CN109456952A - A kind of ω being catalyzed sitafloxacin five-membered ring key intermediate-transaminase mutant - Google Patents

A kind of ω being catalyzed sitafloxacin five-membered ring key intermediate-transaminase mutant Download PDF

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CN109456952A
CN109456952A CN201811449197.1A CN201811449197A CN109456952A CN 109456952 A CN109456952 A CN 109456952A CN 201811449197 A CN201811449197 A CN 201811449197A CN 109456952 A CN109456952 A CN 109456952A
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廖祥儒
翟李欣
赖英杰
谢子豪
蔡宇杰
管政兵
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Jiangnan University
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Abstract

The invention discloses ω-transaminase mutant that one kind can be catalyzed sitafloxacin five-membered ring key intermediate, belong to genetic engineering and technical field of enzyme engineering.The present invention is by being mutated into L for the Y at 32 position of ω-transaminase, and the Y at 159 positions is mutated into F, and the T at 252 positions is mutated into A, has obtained a kind of ω-transaminase mutant that can be catalyzed sitafloxacin five-membered ring key intermediate.Mutant Y32L/Y159F/T252A catalytic efficiency Kcat/Km of the present invention is 0.75min‑1·mM‑1When being catalyzed (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine generation 5-benzyl-5-azaspiro [2.4] heptan-7-one, 5-benzyl-5-azaspiro [2.4] the heptan-7-one maximum conversion rate of mutant is 78.95%.The mutant that the present invention obtains is suitable for the application of industrially chiral synthesis sitafloxacin five-membered ring key intermediate.

Description

A kind of ω being catalyzed sitafloxacin five-membered ring key intermediate-transaminase mutant
Technical field
The present invention relates to ω-transaminase mutant that one kind can be catalyzed sitafloxacin five-membered ring key intermediate, belong to base Because of engineering and technical field of enzyme engineering.
Background technique
Broad spectrum quinolone class antimicrobial --- the sitafloxacin of Japanese first pharmacy Sankyo Co., Ltd exploitation in 2008 (Sitafloxacin Hydrate) has good bactericidal effect to many common bacterial strains of resistance to fluoroquinolones of clinic, for treating Serious Difficult infection disease.Compared with other quinolone medicines, the Daily Drug Cost of this product is low, therapeutic effect and safety It is good.But the key technical problems in its synthesis technology are asymmetric syntheses sitafloxacin five-membered ring key intermediates.The technique Technical restriction that is unstable, at high cost, being difficult to amplify production seriously limits the production of sitafloxacin.Compared to chiral amine medicine The chemical synthesis process of object, the enzyme asymmetry catalysis of biological synthesis method is with its Substratspezifitaet, stereoselectivity is strong, reaction The advantages such as mild condition, environmental-friendly attract attention.Therefore the biological synthesis method of enzyme asymmetry catalysis with more research and is answered With value.
Transaminase (aminotransferase, transaminase) belongs to transferase, commonly used in catalytic amino by Amino group donor compound is transferred to amino acceptor compound.According to the difference of different variable regions, by separate sources reported in the literature The protein sequence of transaminase be compared and clustering, superposition and iteration comparison further according to hydrophilic site, Wo Menke Transaminase is divided into 4 classes.Wherein ω-transaminase belongs to the second subtribe, is usually used in preparing Chiral Amine and unnatural amino acid, such as Beta-amino acids.Because the big pocket bond area of ω-transaminase is than aspartate transaminase (AspATs) and aromatic series transaminase (AroATs) slightly larger, so it can combine substrate more biggish than natural amino acid volume.Transaminase-catalyzed reaction master There are asymmetric syntheses and Kinetic Resolution, using Enzyme catalyzed synthesis chirality aminated compounds, can not only greatly improve production Rate can also avoid the waste of raw material, the higher chiral aminated compounds of a large amount of optical purity is made.ω-transaminase is not It can be applied only to the fractionation to racemic modification, it can be with catalytic precursor ketone substrate asymmetric syntheses chirality aminated compounds.ω- Transaminase is wider in the chiral amine compound range of preparation, therefore has more application value.
