence Change encoded amino acid sequence, be usually used in studying shadow of some (a little) amino acid residue to protein structure and function It rings.In the design and rational of enzyme, it can screen to obtain catalytic activity, substrate specificity and/or stability using directed mutagenesis method The mutant enzyme of raising.
Saturation mutation is transformed by the encoding gene to destination protein, and target site Amino acid score is obtained in the short time Not by a kind of method of the mutant of other 19 kinds of amino acid replacements.The method is not only the strong work of protein directional transformation Tool, and be the important means of protein structure-functional relationship research.Saturation mutation tends to obtain than simple point mutation more Preferable evolution body.And for these indeterminable problems of directed mutagenesis method, exactly saturation mutation method is good at Unique distinction.
ω-transaminase (ω-TA) belongs to transferase, the same with others transaminase class, is catalyzed an amino and ketone The process that base exchanges.ω-transaminase, can be efficient by Stereoselective transamination using ketone compounds as raw material Chiral Amine is produced, numerous researchers is subject to pay close attention to and pay attention to.
3- amino-pyrrolidine derivatives and its optical isomer are a kind of chiral amine compounds, are a large amount of chiral drugs of synthesis Or the key intermediate of agricultural chemicals.(S) -1- benzyloxycarbonyl groups -3- amino-pyrrolidines are important 3- with optical activation Amino-pyrrolidine derivatives.J.M.C1992,35,1764 report using (R) -1- benzyl -3- pyrrolidinols as starting material, use (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines are made in the reaction of five steps, and ee values are 96%, and synthetic route is as follows：
The starting material higher price of the synthetic route, and harmful reagent sodium azide has been used in synthesis technology, to behaviour Make that equipment and personnel safety, three-protection design etc. are more demanding, it is larger to the pollution of environment.But that reports at present is given birth to using biological enzyme The method of object catalysis asymmetric syntheses chirality (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines is few.
Although also having been reported that at present, transaminase can be used as biocatalyst, and a step reductone substrate prepares high optical voidness (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines of degree.Compared with traditional chemical routes, the reaction condition of biotransformation method is mild, Avoid the use of strong oxidizer, strong reductant and hazardous agents, mild condition, environmental pollution is small.
But the biotransformation method, in industrial application, still remaining some problems needs further to solve, should Enzymatic activity is not efficient enough in method, and reaction system total volume is 40-60ml/g substrates, and reaction system volume is big, causes The increase of production batch and production cost, and cause the dosage of organic solvent in last handling process big, after reaction being increased The difficulty of processing brings larger burden to environment.In addition it is most with D-alanine or l-Alanine or its salt in the prior art As amino group donor, also need to add in glucose and GDH, the coupled cofactors system such as ammonium formate and FDH in the reaction system.
Therefore, it is still necessary to existing biotransformation method is improved, to improve the catalysis characteristics of transaminase, reduce reaction System total volume reduces production cost, reduces environmental pollution.
The content of the invention
The present invention is intended to provide a kind of transaminase mutant and its application, to improve its catalytic activity.
To achieve these goals, according to the one side of the application, a kind of transaminase mutant is provided, transaminase is dashed forward The amino acid sequence of variant is SEQ ID NO：The amino acid sequence that sequence shown in 1 is undergone mutation, the amino acid sites choosing of mutation From one or more of F89, K193, P243, V234, I262, Q280, V379, R416, A417 and C418.
Further, the mutation that the amino acid sites of mutation occur include it is following any one or more：P243E、F89Y/ W, K193E, V234I, I262V, Q280K, V379L/M/T, R416A/C/H/Q/T/S, A417S and C418A/Q/S, wherein, "/" Represent "or".
Further, mutation, which includes any one following combination, includes：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。
To achieve these goals, according to the second aspect of the application, a kind of DNA molecular is provided, which compiles Any of the above-described kind of transaminase mutant of code.
According to the 3rd of the application the aspect, a kind of recombinant plasmid is provided, which is connected with above-mentioned DNA points Son.
Further, recombinant plasmid is 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-1、pGEX-6p-1、pBV220、pBV221、pBV222、pTrc99A、 PTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.
According to the 4th of the application the aspect, a kind of host cell is provided, which contains any of the above-described kind of weight Group plasmid.
Further, host cell is prokaryotic cell or eukaryocyte；Preferably, eukaryocyte is yeast cells；It is preferred that Ground, host cell are competent cell, and further preferred competent cell is e. coli bl21 cell or Escherichia coli W3110。
According to the 4th of the application the aspect, a kind of method for producing Chiral Amine is provided, this method, which includes using, turns ammonia The step of enzyme carries out catalysis transamination to ketone compounds and amino group donor, transaminase are mutated for any of the above-described kind of transaminase Body.
