CN104099305A - Carbonyl reductase mutant as well as gene and application thereof - Google Patents

Carbonyl reductase mutant as well as gene and application thereof Download PDF

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CN104099305A
CN104099305A CN201410345443.4A CN201410345443A CN104099305A CN 104099305 A CN104099305 A CN 104099305A CN 201410345443 A CN201410345443 A CN 201410345443A CN 104099305 A CN104099305 A CN 104099305A
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carbonyl
mutant
reduction enzyme
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carbonyl reductase
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郁惠蕾
黄磊
许建和
李春秀
潘江
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East China University of Science and Technology
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)

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Abstract

The invention relates to a carbonyl reductase CgKR1 mutant, a coding gene of the mutant, a recombinant expression vector containing the gene of the carbonyl reductase mutant, a recombinant expression transformant, a recombinase, a preparation method of the recombinase, and an application of the carbonyl reductase mutant to asymmetric reduction of ketonic ester for preparation of optically pure chiral hydroxyl ester, such as catalysis of o-cyano methyl phenylglyoxylate for asymmetric reduction to prepare (R)-o-chloro mandelic acid methyl ester. Compared with wild enzymes, the carbonyl reductase mutant has the advantages that the thermal stability is substantially improved, and the catalytic activity of part of the mutant to the o-chlorobenzoic acid formyl methyl ester is also obviously improved. The multiple mutants can be applied to catalysis of the ketonic ester for asymmetric reduction to prepare the optical purely-chiral hydroxyl ester, such as catalysis of the o-cyano methyl phenylglyoxylate for asymmetric reduction to prepare the optically pure (R)-o-chloro mandelic acid methyl ester. The carbonyl reductase mutants have the very good industrial application prospect.

Description

A kind of carbonyl reduction enzyme mutant and gene and application
Technical field
The invention belongs to technical field of bioengineering, be specifically related to carbonyl reductase CgKR1 mutant and encoding gene thereof, the recombinant expression vector that contains described carbonyl reductase mutant gene and recombinant expressed transformant, the preparation method of its recombinase and this recombinase, and this carbonyl reduction enzyme mutant is at asymmetric reduction ketone ester, prepare optical homochiral hydroxy ester, such as the application in the adjacent chlorobenzoyl methyl-formiate of catalysis asymmetric reduction preparation (R)-o-Chloromelic acid methyl esters.
Background technology
Chiral alcohol is widely used in the preparation of chiral medicinal, agricultural chemicals and polytype chiral material.Wherein (molecular formula is o-Cl-C to (R)-o-Chloromelic acid methyl esters [(R)-CMM] 6h 4cH (OH) COOCH 3, No. CAS: 32345-59-8) and be the important chiral intermediate of synthetic anticoagulant clopidogrel.Clopidogrel is a kind of adenosine diphosphate (ADP) (ADP) receptor-blocking agent, can with platelet membrane surfaces A DP receptors bind, make Fibrinogen cannot with glycoprotein GP IIb/IIIa receptors bind, thereby suppressing thrombocyte assembles mutually, be mainly used in treating acute myocardial infarction, there is at home market widely.
Researchist has developed the synthetic method of multiple optical activity chirality alcohol, comprises kinetic resolution and asymmetric synthesis.Wherein, utilize the approach of the asymmetric reduction synthesis of optically active chiral alcohol of prochiral carbonyl compounds, productive rate that can realization theory 100%, is the important method of production optical activity (R)-o-Chloromelic acid methyl esters.Chiral metal derivative can be used as the asymmetric reduction of catalyzer for carbonyl, although this chemical process has been partly used for industrial production, but the method operation easier is large, severe reaction conditions, and may residual heavy metal in product, therefore its application is restricted.Biocatalysis method is reaction conditions gentleness, environmentally friendly not only, there is regioselectivity and the stereoselectivity of height, and avoid heavy-metal residual in product, just made up the weak point of chemical process, therefore the application of the carbonyl asymmetric reduction reaction of biocatalysis in chiral alcohol asymmetric synthesis in recent years more and more comes into one's own.
(R) the biological method of asymmetric synthesis of-o-Chloromelic acid or derivatives thereof ester mainly contains the asymmetric hydrolysis of adjacent chlorine mandelonitrile of nitrilase catalysis and the asymmetric reduction of the adjacent chlorobenzoyl manthanoate of carbonyl reduction enzyme catalysis.Dynamic Kinetic Hydrolysis Resolution preparation (the R)-o-Chloromelic acid of nitrilase adjacent chlorine mandelonitrile of catalytic racemization in toluene-water two-phase system in Bacillus foecalis alkaligenes (Alcaligenes sp.ECU0401) source for Zhang Zhi's an ancient unit of weight etc., concentration of substrate is 200mM, the enantiomeric excess value (ee) of product is 90.4%, productive rate is 76.5% (J.Biotechnol.2011,152:24-29).The adjacent chlorobenzoyl methyl-formiate of carbonyl reductase Gre2p catalysis asymmetric reduction preparation (R)-o-Chloromelic acid methyl esters in bread yeast source for Ema etc., concentration of substrate is 200g/L, the ee value of product is higher than 99%, separating yield is 89% (Adv.Synth.Catal.2008,350:2039-2044).For realizing high productive rate, the NADP of 1g/L has additionally been added in this reaction +coenzyme, because coenzyme is expensive, the interpolation of a large amount of coenzyme will certainly increase the cost of reaction.