In protein molecule transformation, currently used method rationality design, nonideal explosives and half design and rational.Three Whether the main distinction of kind method is whether to fully understand zymoprotein molecular structure and need using bioinformatics software It is calculated and is predicted.Wherein design and rational has many advantages, such as that experimental cost is low, the simple and convenient and time is short.
Although ω-transaminase has huge application and researching value, ω-transaminase for being filtered out from wild mushroom Can not specific catalytic sitafloxacin five-membered ring key intermediate, be allowed to can not further by the ω-transaminase be applied to industry In production, exploitation and utilization are greatly reduced.
Summary of the invention
To solve the above-mentioned problems, by ω-transaminase from Bacillus pumilus W3, (one kind comes from the present invention ω-transaminase of bacillus pumilus and the application .201811219769.7 in biological amination) carry out heterogenous expression and fixed point Mutation transformation, enables improved ω-transaminase specific catalytic sitafloxacin five-membered ring key intermediate, turns ammonia to ω- The industrial applications of enzyme and popularization have far-reaching technological guidance's meaning.
ω of the invention-transaminase mutant, can be five yuan of specific catalytic sitafloxacin compared with parent ω-transaminase Ring key intermediate.The parental gene and bacillus pumilus (Bacillus pumilus W3) ω-aminotransferase gene (Sequence ID:MH196528) unanimously, the plasmid template as mutation is to carry natural ω-transaminase coding base The carrier ota3/pCold II (application number: CN201811219769.7) of cause.
The first purpose of the invention is to provide one kind to be capable of specific catalytic sitafloxacin five-membered ring key intermediate ω-transaminase mutant, the amino acid sequence of the mutant include: on the basis of the amino acid sequence of SEQ ID NO:1 The tyrosine of the 32nd position is mutated into leucine simultaneously, the tyrosine of the 159th position is mutated into phenylalanine, the 252nd position Threonine be mutated into alanine, and obtained amino acid sequence is named as Y32L/Y159F/T252A.
In one embodiment, the ω-transaminase mutant amino acid sequence, as shown in SEQ ID NO:5 Sequence.
In one embodiment, the ω-transaminase mutant nucleotide sequence, including shown in SEQ ID NO:2 Sequence.
Second object of the present invention is a kind of method for preparing the mutant, is included the following steps:
(1) rite-directed mutagenesis the primer is designed, is mutated simultaneously using carrying ω-transaminase encoding gene carrier as template Construct the plasmid vector of Y32L/Y159F/T252A mutant；
(2) the correct recombinant plasmid transformed e. coli bl21 (DE3) of sequence is obtained into recombinant bacterium, recombinant bacterium is fermented and is trained It supports, obtained fermented supernatant fluid contains ω-transaminase mutant.
In one embodiment, the fermentation is by recombinant bacterium in 37 DEG C of cultures to OD600=0.6, then it is cooled to 15 DEG C and the IPTG of final concentration 0.4mM be added induced, culture for 24 hours when centrifugation obtain upper clear enzyme solution.
In one embodiment, the preparation method is still further comprised using AKTA protein purification instrument and HisTrap The nickel column of FF crude 1ml purifies ω-transaminase in fermented supernatant fluid.
Third object of the present invention is to provide a kind of recombinant plasmid vectors of amino acid sequence containing the mutant.
In one embodiment, the plasmid vector is appointing in pET series, pGEX series, pCold series or pUB It anticipates one kind.
The genetic engineering bacterium of the gene for encoding the mutant and the expression mutant is also claimed in the present invention.
The claimed mutant of the present invention, coding state the gene work of the gene of mutant, the expression mutant Journey bacterium is in food, chemical industry or the application for catalyzing and synthesizing relevant chiral amine in terms of preparing drug, especially in preparation Xi Tasha Application in star drug.
In one embodiment, the application, including catalytic amino are transferred to amino acceptor by amino group donor compound Compound.
The application, including the property of can choose catalysis and can chirality synthesis of chiral amine, such as (S) -5-benzyl-5- Azaspiro [2.4] heptan-7-amine and other unnatural amino acids.