Further, ketone compounds areWherein, R1 and R2 be each independently for C1~C8 alkyl, C5~ It is miscellaneous that with the carbon on carbonyl C5~C10 is collectively formed in C10 cycloalkyl, C6~C10 aryl or C5~C10 heteroaryls or R1 and R2 Ring group, C5~C10 carbocylic radicals or C5~C10 heteroaryls, the hetero atom in C5~C10 heterocycles and C5~C10 heteroaryls is each The heteroaryl in aryl, C5~C10 heteroaryls at least one of nitrogen, oxygen and sulphur, C6~C10 aryl, The heterocycle in carbocylic radical or C5~C10 heterocycles in C5~C10 carbocylic radicals is unsubstituted independently or by halogen, alkane At least one group in oxygroup or alkyl is substituted, it is preferable that ketone compounds are It is anti-to turn amino The product is answered to bePreferably, amino group donor is isopropylamine or isopropyl amine salt.
Apply the technical scheme of the present invention, by using the method for rite-directed mutagenesis and/or saturation mutation to ω-transaminase into Row improves, and obtains high catalytic efficiency and/or ω-transaminase mutant of high stability.The part ω that the application is obtained-turn Ammonia enzyme mutant, in (S) -1- benzyloxycarbonyl group -3- amino-heterocyclic compounds (particularly (S) -1- benzyloxycarbonyl group -3- amino-pyrroles Alkane and (S) -1- benzyloxycarbonyl group -3- aminos piperidines) in synthesis, the dosage of enzyme is reduced to 0.3wt~0.5wt, reaction volume 10V-20V is reduced to, substantially increases the utilization rate of enzyme and the utilization rate of reaction ax, reduces the use of enzyme solution production batch and material Amount effectively reduces the usage amount of the organic solvent in post processing so that the difficulty of post processing and the discharge capacity of the three wastes reduce, contracting Subtract cost of labor.Moreover, (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines of the high-optical-purity obtained and (S) -1- benzyloxy carbonyls Base -3- amino piperidines are greatly lowered the industrial production cost of the compound so that the enzyme has in the industrial production Better application value.
Specific embodiment
It should be noted that in the case where there is no conflict, the feature in embodiment and embodiment in the application can phase Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
Name Resolution：
Catalytic activity：Refer to that per unit volume (or quality) catalyst converts the number of raw material reactant within the unit interval Amount.In the present invention, the catalytic activity height of transaminase and the conversion ratio of heretofore described reaction raw materials are proportionate.
It evolves：The diversity of establishment molecule is gone with the means such as mutation or restructuring, then this diversity is screened, is obtained Gene or DNA with new function.In the present invention, wild type transaminase is transformed by the means such as being mutated or recombinating, with Obtain the transaminase that performance improves.
Wild type：Refer to what is obtained from the Nature, do not transformed without induced mutations or.In the present invention, wild type ω-transaminase, it is natural to refer to screen obtain a kind of from Genebank, turns without what artificial reconstructed gene order encoded Ammonia enzyme.
Immobilised enzymes：Refer to its catalytic action in certain spatial dimension, and can repeatedly with continuous use enzyme.Usually Enzymic catalytic reaction all carries out in aqueous solution, and immobilised enzymes is to make water-soluble enzyme with either physically or chemically handling Become it is not soluble in water, but still with enzymatic activity state.After enzyme immobilization, general stability increases, easily from reactant It is separated in system, it is easily controllable, it can be used for multiple times, be readily transported and store, be conducive to automated production, but activity reduces, and uses Scope reduces.
Immobilized cell：It is for obtaining a kind of method of the enzyme of cell and metabolite, is on the basis of immobilised enzymes On grow up.Immobilized cell, which refers to, to be fixed on insoluble carrier, and the thin of vital movement is carried out in certain spatial dimension Born of the same parents.Since immobilized cell can normally be grown, be bred and metabolism, so also known as immobilized growing cell or immobilization Proliferative cell.
In the present invention, involved 1wt, which refers both to conversion 1g main materials, needs 1g transaminases mutant to recombinate wet cell.
In the present invention, involved 1V is equal to the quality of volume/substrate of reaction system.
Transaminase is transformed using the method for rite-directed mutagenesis and/or saturation mutation, improves its catalytic activity, substrate specificity And/or stability, help to solve in the prior art such as the problems such as enzyme solution dosage is larger, reaction system is big, production cost is high.This The main purpose of invention is to be improved ω-transaminase using enzyme molecule remodeling method, obtains high catalytic efficiency and/or height The ω of stability-transaminase mutant to solve deficiency of the prior art, improves its industrial application value.