Clone the highly-solid selectively asymmetric reduction of the carbonyl compound that the carbonyl reductase CgKR1 that obtains can the multiple high density of catalysis from Candida glabrata (Candida glabrata) CGMCC2.234 and prepare optics chiral alcohol, this carbonyl reductase is restructuring overexpression (Adv.Synth.Catal.2012 in intestinal bacteria (E.coli), 354,1765-1772; CN102618513A).This enzyme has higher vigor to multiple carbonyl compound, and ketone ester class substrate that can the multiple high density of catalysis transforms completely, but the thermostability of this carbonyl reductase is poor, only have 41.8 DEG C, catalysis 0.5M substrate conversion, at 30 DEG C, reaction is complete deactivation less than 10h, and the thermostability of enzyme is the key character of industrial enzymes.Means by the protein engineering such as random mutation, rite-directed mutagenesis are carried out thermostability transformation to CgKR1, improve its thermostability, will have higher industrial application value.
Summary of the invention
Technical problem to be solved by this invention is, for the problem of the carbonyl reductase poor heat stability of having found at present, a kind of carbonyl reductase mutein of thermostability raising, the nucleotide sequence of this mutein of encoding are provided, the recombinant expression vector that contains this nucleotide sequence and recombinant expressed transformant, the preparation method of the whole cell of recombinant expressed this carbonyl reduction enzyme mutant and at catalysis of carbonyl asymmetric reduction, prepares the application in optical homochiral alcohol.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: the carbonyl reductase CgKR1 mutein that provides a kind of thermostability to improve.
The protein with aminoacid sequence as shown in SEQ ID No.2 in sequence table is suddenlyd change, the aspartic acid of the 138th is replaced with to l-asparagine, or on this basis, the phenylalanine of the 92nd, the phenylalanine of the 94th, the Isoleucine of the 99th, the glycine of 174 are carried out respectively or jointly to amino-acid residue replacement, obtain mutein that derived by CgKR1 and that there is carbonyl reduction enzymic activity.
Mutant CgKR1 d138Nthe protein of shown aminoacid sequence composition is to screen and obtain the random mutation storehouse from being built by CgKR1.Obtain in screening the mutant CgKR1 that thermostability is significantly improved d138Nbasis on, we continue CgKR1 d138Ntransform, the amino-acid residue that is other by some non-conservative site mutations in enzyme surface and active centre, further to strengthen the thermostability of this enzyme.
Preferably, described carbonyl reduction enzyme mutant, for there is the mutant that the sudden change of any one situation obtains as follows in the amino-acid residue of wild carbonyl reductase CgKR1:
(1) in the aminoacid sequence of wild-type carbonyl reductase CgKR1, the 138th aspartic acid replaces with l-asparagine, called after CgKR1 d138N;
(2) in the aminoacid sequence of wild-type carbonyl reductase CgKR1, the 138th aspartic acid replaces with l-asparagine, and the 99th Isoleucine replace with tyrosine, and the 174th glycine replaces with L-Ala, called after CgKR1 199Y/D138N/G174A;
(3) in the aminoacid sequence of wild-type carbonyl reductase CgKR1, the 138th aspartic acid replaces with l-asparagine, and the 92nd phenylalanine replaces with leucine, and 94 phenylalanines replace with α-amino-isovaleric acid, the 99th Isoleucine replaces with tyrosine, and the 174th glycine replaces with L-Ala, called after CgKR1 f92L/F94V/199Y/D138N/G174A.
The preparation method of described mutant is specially: to derive from the wild enzyme CgKR1 of carbonyl reductase gene in Candida glabrata (Candida glabrata) CGMCC2.234 as template, utilize random mutation primer to carry out fallibility PCR and build random mutation storehouse, high flux screening is carried out in obtained mutator gene storehouse, obtain the mutant CgKR1 that thermostability improves d138N( it is 48.8 DEG C); With mutant CgKR1 d138Ngene be template, the mutant primer that utilization contains catastrophe point (is chosen one section of base sequence of the each 15～20bp of amino acid sites upstream and downstream that need to suddenly change, be the rear amino acid whose codon of sudden change by the Substitution in mutational site, as PCR forward primer, its reverse complementary sequence is PCR reverse primer), increase by PCR method, obtain mutant gene, express and obtain CgKR1 199Y/D138N/G174A( be 53.4 DEG C) and CgKR1 f92L/F94V/199Y/D138N/G174A( it is 54.6 DEG C).
Wherein said fallibility pcr amplification is this area routine techniques, and (50 μ are l) system of PCR reaction: template 0.5～20ng, 5 μ l10 × rTaq buffer, 5 μ l dNTP (each 2.0mM), 2 μ l MgSO 4(25mM), 5 μ l MnCl 2(100 μ M), the each 1 μ l of a pair of mutant primer (20 μ M), the rTaq enzyme (TaKaRa, Japan) of 1 unit, adds sterile purified water to 50 μ l.
The program of described fallibility pcr amplification is: (1) 94 DEG C of sex change 3min; (2) 94 DEG C of sex change 10sec, (3) 60 DEG C of annealing 30sec, (4) 68 DEG C are extended 90sec, and step (2)～(4) are carried out 30 circulations altogether, and last 72 DEG C are extended 10min, 4 DEG C of preservation products.