Beneficial effects of the present invention:
ω of the invention-transaminase mutant is based on from R type ω-transaminase of Bacillus pumilus It is mutated, ω-transaminase mutant Y32L/Y159F/T252A of building has specific catalytic sitafloxacin five-membered ring The performance of key intermediate.Enzyme kinetic analysis shows, the K of mutant Y32L/Y159F/T252A of the inventionmValue is 4.12 ±0.31mM；Catalytic efficiency Kcat/KmFor 0.75min-1mM-1.It is catalyzed (S) -5-benzyl-5-azaspiro [2.4] heptan- When 7-amine generates 5-benzyl-5-azaspiro [2.4] heptan-7-one, the 5-benzyl-5- of mutant of the present invention Azaspiro [2.4] heptan-7-one maximum conversion rate is 78.95%.And ω-transaminase parent can not be catalyzed the west he Husky star five-membered ring key intermediate (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine.Therefore, of the invention ω-transaminase mutant is more suitable for ω-transaminase in catalysis (S) -5-benzyl-5-azaspiro [2.4] heptan- than parent Application during the Chiral Amines such as 7-amine.
Detailed description of the invention
Fig. 1: natural ω-transaminase three-dimensional simulation structure；
Fig. 2: the chemical structure of sitafloxacin five-membered ring key intermediate；
Fig. 3: natural ω-transaminase and the pure enzyme PAGE gel electrophoresis of mutant；Wherein, swimming lane 1 represents protein molecular Amount standard, swimming lane 2 are mutant Y32L/Y159F/T252A, and swimming lane 3 is natural ω-transaminase.
Specific embodiment
The preparation and building of embodiment 1: ω-transaminase directed mutants
From a kind of directed mutants Y32L/Y159F/T252A of ω-transaminase of Bacillus pumilus W3:
In the present invention, with the highest thermophilic archaea transaminase crystal structure of similarity (PDB ID: It is 5E25) template, Bacillus pumilus W3 ω-transaminase (ω-is constructed by Swiss-Model line server BPAT three-dimensional simulation structure (Fig. 1)).It is compared and is found by amino acid primary sequences, thermophilic archaea turns ammonia Similarity reaches 51.21% between the two by enzyme and ω-BPAT, meets the parameter of homologous modeling, it can be considered that ω-BPAT has The similar three-dimensional structure of thermophilic archaea transaminase.According to software analysis prediction as a result, being mediated using PCR Directed mutagenesis method construct mutant Y32L/Y159F/T252A.
The preparation method of directed mutants, according to Bacillus pumilus W3 ω-transaminase sequence (amino acid sequence Column are as shown in SEQ ID NO:1), separately design and synthesize the primer of introducing rite-directed mutagenesis, to ω-transaminase Y32, Y159 and The position T252 carries out rite-directed mutagenesis simultaneously, measures DNA encoding sequence, respectively sequencing confirmation ω-transaminase mutant coding base Because whether correct；Mutant gene is connected to expression vector appropriate (in pET series, pGEX series, pCold series or pUB Any one) in and import in Escherichia coli and expressed, obtain corresponding ω-transaminase directed mutants.
The PCR amplification of directed mutants encoding gene: round pcr is utilized, is template with expression vector ota3/pCold II.
Introduce the mutant primer of Y32L/Y159F/T252A rite-directed mutagenesis are as follows:
BPTA-F3:5 '-CATGGGTTCTTACTTGGCGATGGGGTCTTCGAAGGC-3 ' (SEQ ID NO:3)
BPTA-R3:5 '-CCCCATCGCCAAGTAAGAACCCATGGTCGTATACAG-3 ' (SEQ ID NO:4)
BPTA-F4:5 '-AAAATCTCTTAATTTTCTGAACAACATCCTTGTTCGC-3 ' (SEQ ID NO:6)
BPTA-R4:5 '-GTTGTTCAGAAAATTAAGAGATTTTACTTTTGGCGA-3 ' (SEQ ID NO:7)
BPTA-F5:5 '-TTTCTTACCGGTGCGGCTGCTGAGGTCATCGCAGTT-3 ' (SEQ ID NO:8)
BPTA-R5:5 '-CCTCAGCAGCCGCACCGGTAAGAAAAACTTCCTCTG-3 ' (SEQ ID NO:9)
PCR amplification program setting are as follows: firstly, 95 DEG C of initial denaturation 3min；It is recycled subsequently into 25: 95 DEG C of denaturation 20s, 60 DEG C annealing 20s, 72 DEG C of extension 1min 40s；Last 72 DEG C of extensions 10min, 4 DEG C of heat preservations.PCR product 1% Ago-Gel electricity Swimming is detected.