The present invention is to derive from wild type ω-transaminase base of chromabacterium biolaceum (Chromobacterium violaceum) Because setting out gene, since the transaminase of wild type gene coding (has SEQ ID NO:Amino acid sequence shown in 1) this Body activity is relatively high, and using 3wt wild type transaminase bacterium muds, reaction can substantially convert completion.And with the catalytic activity phase Based on the transaminase of higher wild type, enzyme molecule evolution transformation is carried out, to obtain turn that catalytic activity further improves Ammonia enzyme is relatively difficult.Therefore the present invention has done the rite-directed mutagenesis in 36 sites, and saturation mutation has screened 1500 mutant bacterias Strain, just obtains following catalytic activity and/or stability-enhanced ω-transaminase mutant.
Make ω-transaminase mutant catalytic activity and/or the amino acid sites of stability-enhanced mutation be selected from F89, One or more of K193, P243, V234, I262, Q280, V379, R416, A417 and C418.Wherein, put forward catalytic activity High site is selected from：F89, K193, P243, V234, I262, Q280, V379, R416, A417 and C418, these points are urged positioned at enzyme Change immediate vicinity, may enter or combine related to substrate.
Above-mentioned transaminase, by selecting with SEQ ID NO:1 is basic sequence, and passes through genetic engineering means and transform to obtain Comprising the mutant that single or multiple amino acid residues change, catalytic activity and/or stability significantly improve.
On the basis of undergoing mutation in above-mentioned site, inventor by by these site mutations into different amino acid and inspection Survey the variation of its transaminase activity, find when these amino acid sites sport it is following in any one or the combination of several of them it Afterwards, the activity of transaminase and/or stability are further enhanced.Mutation include it is following any one or it is several：P243E、 F89Y/W, K193E, V234I, I262V, Q280K, V379L/M/T, R416A/C/H/Q/T/S, A417S and C418A/Q/S, In, "/" represents "or".
Above-mentioned these have catalytic activity and/or stability the site progress multi-point combination of positive acting to dash forward by the present inventor Become, ω-transaminase mutant that catalysis characteristics further improves obtained by the method for directed screening, the mutant with it is wild Type ω-transaminase is significantly improved compared to catalytic activity and/or stability.
In a kind of preferred embodiment, above-mentioned mutation includes any one following combination：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+V 234I, but not limited to this.
The specific screening process of above-mentioned mutant and combinations thereof is as follows：
36 sites are carried out using 36 pairs of rite-directed mutagenesis primers rite-directed mutagenesises (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).And carry out saturation mutation using 9 saturation mutation primer pairs, 9 sites (W60, T321, V379, F89, Y153, A231, Y322, R416, A417, C418), wherein rite-directed mutagenesis primer are utilized The primer sequence that QuikChange Primer Design webpage designs obtain is used for this experiment, and saturation mutation primer sequence is seen below Table 1 obtains complete linear fragment by full plasmid PCR, above-mentioned PCR product is digested to the female parent for removing the gene that sets out through Dpn I It after masterplate, is transformed into e. coli bl21 (DE3), is coated in the LB culture dishes containing 50 μ g/ml ampicillins, 37 DEG C Overnight incubation.Rite-directed mutagenesis determines mutational site using gene sequencing, and saturation mutation passes through gene sequencing again after high flux screening Determine mutational site.
Table 1：Saturation mutation primer：
(1) high flux screening is carried out with the following method
1st, 96 orifice plate induced expression：Picking monoclonal is inoculated in the LB fluid nutrient mediums containing 100 μ g/ml ampicillins In, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG to final concentration of 0.2mM is added in, induced expression mistake is carried out at 25 DEG C Night.
2nd, enzyme solution preparation method：Supernatant culture medium is removed in the centrifugation of 96 orifice plates, and 200 μ l enzymolysis solution (lysozymes are added in per hole 2mg/mL, polymyxin 1mg/mL, pH=7.0), the broken 2h of 37 DEG C of heat preservations.Clasmatosis liquid 4000rpm centrifugations after enzymolysis 10min takes supernatant to obtain crude enzyme liquid.
3rd, according to table 2 shown in system, pass through microplate reader and carry out active primary dcreening operation.
Table 2：
System Addition
N-Cbz- pyrrolidones (9.26mg/ml DMSO) 48μl
P-nitrophenyl ethamine (8.11mg/ml) 36μl
PLP(0.2mg/ml) 12μl
Phosphate buffer pH7.0 48μl
Enzyme solution 96μl
As shown in upper table 2 system by other components in addition to enzyme solution the mixing in 96 shallow bore hole plates, in 430nm carry out this Bottom is detected, and then adds in the 96 μ L of mutant enzyme solution prepared into each hole respectively, and mixed system is placed in 40 immediately DEG C, the reaction of 200rpm shaking tables, detect OD with microplate reader after 30~40min430Light absorption value variation.