Wherein said rite-directed mutagenesis pcr amplification is this area routine techniques, and (50 μ are l) system of PCR reaction: template 0.5～20ng, 5 μ l10 × KOD plus buffer, 5 μ l dNTP (each 2.0mM), 2 μ l MgSO 4(25mM), the each 1 μ l of a pair of mutant primer (20 μ M), the KOD enzyme (TOYOBO, Japan) of 1 unit, adds sterile distilled water to 50ul.
The program of described rite-directed mutagenesis pcr amplification is: (1) 94 DEG C of sex change 5min; (2) 94 DEG C of sex change 30sec, (3) 60 DEG C of annealing 1min, (4) 68 DEG C are extended 4min, and step (2)～(4) are carried out 30 circulations altogether, and last 68 DEG C are extended 10min, 4 DEG C of preservation products.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of nucleic acid of separation, described nucleic acid is the nucleic acid of coding mutein as described in technical scheme one.
The preparation method of nucleic acid of the present invention is the conventional preparation method in this area, described preparation method preferably comprises: obtain the nucleic acid molecule of coding carbonyl reduction enzyme mutant by gene clone technology, or by the synthetic method of artificial complete sequence obtain the encoding nucleic acid molecule of carbonyl reduction enzyme mutant.
Nucleic acid of the present invention is preferably for having the nucleic acid molecule of point mutation, the preparation method of the described nucleic acid molecule with point mutation is the preparation method of this area routine, described preparation method is preferably: the mutant DNA sequence dna obtaining taking technical scheme one is template, utilize primer to carry out the required goal gene of pcr amplification, obtain the nucleic acid molecule with point mutation: CgKR1 d138N, CgKR1 199Y/D138N/G174Aand CgKR1 f92L/F94V/199Y/D138N/G174A.
Wherein said pcr amplification is this area routine techniques, and (50 μ are l) system of PCR reaction: template 0.5420ng, 5 μ l10 × rTaq buffer, 5 μ l dNTP (each 2.0mM), 2 μ l MgSO 4(25mM), the each 1 μ l of pair of primers (20 μ M), the KOD enzyme (TOYOBO, Japan) of 1 unit, adds sterile purified water to 50 μ l.
The program of described fallibility pcr amplification is: (1) 94 DEG C of sex change 3min; (2) 94 DEG C of sex change 10sec, (3) 60 DEG C of annealing 30sec, (4) 68 DEG C are extended 90sec, and step (2)～(4) are carried out 30 circulations altogether, and last 72 DEG C are extended 10min, 4 DEG C of preservation products.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of recombinant expression vector that comprises above-mentioned nucleic acid.
Wherein said recombinant expression vector can obtain by this area ordinary method, that is: the nucleic acid molecule of carbonyl reductase mutant gene of the present invention is connected on various expression vectors and builds and form.Carrier of the present invention is preferably plasmid pET-28a (+).Preferably, can make recombinant expression vector of the present invention by following method: the nucleic acid product by pcr amplification gained and expression vector pET28a are used respectively to restriction enzyme Sal I and Not I double digestion, form complementary sticky end, connect through T4DNA ligase enzyme, form the recombinant expression plasmid pET28a-CgKR1 that contains carbonyl reductase gene of the present invention d138N, pET28a-CgKR1 199Y/D138N/G174Aand pET28a-CgKR1 f92L/F94V/199Y/D138N/G174A.
For solving the problems of the technologies described above, four of the technical scheme that the present invention takes is: a kind of recombinant expressed transformant that comprises above-mentioned recombinant expression vector.
The preparation method of wherein said recombinant expressed transformant is preferably: above-mentioned recombinant expression vector is converted in host microorganism and is made.Wherein said host microorganism is preferably: intestinal bacteria (E.coli), better is e. coli bl21 (DE3) or bacillus coli DH 5 alpha.Aforementioned recombinant expression plasmid is converted in E.coli BL21 (DE3), gets final product to obtain the preferred engineering strain of the present invention, i.e. E.coli BL21 (DE3)/pET28a-CgKR1 d138N, E.coli BL21 (DE3)/pET28a-CgKR1 199Y/D138N/G174A, E.coli BL21 (DE3)/pET28a-CgKR1 f92L/F94V/199Y/D138N/G174A.Method for transformation can be selected this area ordinary method, and as electricity turns method, heat shock method etc., preferably select heat shock method to transform, hot shock condition preferably: 45 DEG C, heat shock 90 seconds.
For solving the problems of the technologies described above, five of the technical scheme that the present invention takes is: a kind of preparation method of carbonyl reduction enzyme mutant, and comprising following steps: cultivate above-mentioned recombinant expressed transformant, obtain restructuring carbonyl reduction enzyme mutant from culture.
Wherein said preparation method is preferably: above-mentioned recombination bacillus coli is seeded in the LB substratum that contains kantlex (50 μ g/ml), 30～40 DEG C, 150～200rpm cultivates, the extinction density OD of nutrient solution 600reach 0.5～1.0 (preferably 0.8), add sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) of 0.05～1.0mM (preferably 0.1mM) to induce, inducing temperature is 16 DEG C, and induction can obtain the restructuring carbonyl reduction enzyme mutant of high efficient expression for 24 hours.
In the present invention, catalysis prochiral carbonyl compounds carries out the catalyzer of asymmetric reduction reaction formation optical activity chirality alcohol, can be above-mentioned restructuring carbonyl reductase mutant protein, or the transformant cell that comprises restructuring carbonyl reduction enzyme mutant.