After purification, DPn I is added, 37 DEG C, water-bath 1h, then template of degrading assembles mutant fragments, later in PCR product Transformed E .coli JM109, picking positive colony, LB liquid medium culture 8-10h protect glycerol tube, send to sequencing.Sequencing is just True mutant (amino acid sequence as shown in SEQ ID NO:5, nucleotide sequence as shown in SEQ ID NO:2), from glycerol tube It is seeded to LB culture medium, is incubated overnight, plasmid is extracted, it is thin that plasmid is converted expression host e. coli BL21 (DE3) competence Born of the same parents obtain that mutant Y32L/Y159F/T252A recombinant bacterial strain can be expressed.
Embodiment 2: the expression and purification of natural ω-transaminase and its directed mutants
The positive monoclonal that picking is transferred to expressive host e. coli bl21 (DE3) (contains 30 μ g/mL in LB liquid medium Ampicillin) 8~10h of growth, seed fermentation liquid is connected to LB liquid medium (containing 30 μ g/mL ammonia benzyls blueness by 5% inoculum concentration Mycin)；Escherichia coli are in 37 DEG C of shaking table culture 2h, until OD600=0.6 or so, mutant Y32L/Y159F/T252A recombinant bacterial strain The IPTG that 0.05mM final concentration is added induces extracellular expression, and after 15 DEG C of shaking tables continue cultivation and fermentation for 24 hours, by fermentation liquid in 4 DEG C, 8000g centrifugation 10min remove thallus, collect centrifugation fermented supernatant fluid.In the case where magnetic stir bar stirs at low speed, sent out to mutant 60% (NH is slowly added in ferment supernatant4)2SO4, place for 4 DEG C and saltout overnight.4 DEG C, 10000g centrifugation 20min, collect precipitating. After redissolving precipitating with 5.3 citrate-phosphate disodium hydrogen buffer of 50mmol/L pH, in 5.3 citric acids of 50mmol/L pH-phosphorus During which dialysed overnight in sour disodium hydrogen buffer replaces 2-3 elution buffer, be made after being filtered by 0.22 μm of film upper all Product.The purifying of recombinant protein is carried out using AKTA avant protein purification instrument, the control of whole process of purification temperature is 4 DEG C.Sun from Sub- exchange chromatography purification step: (1) it balances: with the 5.3 citrate-phosphate disodium hydrogen buffer of 50mmol/L pH of 5 times of volumes Balance strong cation exchange chromatography column；(2) loading: the sample pre-processed is with the flow velocity loading of 1mL/min；(3) it elutes: packet The unadsorbed substance of elution, foreign protein and destination protein, flow velocity 1.0mL/min are included, eluent is the 50mmol/L containing 1M NaCl The citrate-phosphate disodium hydrogen buffer of pH 5.3, carries out linear elution, and Detection wavelength 280nm is collected different containing sucrose in batches The eluent of structure enzyme enzyme activity；Only there is a destination protein eluting peak, subsequent survey enzyme activity and SDS-PAGE albumen electricity in elution process Swimming discovery, either wild type or mutant, the enzyme solution that summit is collected are most pure part.As shown in Figure 3.
Embodiment 3: enzyme activity analysis method
ω-method the reference of transaminase enzyme activity determination Gao, S. (Gao, S., Su, Y., Zhao, L., Li, G., Zheng, G., 2017.Characterization of a(R)-selective amine transaminase from Fusarium oxysporum.Process.Biochem.63,130-136.)。
Appropriate thallus supernatant (or purifying dilution enzyme solution) is taken, 500 μ L sodium dihydrogen phosphates/disodium hydrogen phosphate buffering is added Liquid (100mM, pH7.0) contains 20mM (R)-α-phenethylamine (or (S)-α-in the buffer Phenethylamine), 20mM Sodium Pyruvate, 5 '-phosphate of 0.1mM pyridoxal (PLP) are mixed, 45 DEG C of difference 15min is reacted, the ethyl acetate that isodose is then added terminates reaction.Absorbance of the measurement reaction front and back solution at 254nm.