Enzyme activity calculation formula：Enzyme activity (u/mL)=(Δ A × 60 × V1)/(6.22×t×V2)
ΔA：Absorption photometric value variable quantity in reaction process；
V1：The total volume of reaction system；
6.22：Extinction coefficient；
t：The detection time of Δ A；
V2：The enzyme solution volume of addition.
By being compared with the enzyme activity of wild type transaminase, the higher mutant strain of activity is filtered out, secondary screening is carried out and gene is surveyed Sequence.
(2) secondary screening of transaminase mutant
Above-mentioned primary dcreening operation enzyme activity is inoculated in 500ml containing 100 μ g/ml ampicillins higher than the mutant of wild type transaminase LB fluid nutrient mediums in, 37 DEG C of shaken cultivations to OD600When=0.6, add in IPTG to final concentration of 0.2mM, at 25 DEG C into Row induced expression.After inducing 16h, 6000g centrifugations 10min collects thalline.Thalline is with Ultrasonic Cell Disruptor (JY92-2D, the new sesame in Ningbo Biotech inc) smudge cells, 4 DEG C, 10000g centrifugations 20min obtains supernatant, for Activity determination.
(1) reactivity of transaminase mutant is examined using N-Cbz-3- pyrrolidones as substrate.Using following system：
0.2g N-Cbz-3- pyrrolidones substrates are dissolved in mixing in 0.5ml DMSO, add the isopropylamine hydrochloride of 4.3M The thick enzyme of restructuring of the PLP 0.2ml, 0.3~3wt of 1.25ml, 0.01g/ml, are supplied instead with 100mM phosphate buffers pH7.0 20~30V of system is answered, adjusts pH7.0,30 DEG C of constant-temperature table 200rpm reactions.3h, 16h take 200 μ L of system, add in 400 μ L methanol Mixing, 12000rpm are centrifuged 3 minutes, and 200 μ L supernatants is taken to add in 800 μ L methanol mixings, and HPLC is sent to detect conversion ratio.It determines to urge Change the mutant that activity improves.
Partial detection is shown in Table 3.
Table 3：
From table 3 it is observed that the reaction speed of transaminase is very fast, it is basically identical in 3h and the changing effect for staying overnight 16h, Conversion ratio difference is no more than 10%, but sampling operation for convenience, and experiment is using reaction overnight afterwards.Part in the present invention ω-transaminase mutant, in (S) -1- benzyloxycarbonyl group -3- amino-heterocyclic compounds (particularly (S) -1- benzyloxycarbonyl group -3- amino Pyrrolidines) synthesis in, the dosage of enzyme is further reduced to 0.3-0.5wt, reaction volume 20V, (the S) -1- benzyloxies obtained The ee values of carbonyl -3- amino-pyrrolidines are more than 99%, which greatly enhances the utilization rate of enzyme, make the industrial production of the compound into Originally it is greatly lowered.
In addition, it the results are shown in Table using the reaction condition and partial reaction synthesized to (S) -1- benzyloxycarbonyl group -3- amino piperidines 4。
Table 4：
As can be seen from Table 4, part ω-transaminase mutant in the present invention, in (S) -1- benzyloxycarbonyl group -3- amino In piperidines synthesis, the dosage of enzyme is reduced to 0.5wt, and reaction volume is reduced to 10V, and conversion ratio improves 8%~30%, ee values ＞ 98%.Which greatly enhances the utilization rates of enzyme and reaction kettle, effectively reduce the usage amount of enzyme solution production batch and material, reduce Production cost.
(2) tolerance of transaminase mutant is examined using N-Cbz-3- pyrrolidones as substrate.Using following system：
0.2g N-Cbz-3- pyrrolidones substrates are dissolved in mixing in 0.5ml DMSO, add the isopropylamine hydrochloride of 4.3M The PLP 0.2ml of 1.25ml, 0.01g/ml, through 30 DEG C, pH9.5,50%DMSO handle the thick 0.5~2wt of enzyme of restructuring of 1h, use 100mM NaHCO320~30V of reaction system is supplied, adjusts pH7.0,30 DEG C of constant-temperature table 200rpm reactions are overnight.16h takes system 200 μ L add in 400 μ L methanol mixings, and 12000rpm is centrifuged 3 minutes, and 200 μ L supernatants is taken to add in 800 μ L methanol mixings, are sent HPLC detects conversion ratio.Determine stability-enhanced mutant.Partial results are shown in Table 5.