For solving the problems of the technologies described above, six of the technical scheme that the present invention takes is: above-mentioned protein or the whole cell of above-mentioned recombination bacillus coli carry out asymmetric reduction reaction as catalyzer at catalysis prochiral carbonyl compounds, forms the application in chiral alcohol.
The preferred carbonyl reduction enzyme mutant of the present invention of described protein or restructuring carbonyl reduction enzyme mutant.Described prochiral carbonyl compounds is preferably α-one ester, 'beta '-ketoester compounds, is respectively the compound shown in formula 1 or 2:
Wherein,
R 1for-CH 3,-C 6h 5,-C 6h 4-o-Cl or-(CH 2) 2c 6h s;
R 2for-CH 3or-CH 2cH 3;
R 3for-CH 3,-CH 2cH 3,-CH 2cl or-C 6h 5.
Preferably, R 1for-C 6h 4-o-Cl, R 2for-CH 3, formula 1 is adjacent chlorobenzoyl methyl-formiate.
Preferably, R 1for-(CH 2) 2c 6h 5, R 2for-CH 2cH 3, formula 1 is 2-carbonyl-4-phenylbutyrate ethyl ester.
Preferably, R 3for-CH 2cl, formula 2 is the chloro-3-carbonyl of 4-ethyl butyrates.
Each condition of asymmetric reduction reaction of the present invention can be selected by the normal condition of this type of reaction of this area, preferably, described application comprises the steps: that described carbonyl reduction enzyme mutant consumption is 5～60kU/L, the concentration of described prochiral carbonyl compounds is 10mmol/L～2.0mol/L, the vigor of Hexose phosphate dehydrogenase is 1～2 times of carbonyl reduction enzyme activity, the consumption of glucose is 1.0～1.5 times of substrate molar weight, the NADP additionally adding +amount be 0～0.5mmol/L, reaction buffer pH is 5.5～7.0, temperature of reaction is 20～35 DEG C.
The preferred concentration of wherein said prochiral carbonyl compounds (except adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate ethyl ester and the chloro-3-carbonyl of 4-ethyl butyrate) in reaction solution is 10～100mmol/L.Carbonyl reduction enzyme dosage of the present invention is catalytically effective amount, is preferably 5～60kU/L.The vigor of Hexose phosphate dehydrogenase is preferably 1 times of reductase vitality.The consumption of glucose is preferably 1.5 times of substrate molar weight.The NADP additionally adding +consumption be preferably 0～0.5mmol/L.Described damping fluid can be the conventional damping fluid in this area, needs only its pH scope 5.5～7.0, and preferably phosphoric acid salt buffer, as phosphoric acid-sodium phosphate buffer.The concentration of phosphate buffered saline buffer is preferably 0.05～0.1mol/L, and described concentration refers to the total concn of conjugate acid and base in buffered soln.Described asymmetric reduction reaction preferably carries out under vibration or agitation condition.The temperature of described asymmetric reduction reaction is preferably 20～35 DEG C.The described asymmetric reduction reaction time, the time that production concentration no longer continues to improve was as the criterion preferably with in reaction process.After asymmetric reduction reaction finishes, can from reaction solution, extract chiral alcohol product by this area ordinary method.
For adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate ethyl ester and the chloro-3-carbonyl of 4-ethyl butyrate, its concentration of substrate best result can not reach 1.5mol/L, 2.0mol/L and 2.0mol/L.Carbonyl reduction enzyme mutant consumption of the present invention is catalytically effective amount, and the amount ratio of carbonyl reduction enzyme mutant and adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate ethyl ester or the chloro-3-carbonyl of 4-ethyl butyrate is preferably 10～100kU carbonyl reductase/mol substrate.The amount ratio of glucose and adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate ethyl ester or the chloro-3-carbonyl of 4-ethyl butyrate is preferably 200～300g glucose/mol substrate.The vigor of Hexose phosphate dehydrogenase is preferably 1 times of reductase vitality, the NADP additionally adding +consumption be preferably 0～1.0mmol/L.Described damping fluid can be the conventional damping fluid in this area, is 6.0 phosphate buffered saline buffer, as phosphoric acid-sodium phosphate buffer as long as its pH scope 5.5～7.0, is preferably selected pH.The concentration of phosphate buffered saline buffer is preferably 0.05～0.1mol/L, and described concentration refers to the total concn of conjugate acid and base in buffered soln.Described asymmetric reduction reaction preferably carries out under agitation condition.The temperature of described asymmetric reduction reaction is preferably 20～35 DEG C, is more preferably 30 DEG C.Compare with wild enzyme CgKR1, CgKR1 mutant under equal conditions deactivation rate is slower, and reaction required time is shorter.The time of described asymmetric reduction reaction, the time that production concentration no longer improves was as the criterion preferably with in reaction process.After asymmetric reduction reaction finishes, can from reaction solution, extract chiral alcohol product by this area ordinary method.
In table 1, contrast CgKR1 mutant (CgKR1 of the present invention f92L/F94V/199Y/D138N/G174A) with the effect of the adjacent chlorobenzoyl methyl-formiate of some the carbonyl reduction enzyme catalysiss asymmetric reduction reactions of having reported, compared with the carbonyl reductase of reporting, CgKR1 mutant can catalysis within a short period of time high concentration substrate conversion completely, and without additionally adding coenzyme, there is significant advantage.