Under the above conditions, it is catalyzed enzyme amount required for 1 μm of ol correlation ketone in 1 minute, is defined as an enzyme-activity unit (U/ml).Pass through △ A254Calculate ω-transaminase enzyme activity.U/ml=(△ A/min) * (V*/rvb)；△ A/min- absorbance becomes Change；V- reaction system volume (ml)；；R- molar extinction coefficient (cm2/umol)；V- sample size (ml)；B- cuvette light path (cm), above-mentioned dosage can be scaling up or reduce.
After measured, native enzyme is 1.1760U/mL to the thick enzyme activity of substrate (R)-α-phenethylamine, and to (S)- α-phenethylamine does not have enzyme activity；Recombination mutation ω-transaminase of the invention is then to (R)-α-phenethylamine (or (S)-α-phenethylamine) all without activity.
Embodiment 4: mutant is in sitafloxacin five-membered ring key intermediate (S) -5-benzyl-5-azaspiro [2.4] Application in heptan-7-amine production
ω-transaminase can be divided into R- ω-transaminase and S- ω-transaminase, and different types of ω-transaminase can be catalyzed Substrate type it is different, therefore can produce the product with different optical characteristics.
The present embodiment is with (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine (or (R) -5-benzyl- 5-azaspiro [2.4] heptan-7-amine) it is amino group donor, Sodium Pyruvate is amino acceptor, detects recombination obtained Enzyme and mutation recombinase are to its catalytic capability, to judge whether the enzyme can be catalyzed (S) -5-benzyl- with specific efficient 5-azaspiro [2.4] heptan-7-amine, to determine it in sitafloxacin five-membered ring key intermediate commercial synthesis Using.
Chiral synthesis catalytic experiment: appropriate purifying dilution enzyme solution is taken, it is slow that 500 μ L sodium dihydrogen phosphates/disodium hydrogen phosphate is added Fliud flushing (100mM, pH7.0) contains 20mM (S) -5-benzyl-5-azaspiro [2.4] heptan-7- in the buffer Amine (or (R) -5-benzyl-5-azaspiro [2.4] heptan-7-amine), 20mM Sodium Pyruvate, 0.1mM 5 '-phosphate of pyridoxal (PLP) is mixed, and 45 DEG C are reacted 15min respectively, and the ethyl acetate that isodose is then added is whole Only react.12,000 × g is centrifuged 1min, takes upper organic phase, and 0.22 μm of membrane filtration carries out efficient liquid phase (HPLC) detection, Detection product is 5-benzyl-5-azaspiro [2.4] heptan-7-one.
Testing conditions:
Pillar: Agilent C18column (250*4.6mm, Agilent, USA)；Mobile phase: acetonitrile/water (95/5, v/ v)；Flow velocity: 1mL/min；Detection wavelength: 220nm.
Table 1
Control sample: using (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine as substrate, in above-mentioned reaction Under the conditions of detect natural ω-transaminase to its catalytic capability.
Test sample: using (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine as substrate, in above-mentioned reaction Under the conditions of detection ω-transaminase Y32L/Y159F/T252A mutant to its catalytic capability.
Statistics indicate that the recombinase is to experimental group (using ω-transaminase Y32L/Y159F/T252A mutant as catalyst) Catalytic activity is better than control group (using natural ω-transaminase as catalyst).It is above-mentioned statistics indicate that recombination ω-transaminase Y32L/ Y159F/T252A mutant has the function of that efficient selective synthesizes sitafloxacin five-membered ring key intermediate, there is larger application Potentiality (are shown in Table 2).