Table 5：
Transaminase Enzyme solution treatment conditions Relative residual vigor
Wild type 30 DEG C, pH9.5,50%DMSO processing 1h 21.84%
V379T+R416A 30 DEG C, pH9.5,50%DMSO processing 1h 62.14%
V379T+R416A+C418A 30 DEG C, pH9.5,50%DMSO processing 1h 27.13%
V379T+R416A+C418S 30 DEG C, pH9.5,50%DMSO processing 1h 58.05%
Enzyme can lose activity under the extreme conditions such as high temperature, strong acid, highly basic, organic solvent for a long time, residual enzymic activities It is exactly the gross activity for the enzyme that activity is also kept under the environment such as high temperature, strong acid, highly basic or organic solvent.Relative residual vigor is Refer to through appropriate treated the enzyme activity that enzyme solution measures of the extreme conditions such as high temperature, alkalescence, organic solvent, and without extreme environment The percentage of enzyme activity of the enzyme solution of lower processing under optimum condition.Under identical treatment conditions, relative residual vigor is high, says The stabilization of the bright enzyme with this condition is higher.
As can be seen from Table 5, under identical extreme condition, the relative residual vigor of mutant is than wild type transaminase It is twice to 3 times high.Therefore, the stability that ω-transaminase mutant is obtained in the present invention is greatly improved, after this is Preferable precondition is created in continuous immobilization and continuous flow reaction.
In summary, the present invention is obtained by the method for directed screening ω-transaminase mutant catalytic activity and/or steady It is qualitative to improve a lot so that its enzyme dosage in transamination reaction reduces, reaction system reduction, especially with cheap different Propylamine is amino group donor, catalyzes and synthesizes (S) -1- benzyloxycarbonyl group -3- amino piperidines and (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines Reaction.
In second of typical embodiment of the present invention, a kind of DNA molecular is provided, DNA molecular coding is above-mentioned Any ω-transaminase mutant.The above-mentioned transaminase mutant of coding is significantly improved with catalytic activity and/or stability Advantage.
In the third typical embodiment of the present invention, a kind of recombinant plasmid is provided, recombinant plasmid is connected with State DNA molecular.
In above-mentioned recombinant plasmid, the recombinant plasmid of any DNA molecular that can be used in expressing above-mentioned transaminase mutant is equal Suitable for the present invention.In a preferred embodiment of the invention, recombinant plasmid is selected from one of following：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-1、pGEX-6p-1、 PBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.
In the 4th kind of typical embodiment of the present invention, a kind of host cell is provided, which contains State any recombinant plasmid.Specific host cell is prokaryotic cell or eukaryocyte, and preferably eukaryocyte is yeast cells.More Preferably, above-mentioned host cell is competent cell, and further preferred competent cell is e. coli bl21 cell or large intestine Bacillus W3110.
In the 5th kind of typical embodiment of the present invention, a kind of method for producing Chiral Amine is provided, including using The step of transaminase carries out catalysis transamination to ketone compounds and amino group donor, wherein, transaminase is any of the above-described kind Transaminase mutant.
Ketone compounds areWherein, R1And R2It is each independently as C1~C8 alkyl, C5~C10 cycloalkyl, C6 ~C10 aryl or C5~C10 heteroaryls or R1And R2C5~C10 heterocycles, C5~C10 carbon is collectively formed with the carbon on carbonyl Ring group or C5~C10 heteroaryls, the hetero atom in C5~C10 heterocycles and C5~C10 heteroaryls are each independently selected from nitrogen, oxygen At least one of with sulphur, in the aryl in C6~C10 aryl, the heteroaryl in C5~C10 heteroaryls, C5~C10 carbocylic radicals Carbocylic radical or C5~C10 heterocycles in heterocycle it is unsubstituted independently or by halogen, alkoxy or alkyl At least one group is substituted, it is preferable that ketone compounds areTransamination product isPreferably, amino group donor is isopropylamine or isopropyl amine salt.
The transaminase mutant being significantly improved using these catalytic activity and/or stability may be such that (S) -1- The dosage and reaction volume of enzyme in the synthesis of benzyloxycarbonyl group -3- amino-heterocyclic compounds are significantly reduced, and are greatly dropped Low production batch and manufacturing cost so that the enzyme has better application value in the industrial production.The present invention is using cheap Amino group donor (such as isopropylamine and its hydrochloride) reacted, coupled cofactor system, reaction mass can be not required in reaction Species is few, and operation is simpler.