The adjacent chlorobenzoyl methyl-formiate of table 1 different sources carbonyl reduction enzyme catalysis asymmetric reduction
Wherein carbonyl reductase Gre2p preparation method is referring to reference Adv.Synth.Catal.2008,350,2039-2044; Carbonyl reductase YtbE preparation method is referring to reference Tetrahedron Letters2012,53,4715-4717; Carbonyl reductase CgKR1 preparation method is referring to reference Adv.Synth.CataL2012,354,1765-1772.
In the present invention, carry out, in the reaction of asymmetric reduction reaction formation chiral alcohol, if use pure carbonyl reduction enzyme mutant, needing to add coenzyme NAD P at described carbonyl reduction enzyme mutant catalysis prochiral carbonyl compounds +if what use is the carbonyl reductase crude enzyme liquid extracting from microorganism cells or the resting cell of expressing carbonyl reductase, does not need additionally to add coenzyme, only need utilize contained coenzyme in cell.
Agents useful for same of the present invention and raw material be commercially available obtaining all.Positive progressive effect of the present invention is: compared with wild-type carbonyl reductase, carbonyl reduction enzyme mutant of the present invention has higher thermostability, and fractional mutant is lived and also had large increase the reductase enzyme of adjacent chlorobenzoyl methyl-formiate.Multiple carbonyl reduction enzyme mutants that the present invention obtains are specially adapted to the adjacent chlorobenzoyl methyl-formiate preparation of asymmetric reduction (R)-o-Chloromelic acid methyl esters, have good prospects for commercial application.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
Random mutation
Utilize fallibility round pcr to introduce random nucleotide sudden change to carbonyl reductase gene, wherein the primer is as follows:
Upstream primer: ACGCGTCGACAAATGGCTTCTGATAACAGCAAC
Downstream primer: ATTTGCGGCCGCTTAATTAGAGTTCTTCTCGGC
Wherein template is: the wild enzyme gene recombination plasmid of carbonyl reductase, gene order is as SEQ ID No:1.
Pcr amplification system (50 μ l): template 0.5～20ng, 5 μ l10 × rTaq buffer, 5 μ l dNTP (each 2.0mM), 2 μ l MgSO 4(25mM), 5 μ l MnCl 2(100 μ M), the each 1 μ l of a pair of mutant primer (20 μ M), the rTaq enzyme (TaKaRa, Japan) of 1 unit, adds sterile purified water to 50 μ l.
Pcr amplification program: (1) 94 DEG C of sex change 3min; (2) 94 DEG C of sex change 10sec, (3) 60 DEG C of annealing 30sec, (4) 68 DEG C are extended 90sec, and step (2)～(4) are carried out 30 circulations altogether, and last 72 DEG C are extended 10min, 4 DEG C of preservation products.
After pcr amplification product purifying, respectively fallibility pcr amplification product and carrier pET28a are carried out to double digestion with Sal I and Not I, both recovery products, connect 5h with at 16 DEG C of T4 ligase enzymes, Transformed E .coli BL21 (DE3) competent cell, and evenly coat the LB agar plate that contains 50 μ g/ml kantlex, 37 DEG C of incubated overnight, build random mutation body library, the row filter of going forward side by side.Screening obtains mutant that a thermostability is improved largely, and (remnant enzyme activity being incubated at 45 DEG C after 15min is 75%, and the remaining vigor of wild enzyme CgKR1 is only 0.4% under the same terms), find that through DNA sequencing its mutational site is D138N, called after CgKR1 d138N.
Embodiment 2
Rite-directed mutagenesis
Rite-directed mutagenesis adopts iI Site-Directed Mutagenesis Kit (Stratagene, Catalog#200522) scheme operates.First the mutant primer that design contains catastrophe point is as follows:
Sudden change F92L/F94V:
Upstream primer: CGCCTCTCCACTACACGTCAACACCACTGA
Downstream primer: CAGTGGTGTTGACGTGTAGTGGAGAGGCGGTG
Sudden change 199Y:
Upstream primer: CACCACTGACTACGAAAAGGATCTATTGATCCC
Downstream primer: GATCCTTTTCATAGTCAGTGGTGTTGAAGTGGAATG
G174A sudden change:
Upstream primer: TCCAATCAGAGGATACTGTGGTTCAAAGAAGTT
Downstream primer: CCACAGTAAGCTCTGATTGGATCGGATTGAC
PCR reaction system (50 μ l): template 0.5～20ng, 5 μ l10 × KOD plus buffer, 5 μ l dNTP (each 2.0mM), 2 μ l MgSO 4(25mM), the each 1 μ l of a pair of mutant primer (20 μ M), the KOD enzyme (TOYOBO, Japan) of 1 unit, adds sterile distilled water to 50 μ l.
Pcr amplification program: (1) 94 DEG C of sex change 5min; (2) 94 DEG C of sex change 30sec, (3) 60 DEG C of annealing 1min, (4) 68 DEG C are extended 4min, and step (2)～(4) are carried out 30 circulations altogether, and last 68 DEG C are extended 10 min, 4 DEG C of preservation products.