Table 2
Enzyme activity (U/mg): it is catalyzed enzyme amount required for 1 μm of ol correlation ketone in 1 minute, is defined as an enzyme-activity unit (U/ mg)。
The Determination of Kinetic Parameters of embodiment 5: ω-transaminase mutant
The method reference of ω-transaminase Determination of Kinetic Parameters Gao, S. (Gao, S., Su, Y., Zhao, L., Li, G., Zheng,G.,2017.Characterization of a(R)-selective amine transaminase from Fusarium oxysporum.Process.Biochem.63,130-136.)。
The present embodiment determines the purifying of embodiment 2 and obtains enzyme mutant Y32L/Y159F/T252A (amino acid sequence such as SEQ Shown in ID NO:5) kinetic parameter at 45 DEG C.Specific implementation method are as follows: take appropriate thallus supernatant (or purifying dilution Enzyme solution), 500 μ L sodium dihydrogen phosphates/disodium hydrogen phosphate buffer (100mM, pH7.0) is added, contains in the buffer different dense (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine (specific concentration gradient is referring to above-mentioned document), 20mM of degree Sodium Pyruvate, 5 '-phosphate of 0.1mM pyridoxal (PLP) are mixed, and 45 DEG C are reacted 15min respectively, are then added same The ethyl acetate of equivalent terminates reaction.The results are shown in Table 3 for dynamics research.
The results show that the K of mutant Y32L/Y159F/T252AmValue is 4.12mM.In addition, Y32L/Y159F/T252A Catalytic constant KcatFor 3.1min-1.The catalytic efficiency K of mutant Y32L/Y159F/T252Acat/KmFor 0.75min-1·mM-1。
As shown in Figure 1, Y32L/Y159F/T252A is in ω-transaminase catalytic center and isomerizing zone, mutation may The structure that will lead to the other parts region of catalytic center and isomerizing zone expands, and bigger substrate is entered from urging Change center, consequently, it is possible to the substrate specificity and kinetic parameter to enzyme generate active influence.
3 ω of table-transaminase mutant kinetic parameter
Conversion ratio, which refers to, becomes 5-benzyl-5- for (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine The transformation efficiency of azaspiro [2.4] heptan-7-one.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of ω-transaminase mutant that can be catalyzed sitafloxacin five-membered ring key intermediate
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 304
<212> PRT
<213>artificial sequence
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Met Glu Asp Gln Lys Glu Gln Trp Ile Phe Leu Asn Asp Glu Leu Val
1 5 10 15
Lys Lys Glu Asp Ala Lys Ile Ser Val Tyr Asp His Gly Phe Leu Tyr
20 25 30
Gly Asp Gly Val Phe Glu Gly Ile Arg Val Tyr Asn Gly Asn Ile Phe
35 40 45
Arg Met Lys Glu His Leu Asp Arg Leu Tyr Asp Ser Ala Arg Ser Ile
50 55 60
Met Leu Asn Ile Pro Tyr Ser Leu Glu Glu Leu Thr Glu Lys Met Ile
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His Thr Val Glu Arg Asn Gly Leu Lys Asp Ala Tyr Ile Arg Leu Val
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Val Ser Arg Gly Ala Gly Asp Leu Gly Leu Asp Pro Asn Asn Cys Gly
100 105 110
Arg Ala Asn Thr Val Ile Ile Val Glu Pro Leu Ala Ile Phe Pro Lys
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His Leu Tyr Glu Thr Gly Ile Asp Ile Val Thr Val Pro Thr Arg Arg
130 135 140
Asn Arg Pro Asp Val Leu Ser Pro Lys Val Lys Ser Leu Asn Tyr Leu
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Asn Asn Ile Leu Val Arg Ile Glu Ala His Met Ala Gly Val Ser Glu
165 170 175
Ala Leu Met Leu Asn Asp Gln Gly Tyr Val Ala Glu Gly Ser Ala Asp
180 185 190
Asn Val Phe Ile Tyr Lys Lys Gly Lys Leu Tyr Thr Pro Pro Gly Tyr