By the use of the mutation of the present invention above-mentioned transaminase as catalyst preparation (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines or (S) during -1- benzyloxycarbonyl groups -3- amino piperidines etc. (S) -1- benzyloxycarbonyl group -3- amino-heterocyclic compounds, due to urging for enzyme Change activity and/or stability significantly improves, thus the dosage of enzyme is substantially reduced, reaction volume tapers to 10-20ml/g substrates, bright Aobvious reaction volume than in the prior art is small.On this basis, the temperature of above-mentioned reaction, time and pH value can be existing Reasonable adjusting and optimizing is carried out on the basis of reaction condition to obtain.Using the preferred reaction condition of the application, reaction efficiency higher.
It will be further illustrated the present invention below by following non-limiting examples, it is well known to those skilled in the art, not In the case of spirit of the invention, many modifications can be made to the present invention, such modification also falls into the scope of the present invention.
Following experimental methods are conventional method unless otherwise instructed, used experiment material unless otherwise instructed, It can easily be obtained from commercial company.
Embodiment 1
Utilize 9 shown in 36 pairs of rite-directed mutagenesis primers of QuikChange Primer Design webpage designs and table 1 Saturation mutation primer obtains complete linear fragment by full plasmid PCR, above-mentioned PCR product is digested removing through Dpn I and is set out It after the maternal masterplate of gene, is transformed into e. coli bl21 (DE3), is coated on the LB trainings containing 50 μ g/ml ampicillins It supports in ware, 37 DEG C of overnight incubations.Saturation mutation passes through high flux screening.Height is specifically carried out with the following method to above-mentioned mutant Flux screening：
(1) 96 orifice plate induced expression：Picking monoclonal is inoculated in the LB fluid nutrient mediums containing 100 μ g/ml ampicillins In, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG to final concentration of 0.2mM is added in, induced expression mistake is carried out at 25 DEG C Night.
(2) enzyme solution preparation method：Supernatant culture medium is removed in the centrifugation of 96 orifice plates, and 200 μ l enzymolysis solution (lysozymes are added in per hole 2mg/mL, polymyxin 1mg/mL, pH7.0), the broken 2h of 37 DEG C of heat preservations.Clasmatosis liquid 4000rpm centrifugations after enzymolysis 10min takes supernatant to obtain crude enzyme liquid.
Embodiment 2：Transaminase mutant is to the activity assay of N-Cbz-3- pyrrolidones substrates
Above-mentioned enzyme activity is inoculated in LBs of the 500ml containing 100 μ g/ml ampicillins higher than the mutant of wild type transaminase In fluid nutrient medium, 37 DEG C of shaken cultivations to OD600When=0.6, IPTG to final concentration of 0.2mM is added in, is lured at 25 DEG C Lead expression.After inducing 16h, 6000g centrifugations 10min collects thalline.Thalline Ultrasonic Cell Disruptor (JY92-2D, the new sesame biology in Ningbo Science and Technology Co., Ltd.) smudge cells, 4 DEG C, 10000g centrifugations 20min obtains supernatant, for Activity determination.
0.2g N-Cbz-3- pyrrolidones substrates are dissolved in mixing in 0.5ml DMSO, add the isopropylamine hydrochloride of 4.3M The thick enzyme of restructuring of the PLP 0.2ml, 0.3~2wt of 1.25ml, 0.01g/ml, are supplied instead with 100mM phosphate buffers pH7.0 20~30V of system is answered, adjusts pH7.0,30 DEG C of constant-temperature table 200rpm reactions are overnight.16h takes 200 μ L of system, adds in 400 μ L methanol Mixing, 12000rpm are centrifuged 3 minutes, and supernatant send HPLC to detect conversion ratio.Determine the mutant that catalytic activity improves.
The mutant that catalytic activity improves the results are shown in Table 6.Table 6：
" 1 " represents the thick enzyme of 3wt ketoreductases, during reaction system 30V, 16h conversion ratios ＞ 93%；"+" represents the reduction of 1wt ketone The thick enzyme of enzyme, during reaction system 30V, 16h conversion ratios are more than 93%；" ++ " represents the thick enzyme of 1wt ketoreductases, during reaction system 20V, 16h conversion ratios ＞ 93%；" +++ " represents the thick enzyme of 0.5wt ketoreductases, during reaction system 20V, 16h conversion ratios ＞ 93%；“+++ + " representing the thick enzyme of 0.3wt~0.5wt ketoreductases, during reaction system 20V, 16h conversion ratios are 94%-100%.
Embodiment 3：
By mutant no for 1 to 21,33 to 43 and 54 induced expressions 33 transaminases for (S) -1- benzyloxycarbonyl groups - The screening of 3- amino piperidine synthetic reactions, using following reaction system：0.2g N-BOC- piperidones substrates are dissolved in 0.3ml DMSO Middle mixing, the thick enzyme of restructuring of isopropylamine hydrochloride 698ul, the 2mg PLP, 0.5~3wt of 4.3M, with 100mM phosphate buffers PH7.0 supplies 10~16V of reaction system, adjusts pH7.0,30 DEG C, 200rpm reactions are overnight.Reacted screening, activity are relatively preferable Strain reaction result be shown in Table 7.The catalytic activity of 21 transaminase mutant of residue in addition to table 7 is lived with wild type transaminase Property compared to having no increase.