The PCR product that amplification obtains is Transformed E .coli BL21 (DE3) competent cell after restriction endonuclease DpnI37 DEG C of digestion 2h, and evenly coats the LB agar plate that contains 50 μ g/ml kantlex.After 37 DEG C of incubated overnight, select mono-clonal, obtain E.coli BL21 (DE3) bacterial strain containing mutant expression plasmid, serve Hai Sai sound Bioisystech Co., Ltd and check order.Sequencing result is compared by DNAMAN software and wild-type carbonyl reductase gene order, confirms the difference of sudden change front and back gene order and corresponding aminoacid sequence.
Wherein F92L/F94V/199Y/D138N/G174A sudden change building process is: the first full-length gene order taking carbonyl reduction enzyme mutant D138N is as template, with the PCR primer structure 199Y/D138N of sudden change 199Y, then sport template with this, with the PCR primer structure 199Y/D138N/G174A of sudden change G174A, finally sport template with this, with sudden change F92L/F94V PCR primer be built into F92L/F94V/199Y/D138N/G174A.
Correct through DNA sequencing, gained target stripe is the gene of mutant protein of the present invention, and its title is respectively:
CgKR1 199Y/D138N/G174A, the 99th Isoleucine of aminoacid sequence SEQ ID No.2 replaces with tyrosine, and the aspartic acid of the 138th replaces with l-asparagine, and the 174th glycine replaces with L-Ala;
CgKR1 f92L/F94V/199Y/D138N/G174Athe 92nd phenylalanine of aminoacid sequence SEQ ID No.2 replaces with leucine, and the 94th phenylalanine replaces with α-amino-isovaleric acid, and the 99th Isoleucine replaces with tyrosine, the aspartic acid of the 138th replaces with l-asparagine, and the 174th glycine replaces with L-Ala.
Embodiment 3
The structure of recombinant expression vector (plasmid) and the preparation of recombinant expressed transformant
By the carbonyl reductase mutant gene DNA fragmentation of gained in embodiment 1 and 2 at 37 DEG C with restriction enzyme Sal I and Not I double digestion 12h, through agarose gel electrophoresis purifying, utilize sepharose DNA reclaim test kit reclaim target fragment.By target fragment, under the effect of T4DNA ligase enzyme, with same plasmid pET28a after Sal I and Not I double digestion, at 4 DEG C, connection is spent the night and is obtained recombinant expression plasmid.
Above-mentioned recombinant expression plasmid is transformed in E.coli DH5 α competent cell, and conversion condition is 45 DEG C of heat shocks 90 seconds, in the resistant panel that contains kantlex, positive recombinant chou is screened, and picking mono-clonal, bacterium colony PCR verifies positive colony.Cultivate recombinant bacterium, after plasmid amplification, extract plasmid, again be converted in E.coli BL21 (DE3) competent cell, conversion fluid is applied on the LB flat board that contains kantlex, be inverted overnight incubation for 37 DEG C, obtain positive recombinant conversion body E.coli BL21 (DE3)/pET28a-CgKR1 mutant, bacterium colony PCR verifies positive colony.
Embodiment 4
The expression and purification of carbonyl reduction enzyme mutant and enzymic activity and Stability Determination
The expression strain of gained carbonyl reduction enzyme mutant in embodiment 3 is inoculated in to 100ml containing in the LB liquid nutrient medium of 50 μ g/ml kantlex, and 37 DEG C of 200rpm are cultured to OD 600reach 0.6～0.8, be cooled to 16 DEG C and to add final concentration be the IPTG of 0.1mM, continue to cultivate 20h and carry out abduction delivering.The centrifugal 10min of 6000 × g collects thalline, and uses physiological saline washed twice, obtains resting cell.The resting cell of gained is suspended in the damping fluid of pH7.0, ultrasonication in ice bath, centrifugal collection supernatant liquor, is the crude enzyme liquid of restructuring carbonyl reduction enzyme mutant.By crude enzyme liquid freeze-drying, be the thick enzyme powder of freeze-drying.
With the resuspended gained resting cell of sodium phosphate buffer (50mM, pH8.0, containing 500mM NaCl and 5mM beta-mercaptoethanol), ultrasonication, gets supernatant after the centrifugal 45min of 30,000 × g is centrifugal, can obtain the crude enzyme liquid of carbonyl reductase mutant protein.Use Ni affinity column (1ml) purifying carbonyl reductase mutant protein, concrete grammar is as follows:
(1) with sodium phosphate buffer (50mM, pH8.0 contain 500mM NaCl and 5mM beta-mercaptoethanol) balance Ni post;
(2) crude enzyme liquid of the carbonyl reductase mutant protein of aforesaid method gained is passed through to Ni post with the flow velocity of 1ml/min, target protein is mounted on Ni post;
(3) sodium phosphate buffer (50mM, pH8.0 contain 500mMNaCl and the 5mM beta-mercaptoethanol) wash-out that use contains 0～50mM imidazoles and Ni post do not have the foreign protein of binding ability;
(4) with sodium phosphate buffer (50mM, pH8.0 contain 500mMNaCl and 5mM beta-mercaptoethanol) the wash-out target protein that contains 200mM imidazoles;
(5) with sodium phosphate buffer (50mM, pH8.0 contain 500mM NaCl and 5mM beta-mercaptoethanol) balance Ni post, for subsequent use.