195 200 205
Ile Gly Ala Leu Glu Gly Ile Thr Arg Asn Ala Ile Met Glu Ile Ala
210 215 220
Glu Asp Leu Gly Tyr Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp
225 230 235 240
Val Tyr Thr Ala Glu Glu Val Phe Leu Thr Gly Thr Ala Ala Glu Val
245 250 255
Ile Ala Val Val Lys Val Asp Gly Arg Met Ile Gly Glu Gly Lys Pro
260 265 270
Gly Phe His Thr Asn Lys Leu Leu Glu Gln Phe Arg Lys Arg Val Val
275 280 285
Glu Glu Gly Glu Lys Val Val Phe Thr Asp Glu Asn Ile His Ala Ser
290 295 300
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atggaggatc agaaggaaca gtggatcttt ctgaacgatg agctggtgaa gaaggaagac 60
gcaaaaatct ctgtatacga ccatgggttc ttacttggcg atggggtctt cgaaggcatt 120
cgtgtctaca acggcaatat cttccgtatg aaagaacatt tggaccgcct ttacgatagc 180
gcacgtagta ttatgttgaa tatcccgtat tcacttgaag agctgacaga aaagatgatt 240
cacacagttg agcgtaacgg tttgaaggat gcttacatcc gcttggtcgt gtctcgcgga 300
gcgggcgact tgggattaga cccgaacaat tgtggtcgcg ccaatactgt tatcattgtt 360
gaacctctgg caattttccc gaaacatttg tacgagaccg gcatcgacat tgtaacggtt 420
cccacccgcc gtaatcgtcc cgatgtcctt tcgccaaaag taaaatctct taattttctg 480
aacaacatcc ttgttcgcat tgaggcccac atggcaggtg taagtgaagc gttaatgctt 540
aatgatcaag gatatgtagc cgaagggtcg gctgataatg tttttatcta taaaaaaggt 600
aaactgtaca ctccgccagg ttatattggc gcattagagg gtattacccg caacgcaatt 660
atggagattg cggaagatct tgggtacgag gtcaaggaag agcctttcac gcgccatgac 720
gtctatacag cagaggaagt ttttcttacc ggtgcggctg ctgaggtcat cgcagttgta 780
aaggttgacg gacgcatgat tggcgaagga aagccggggt tccacactaa caaattactt 840
gaacaattcc gcaaacgcgt agtggaggag ggagagaagg ttgtctttac ggatgaaaat 900
atccatgcgt cgtaa 915
<210> 3
<211> 36
<212> DNA
<213>artificial sequence
<400> 3
catgggttct tacttggcga tggggtcttc gaaggc 36
<210> 4
<211> 36
<212> DNA
<213>artificial sequence
<400> 4
ccccatcgcc aagtaagaac ccatggtcgt atacag 36
<210> 5
<211> 304
<212> PRT
<213>artificial sequence
<400> 5
Met Glu Asp Gln Lys Glu Gln Trp Ile Phe Leu Asn Asp Glu Leu Val
1 5 10 15
Lys Lys Glu Asp Ala Lys Ile Ser Val Tyr Asp His Gly Phe Leu Tyr
20 25 30
Gly Asp Gly Val Phe Glu Gly Ile Arg Val Tyr Asn Gly Asn Ile Phe
35 40 45
Arg Met Lys Glu His Leu Asp Arg Leu Tyr Asp Ser Ala Arg Ser Ile
50 55 60
Met Leu Asn Ile Pro Tyr Ser Leu Glu Glu Leu Thr Glu Lys Met Ile
65 70 75 80
His Thr Val Glu Arg Asn Gly Leu Lys Asp Ala Tyr Ile Arg Leu Val
85 90 95
Val Ser Arg Gly Ala Gly Asp Leu Gly Leu Asp Pro Asn Asn Cys Gly
100 105 110
Arg Ala Asn Thr Val Ile Ile Val Glu Pro Leu Ala Ile Phe Pro Lys
115 120 125
His Leu Tyr Glu Thr Gly Ile Asp Ile Val Thr Val Pro Thr Arg Arg
130 135 140
Asn Arg Pro Asp Val Leu Ser Pro Lys Val Lys Ser Leu Asn Phe Leu
145 150 155 160
Asn Asn Ile Leu Val Arg Ile Glu Ala His Met Ala Gly Val Ser Glu
165 170 175
Ala Leu Met Leu Asn Asp Gln Gly Tyr Val Ala Glu Gly Ser Ala Asp
180 185 190
Asn Val Phe Ile Tyr Lys Lys Gly Lys Leu Tyr Thr Pro Pro Gly Tyr
195 200 205
Ile Gly Ala Leu Glu Gly Ile Thr Arg Asn Ala Ile Met Glu Ile Ala
210 215 220
Glu Asp Leu Gly Tyr Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp
225 230 235 240
Val Tyr Thr Ala Glu Glu Val Phe Leu Thr Gly Ala Ala Ala Glu Val
245 250 255
Ile Ala Val Val Lys Val Asp Gly Arg Met Ile Gly Glu Gly Lys Pro
260 265 270
Gly Phe His Thr Asn Lys Leu Leu Glu Gln Phe Arg Lys Arg Val Val
275 280 285
Glu Glu Gly Glu Lys Val Val Phe Thr Asp Glu Asn Ile His Ala Ser
290 295 300
<210> 6
<211> 37
<212> DNA
<213>artificial sequence
<400> 6
aaaatctctt aattttctga acaacatcct tgttcgc 37
<210> 7
<211> 36
<212> DNA
<213>artificial sequence
<400> 7
gttgttcaga aaattaagag attttacttt tggcga 36
<210> 8