Table 7：
Embodiment 4：The tolerance of transaminase mutant is examined using N-Cbz-3- piperidones as substrate
The thick enzyme 0.5wt of transaminase is with 30 DEG C, after pH9.5,50%DMSO handle 1h, for reacting as follows：By 0.2g N- Cbz-3- piperidones substrates are dissolved in mixing in 0.3ml DMSO, add the isopropylamine hydrochloride 698ul of 4.3M, 0.01g/ml's PLP0.2ml, with 100mM NaHCO3Reaction system 11V is supplied, adjusts pH9.5,30 DEG C of constant-temperature table 200rpm reactions are overnight.16h 200 μ L of system are taken, add in 400 μ L methanol mixings, 12000rpm is centrifuged 3 minutes, and supernatant send HPLC to detect conversion ratio.Specific knot Fruit is shown in Table 8.
Table 8：
Transaminase Relative residual vigor
Wild type transaminase 18.13%
V379T+R416A 33.96%
V379T+R416H 27.91%
V379T+R416A+C418A 26.09%
R416T 40.25%
V379T+R416A+C418S+F89Y 50.7%
As can be seen from Table 8, under identical treatment conditions, compared with wild type transaminase, the transaminase of part is dashed forward The stability higher of variant.
Embodiment 5：
The thick enzyme 0.5wt of 4 transaminases is with 30 DEG C, after pH9.5,50%~60%DMSO handle 1h, for reacting as follows：It will 0.2g N-Cbz-3- piperidones substrates are dissolved in mixing in 0.3ml DMSO, add the isopropylamine hydrochloride 698ul, 0.01g/ of 4.3M The PLP 0.2ml of ml, with 100mM NaHCO3Reaction system 2ml is supplied, pH9.5,30 DEG C of constant-temperature table 200rpm is adjusted to react Night.16h takes 200 μ L of system, adds in 400 μ L methanol mixings, and 12000rpm is centrifuged 3 minutes, and supernatant send HPLC to detect conversion ratio, It the results are shown in Table 9.
Table 9：
Embodiment 6：Application of the transaminase mutant in the synthesis of (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines is prepared
Its reaction equation is as follows：
Reaction system is as follows：1g N-Cbz-3- pyrrolidones substrates are dissolved in mixing in 2.5ml DMSO, add the isopropyl of 4.3M The V379T+R416A+C418A+ of PLP (phosphopyridoxal pyridoxal phosphate) 1ml, 0.3~0.5wt of amine hydrochlorate 6.25ml, 0.01g/ml F89Y recombinates thick enzyme, supplies reaction system 20V with 100mM phosphate buffers pH7.0, adjusts pH7.0,30 DEG C of constant-temperature tables 200rpm reacts.HPLC is detected, and 16h conversion ratios are 96.38%, and after reaction, system alkali tune adds in the tertiary ether of first and extracted It takes 3 times, addition magnesium sulfate is dried after merging extraction organic phase, rotary evaporated to dryness, yield 80~86%, and ee values are ＞ 99%.
Embodiment 7：Application of the transaminase mutant in the synthesis of (S) -1- benzyloxycarbonyl group -3- amino piperidines is prepared
Its reaction equation is as follows：
Reaction system is as follows：2g N-BOC- piperidones substrates are dissolved in mixing in 3ml DMSO, the isopropylamine hydrochloride of 4.3M The V379T+R416A+C418S+F89Y of 6.98ml, 20mg PLP (phosphopyridoxal pyridoxal phosphate), 0.5wt recombinate thick enzyme, with 100mM phosphoric acid Salt buffer pH7.0 supplies reaction system 10V, adjusts pH7.0,30 DEG C, 200rpm reactions are overnight.HPLC is detected, and 16h conversion ratios are 98.3%, after reaction, system alkali tune adds in the tertiary ether of first and carries out extraction 3 times, and magnesium sulfate is added in after merging extraction organic phase It is dried, rotary evaporated to dryness, yield ＞ 90%, ee values 99%.