(6) carry out SDS-PAGE with vertical electrophoresis apparatus and detect the protein sample of collecting.Get 20 μ l samples and add 5 μ l5 × SDS sample damping fluids to process, applied sample amount is 10 μ l, and standard molecular weight albumen (Protein Molecular Weight Marker), is purchased from Thermo company of the U.S..SDS-PAGE gum concentration is 12%, selects 110V voltage to concentrate electrophoresis, then changes 200V voltage into and carry out separation electrophoresis.Result shows that the mutant obtaining by aforesaid method can obtain high purity protein by purifying, and molecular size range is in 39kDa left and right.
By detecting the mode of the 340nm place light absorption value variation carbonyl reduction enzyme activity determination of recombinating.Concrete grammar is as follows: in 1ml reaction system (100mM sodium phosphate buffer, pH6.0) in, adding final concentration is the adjacent chlorobenzoyl methyl-formiate of substrate of 2mM, final concentration is the NADPH of 0.1mM, 30 DEG C of insulations added the crude enzyme liquid after appropriate pure enzyme solution or cytoclasis after 2 minutes, mix rapidly, detect the variation of 340nm place light absorption value.The calculation formula of enzyme activity is: enzyme activity (U)=EW × V × 10 3/ (6220 × 1); In formula, EW is the changing value of 340nm place absorbancy in 1min; V is the volume of reaction solution, units/ml; 6220 is the molar extinction coefficient of NADPH, the L/ of unit (molcm); 1 is optical path length, the em of unit.Per unit (U) carbonyl reductase is defined as under these conditions, and per minute catalysis 1 μ mol NADPH is oxidized required enzyme amount.Each mutant is surveyed and lived, and result is referring to table 2.
Carbonyl reduction enzyme mutant stability characterizes: measure carbonyl reductase enzyme according to document (Angew.Chem.Int.Ed.2006,27,7745-7751) value; Measure the t of carbonyl reductase according to document (Bioresource technol.2013,354-360) 1/2.
Table 2CgKR1 and mutant character thereof
Embodiment 5-8
The asymmetric reduction of the adjacent chlorobenzoyl methyl-formiate of carbonyl reduction enzyme mutant catalysis
At 100ml sodium phosphate buffer (100mM, pH6.0) in, the consumption of described carbonyl reduction enzyme mutant is 1000U, (preparation method is referring to J.Ind.Microbiol.Biot.2011 for Hexose phosphate dehydrogenase, 38, consumption 633-641) is 1000U, and adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 100g/L, the glucose of 150g/L and the NADP of 0.2mM +.Reaction is carried out at 30 DEG C, and it is 6.0 that reaction solution pH controls.Regularly sampling, with isopyknic ethyl acetate extraction, uses its transformation efficiency of gas phase analysis after anhydrous magnesium sulfate drying.Reaction finishes to extract by equal-volume ethyl acetate afterwards, extracting twice, and combining extraction liquid, adds the spend the night ee value of post analysis mensuration substrate conversion efficiency and reduzate of anhydrous sodium sulfate drying, and result is as shown in table 3.
The asymmetric reduction result of the adjacent chlorobenzoyl methyl-formiate of table 3CgKR1 and mutant catalysis thereof
Embodiment 9-18
The asymmetric reduction of carbonyl reduction enzyme mutant catalyzed carbonylation compound
The crude enzyme liquid CgKR1 that adds 2U to prepare as embodiment 4 in 0.4ml sodium phosphate buffer (100mmol/L, pH7.0) f92L/F94V/199Y/D138N/G174Awith the Hexose phosphate dehydrogenase of 2U (preparation method referring to: J.Ind.Microbiol.Biot.2011,38,633-641), adding respectively final concentration is the ketone ester of 10mmol/L, and the NADP of final concentration 0.5mmol/L +glucose with 5g/L.At 30 DEG C, 1100rpm oscillatory reaction, the reaction times is 24 hours, now the concentration of reaction product all no longer continues to increase.Reaction finishes to extract by equal-volume ethyl acetate afterwards, extracting twice, and combining extraction liquid, adds the spend the night ee value of post analysis mensuration substrate conversion efficiency and reduzate of anhydrous sodium sulfate drying.The concrete analysis condition of product yield and ee value is with reference to Adv.Synth.Catal.2012,354,1765-1772.The results are shown in Table 4:
The result of table 4CgKR1 mutant catalysis of carbonyl compound asymmetric reduction reaction
Embodiment 19
The asymmetric reduction of the adjacent chlorobenzoyl methyl-formiate of CgKR1 mutant catalysis
The CgKR1 that adds the embodiment 4 of 6000U to prepare in 100ml sodium phosphate buffer (100mmol/L, pH6.0) f92L/F94V/199Y/D138N/G174Athe Hexose phosphate dehydrogenase crude enzyme liquid of crude enzyme liquid and 6000U, adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 1.5mol/L and the glucose of 450g/L.Reaction is carried out at 30 DEG C, and it is 6.0 that reaction solution pH controls, and reaction is to till reacting completely, and the concentration of reaction product no longer continues to increase, and this reaction time is 3 hours.Reaction finishes to extract by equal-volume ethyl acetate afterwards, extracting twice, combining extraction liquid, add anhydrous sodium sulfate drying and spend the night, rotary evaporation is except desolventizing, and underpressure distillation obtains 2.70g (R)-o-Chloromelic acid methyl esters, yield 90%, the ee value of product is 98%.