<211> 36
<212> DNA
<213>artificial sequence
<400> 8
tttcttaccg gtgcggctgc tgaggtcatc gcagtt 36
<210> 9
<211> 36
<212> DNA
<213>artificial sequence
<400> 9
cctcagcagc cgcaccggta agaaaaactt cctctg 36

Claims (10)

1. ω-transaminase mutant that a kind of catalytic efficiency improves, which is characterized in that the amino acid sequence packet of the mutant It includes: being mutated into leucine, the 159th on the basis of the amino acid sequence of SEQ ID NO:1, while by the tyrosine of the 32nd position The tyrosine of position is mutated into phenylalanine, the threonine of the 252nd position is mutated into alanine, and obtained amino acid sequence.
2. ω according to claim 1-transaminase mutant, which is characterized in that the ω-transaminase mutant amino Acid sequence is sequence shown in SEQ ID NO:5.
3. ω according to claim 1-transaminase mutant, which is characterized in that the ω-transaminase mutant nucleosides Acid sequence, including sequence shown in SEQ ID NO:2.
4. a kind of recombinant plasmid vector of the amino acid sequence containing mutant described in claim 1.
5. recombinant plasmid vector according to claim 4, which is characterized in that the recombinant plasmid vector be pET series, Building obtains on the basis of any one plasmid vector in pGEX series, pCold series or pUB.
6. encoding the gene of mutant described in claim 1.
7. expressing the genetic engineering bacterium of mutant described in claim 1.
8. the gene of mutant described in any mutant of claim 1-3, coding claim 1, expression claim 1 institute State the application of the genetic engineering bacterium of mutant.
9. application according to claim 8, which is characterized in that the application includes for food, chemical industry or preparing medicine Object；Especially preparing the application in sitafloxacin drug.
10. application according to claim 8, which is characterized in that the application, including catalytic amino is by amino group donor chemical combination Object is transferred to amino acceptor compound；Optionally, the application includes preparation Chiral Amine or unnatural amino acid；Optionally, institute It states using including catalysis (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017103163A1 (en) * 2015-12-17 2017-06-22 Vito Nv Process for the separation of organic compounds
CN107653233A (en) * 2017-07-06 2018-02-02 泰州学院 A kind of improved transaminase, its encoding gene and the genetic engineering bacterium for expressing the enzyme
CN107805648A (en) * 2017-10-10 2018-03-16 凯莱英生命科学技术(天津)有限公司 The method for preparing the amines with multiple chiral centers
CN108384767A (en) * 2018-02-05 2018-08-10 凯莱英生命科学技术(天津)有限公司 Transaminase mutant and its application
WO2018231462A1 (en) * 2017-06-14 2018-12-20 Codexis, Inc. Engineered transaminase polypeptides for industrial biocatalysis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017103163A1 (en) * 2015-12-17 2017-06-22 Vito Nv Process for the separation of organic compounds
WO2018231462A1 (en) * 2017-06-14 2018-12-20 Codexis, Inc. Engineered transaminase polypeptides for industrial biocatalysis
CN107653233A (en) * 2017-07-06 2018-02-02 泰州学院 A kind of improved transaminase, its encoding gene and the genetic engineering bacterium for expressing the enzyme
CN107805648A (en) * 2017-10-10 2018-03-16 凯莱英生命科学技术(天津)有限公司 The method for preparing the amines with multiple chiral centers
CN108384767A (en) * 2018-02-05 2018-08-10 凯莱英生命科学技术(天津)有限公司 Transaminase mutant and its application

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