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：
Part ω-transaminase mutant that the application is obtained, in (S) -1- benzyloxycarbonyl group -3- amino-heterocyclic compounds In (particularly (S) -1- benzyloxycarbonyl group -3- amino-pyrrolidines and (S) -1- benzyloxycarbonyl group -3- aminos piperidines) synthesis, enzyme Dosage is reduced to 0.3wt~0.5wt, and reaction volume is reduced to 10V-20V, substantially increases the utilization rate of enzyme and the utilization of reaction ax Rate reduces the usage amount of enzyme solution production batch and material, effectively reduces the usage amount of the organic solvent in post processing so that after The difficulty of processing and the discharge capacity of the three wastes reduce, and reduce cost of labor.Moreover, (S) -1- benzyloxy carbonyls of obtained high-optical-purity Base -3- amino-pyrrolidines and (S) -1- benzyloxycarbonyl group -3- amino piperidines, make the industrial production cost of the compound obtain significantly It reduces so that the enzyme has better application value in the industrial production.
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.Within the spirit and principles of the invention, that is made any repaiies Change, equivalent substitution, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Kai Laiying Life Sci-Techs（Tianjin）Co., Ltd
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Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
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Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
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Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
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Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
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Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
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Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
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Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
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<220>
<221> misc_feature
<222> (21)..(21)
<223> m is a,or c
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 18
cagactgggt gaccagaata mnnaaaaccg tggttgaagt cacc 44
<210> 19
<211> 44
<212> DNA
<213>Chromabacterium biolaceum (Chromobacterium violaceum)
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24)..(24)
<223> k is g, or t
<400> 19
ggtgacttca accacggttt tnnktattct ggtcacccag tctg 44
<210> 20
<211> 35
<212> DNA
<213>Chromabacterium biolaceum (Chromobacterium violaceum)
<220>
<221> misc_feature
<222> (17)..(17)
<223> m is a,or c
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, c, g, t or u
<400> 20
ccagcgtgaa cgcctgmnnc ataccaacac cgcgc 35
<210> 21
<211> 35
<212> DNA
<213>Chromabacterium biolaceum (Chromobacterium violaceum)
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (19)..(19)
<223> k is g, or t
<400> 21
gcgcggtgtt ggtatgnnkc aggcgttcac gctgg 35

Claims (10)

1. a kind of transaminase mutant, which is characterized in that the amino acid sequence of the transaminase mutant is SEQ ID NO：1 institute Show the amino acid sequence that sequence is undergone mutation, the amino acid sites of the mutation be selected from F89, K193, P243, V234, I262, One or more of Q280, V379, R416, A417 and C418.
2. transaminase mutant according to claim 1, which is characterized in that the amino acid sites of the mutation occur prominent Become include it is following any one or more：P243E、F89Y/W、K193E、V234I、I262V、Q280K、V379L/M/T、 R416A/C/H/Q/T/S, A417S and C418A/Q/S, wherein, "/" represents "or".
3. transaminase mutant according to claim 1 or 2, which is characterized in that the mutation include it is following any one Combination includes：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。
A kind of 4. DNA molecular, which is characterized in that the transaminase any one of the DNA molecular coding claims 1 to 3 Mutant.
5. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid is connected with the DNA molecular described in claim 4.
6. recombinant plasmid according to claim 5, which is characterized in that the recombinant plasmid is 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-1、pGEX-6p-1、 PBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.
7. a kind of host cell, which is characterized in that the host cell contains recombinant plasmid described in claim 5 or 6.
8. host cell according to claim 7, which is characterized in that the host cell is thin for prokaryotic cell or eucaryon Born of the same parents；
Preferably, the eukaryocyte is yeast cells；
Preferably, the host cell is competent cell, and the further preferred competent cell is thin for e. coli bl21 Born of the same parents or Escherichia coli W3110.
9. a kind of method for producing Chiral Amine turns amino including using transaminase ketone compounds and amino group donor are carried out with catalysis The step of reaction, which is characterized in that transaminase mutant of the transaminase any one of claims 1 to 3.
10. according to the method described in claim 9, it is characterized in that, the ketone compounds areWherein, R1And R2Respectively From independently being as C1~C8 alkyl, C5~C10 cycloalkyl, C6~C10 aryl or C5~C10 heteroaryls or R1And R2With carbonyl C5~C10 heterocycles, C5~C10 carbocylic radicals or C5~C10 heteroaryls, C5~C10 heterocycles is collectively formed in carbon on base At least one of nitrogen, oxygen and sulphur, C6~C10 aryl are each independently selected from the hetero atom in C5~C10 heteroaryls In the aryl, heteroaryl in C5~C10 heteroaryls, miscellaneous in the carbocylic radical in C5~C10 carbocylic radicals or C5~C10 heterocycles Ring group is unsubstituted independently or is substituted by least one group in halogen, alkoxy or alkyl, it is preferable that described Ketone compounds areTransamination product isPreferably, ammonia Base donor is isopropylamine or isopropyl amine salt.
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