Embodiment 20
The asymmetric reduction of CgKR1 mutant catalysis 2-carbonyl-4-phenylbutyrate ethyl ester
The CgKR1 that adds the embodiment 4 of 6000U to prepare in 100ml sodium phosphate buffer (100mmol/L, pH6.0) f92L/F94V/199Y/D138N/G174Athe Hexose phosphate dehydrogenase crude enzyme liquid of crude enzyme liquid and 6000U, adding final concentration is 2-carbonyl-4-phenylbutyrate ethyl ester of 2.0mol/L and the glucose of 600g/L.Reaction is carried out at 30 DEG C, and it is 6.0 that reaction solution pH controls, and reaction is to till reacting completely, and the concentration of reaction product no longer continues to increase, and this reaction time is 8 hours.Reaction finishes to extract by equal-volume ethyl acetate afterwards, extracting twice, combining extraction liquid, add anhydrous sodium sulfate drying and spend the night, rotary evaporation is except desolventizing, and underpressure distillation obtains 3.52g (R)-2-hydroxy-4-phenyl ethyl butyrate, yield 88%, the ee value of product is 98%.
Embodiment 21
The asymmetric reduction of the chloro-3-carbonyl of CgKR1 mutant catalysis 4-ethyl butyrate
The CgKR1 that adds the embodiment 4 of 6000U to prepare in 100ml sodium phosphate buffer (100mmol/L, pH6.0) f92L/F94V/199Y/D138N/G174Athe Hexose phosphate dehydrogenase crude enzyme liquid of crude enzyme liquid and 6000U, adding final concentration is the 4-chloro-3-carbonyl ethyl butyrate of 2.0mol/L and the glucose of 600g/L.Reaction is carried out at 30 DEG C, and it is 6.0 that reaction solution pH controls, and reaction is to till reacting completely, and the concentration of reaction product no longer continues to increase, and this reaction time is 3 hours.Reaction finishes to extract by equal-volume ethyl acetate afterwards, extracting twice, combining extraction liquid, add anhydrous sodium sulfate drying and spend the night, rotary evaporation is except desolventizing, and underpressure distillation obtains 3.05g (R)-4-chloro-3-hydroxyl ethyl butyrate, yield 92%, the ee value of product is 97%.
The above-mentioned description to embodiment is can understand and use invention for ease of those skilled in the art.Person skilled in the art obviously can easily make various amendments to these embodiment, and General Principle described herein is applied in other embodiment and needn't passes through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art are according to announcement of the present invention, and not departing from improvement and the amendment that category of the present invention makes all should be within protection scope of the present invention.
。

Claims (9)

1. a carbonyl reduction enzyme mutant, is characterized in that, it is following (a) or protein (b):
(a) the 138th aspartic acid of the protein with aminoacid sequence as shown in SEQ ID No:2 in sequence table replaced with to the protein that l-asparagine obtains;
(b) in the aminoacid sequence of (a), to one or several site outside 138 through amino-acid residue replace obtain by (a) derivative protein.
2. a kind of carbonyl reduction enzyme mutant according to claim 1, it is characterized in that, described (b) protein be the phenylalanine of the 92nd, the phenylalanine of the 94th, the Isoleucine of the 99th, the glycine of the 174th are replaced with respectively or jointly to other amino-acid residues obtain in the aminoacid sequence of (a) by (a) derivative protein.
3. a kind of carbonyl reduction enzyme mutant according to claim 1 and 2, is characterized in that, described carbonyl reduction enzyme mutant has following sequence:
(1) the 138th aspartic acid in aminoacid sequence as shown in SEQ ID No:2 in sequence table replaced with to l-asparagine;
(2) the 138th aspartic acid in aminoacid sequence as shown in SEQ ID No:2 in sequence table replaced with to l-asparagine, the 99th Isoleucine replaces with tyrosine simultaneously, and the 174th glycine replaces with L-Ala simultaneously;
(3) the 138th aspartic acid in aminoacid sequence as shown in SEQ ID No:2 in sequence table replaced with to l-asparagine, the 92nd phenylalanine replaces with leucine simultaneously, 94 phenylalanines replace with α-amino-isovaleric acid simultaneously, the 99th Isoleucine replaces with tyrosine simultaneously, and the 174th glycine replaces with L-Ala simultaneously.
4. a nucleic acid for separation, is characterized in that, described nucleic acid encoding is as the carbonyl reduction enzyme mutant as described in arbitrary in claim 1-3.
5. a recombinant expression vector that comprises nucleic acid as claimed in claim 4.
6. a recombinant expressed transformant that comprises recombinant expression vector as claimed in claim 5.
7. the recombinate preparation method of carbonyl reduction enzyme mutant, is characterized in that, described preparation method comprises the steps: to cultivate recombinant expressed transformant as claimed in claim 6, obtains restructuring carbonyl reduction enzyme mutant from culture.
8. the carbonyl reduction enzyme mutant as described in as arbitrary in claim 1-3 or recombinant expressed transformant as claimed in claim 6 at a catalysis prochiral carbonyl compounds asymmetric reduction, are prepared the application in optical homochiral alcohol as catalyzer.
9. application according to claim 8, is characterized in that, described prochiral carbonyl compounds be suc as formula 1 or formula 2 shown in compound:
Wherein,
R 1for-CH 3,-C 6h 5,-C 6h 4-o-Cl or-(CH 2) 2c 6h 5;
R 2for-CH 3or-CH 2cH 3;
R 3for-CH 3,-CH 2cH 3,-CH 2cl or-C 6h 5.
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