CN102618513A - Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound - Google Patents
Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound Download PDFInfo
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Abstract
The invention discloses a novel carbonyl reductase, a gene, a mutant thereof, a recombinant expression vector containing the gene and the mutant, a recombinant expression transformant, a recombinase preparation method, and applications of the carbonyl reductase and recombinase to preparation of active chiral alcohols with a chiral carbonyl compound before asymmetrical reduction. The carbonyl reductase is derived from candida glabrata, is applied to preparation of a plurality of optically-active chiral alcohols such as (R)-chloromandelic acid methyl ester, (R)-2-hydroxy-4-phenyl ethyl butyrate, (R)-4-chlorin-3-phenyl ethyl butyrate and the like. Compared with other preparation methods, a product prepared through the method has high concentration, does not require additionally or slightly adding any expensive coenzyme, has high optical purity, and has the advantages of mild reaction conditions, easiness and convenience for operating, easiness for amplifying and the like, and has a good industrial application prospect in the production of clopidogrel, L-carnitine and perindopril antihypertensive medicinal intermediates.
Description
Technical field
The invention belongs to technical field of bioengineering; Be specifically related to a kind of carbonyl reductase and gene thereof and two mutants; The recombinant expression vector and the recombinant expressed transformant that contain this gene; The preparation method of its recombinase and this recombinase, and this carbonyl reductase or its recombinase as catalyzer in the asymmetric reduction chiral carbonyl compounds with the application in the preparation optical activity chirality alcohol.
Background technology
Chiral alcohol is the important intermediate of many situation of selling well medicines and chirality chemical.For example, (R)-(molecular formula is o-Cl-C to the o-Chloromelic acid methyl esters
6H
4CH (OH) COOCH
3, molecular weight is 200.62, CAS number: the important chiral intermediate that 32345-59-8) is synthetic anticoagulant clopidogrel.Clopidogrel is a kind of ADP (ADP) receptor-blocking agent; Can with platelet membrane surfaces A DP receptors bind; Make Fibrinogen can't with gp GP II b/ III a receptors bind; Assemble each other thereby suppress thrombocyte, be mainly used in the treatment acute myocardial infarction, have market widely at home.(R)-(molecular formula is C to 2-hydroxy-4-phenyl ethyl n-butyrate
6H
5(CH
2)
2CH (OH) COOCH
2CH
3, molecular weight is 208.25, CAS number: 90315-82-5) be synthetic multiple angiotensin-convertion enzyme inhibitor (ACEI) Puli medicine, like the important chirality building block of benazepril, Yipingshu etc.Such medicine is mainly used in diseases such as treatment hypertension, congestive heart failure.(R)-(molecular formula is ClCH to 4-chloro-ethyl 3-hydroxybutanoate
2CH (OH) CH
2COOCH
2CH
3, molecular weight is 166.60, CAS number: the important chiral intermediate that 90866-33-4) is the L-carnitine.The L-carnitine is a kind of amino acid that impels fat to be converted into energy; Taking the L-carnitine can be when reducing body fat, reducing body weight; Do not reduce moisture and muscle, regarded as the safest fat-reducing nutrient complementary goods that has no side effect by international fat health tissues in 2003.Therefore, studying the synthetic preparation of the pure chirality of above-mentioned optical homochiral has broad application prospects.
Optical activity chirality alcohol synthetic has two kinds of chemical method and biological processes.Compare with chemical process, the application of carbonyl asymmetric reduction reaction in the chiral alcohol asymmetric synthesis that biological process has multiple advantages, particularly biocatalysis such as reaction conditions gentleness, transformation efficiency height, enantio-selectivity height 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 the catalytic adjacent chlorine melic acid nitrile of nitrilase and the asymmetric reduction of the enzymatic adjacent chlorobenzoyl manthanoate of carbonyl reduction.Zhang Zhi's an ancient unit of weight etc. split preparation (R)-o-Chloromelic acid with the Dynamic Kinetic hydrolysis of nitrilase adjacent chlorine melic acid nitrile of catalysis racemization in water-toluene two-phase system in Bacillus foecalis alkaligenes (Alcaligenes sp.ECU0401) source; Concentration of substrate is 200mM; The enantiomeric excess value of product (ee) is 90.4%; Productive rate be 76.5% (J.Biotechnol.2011,152,24-29).Ema etc. are with the adjacent chlorobenzoyl methyl-formiate of carbonyl reductase Gre2p catalysis asymmetric reduction preparation (R)-o-Chloromelic acid methyl esters in bread yeast source; Concentration of substrate is 200g/L; The ee value of product is higher than more than 99%; Separate yield and be 89% (Adv.Synth.Catal.2008,350,2039-2044).For realizing high productive rate, the extra NADP that adds 1g/L of this reaction
+Coenzyme because coenzyme costs an arm and a leg, makes and should react not fully up to expectations.(R)-preparation of 2-hydroxy-4-phenyl ethyl n-butyrate normally adopts the method for the cell or the resolvase catalysis 2-carbonyl-4-phenylbutyrate asymmetric reduction of wild bacterium or recombinant bacterial strain.In the application of having reported; The highest level of concentration of substrate is 400mM (82g/L); Catalyst system therefor is pretreated bread yeast (Baker ' s yeast), only have behind the 48h 41.9% substrate conversion to become (R)-2-hydroxy-4-phenyl ethyl n-butyrate but react, and enantiomeric excess value (ee) has only 87.5% (Biocatal.Biotransfor.2009; 27,211-218).Can obtain the product of high-optical-purity in other some reports, but production concentration is generally all very low, does not possess the feasibility of industrialization.Zhang etc. are with Ke Lushi candiyeast (Candida krusei SW2026) catalytic preparation (R)-2-hydroxy-4-phenyl ethyl n-butyrate, and when concentration of substrate was 2.5g/L, the ee value reached 99.7%, and separating yield is 95.1%.But after concentration of substrate is brought up to 20g/L, the ee value be reduced to 97.4% (Process Biochemistry 2009,44,1270-1275).Lavandera etc. are with 2-carbonyl-4-phenylbutyrate reduction preparation (R)-2-hydroxy-4-phenyl ethyl n-butyrate of reorganization alcoholdehydrogenase catalysis 5g/L; The ee value of product is higher than 99%; But need external source to add 1mM NADH in the reaction; (ChemSusChem 2008,1,431-436) to have increased substantially its cost.Lin Wenqings etc. are with the Candida boidinii bacterial strains such as (Candida boidinii) of screening; Water and water/organic two mutually in the two-phase system; Catalysis 2-carbonyl-4-phenylbutyrate asymmetric reduction; Product ee value is 84.9%～98.9%, and the highest production concentration of report is merely 50g/L (Chinese patent, publication number CN 101314784A).The aldehyde reductase SSAR in brown shadow yeast (Sporobolomyces salmonicolor) source can prepare (R)-4-chloro-ethyl 3-hydroxybutanoate by catalysis 4-chloro-3-carbonyl ethyl n-butyrate asymmetric reduction; Concentration of substrate can reach 300g/L; But the ee value is 91.7%, and not fully up to expectations.Therefore be directed against three substrates of above narration; Seek a kind of carbonyl reductase; Highly-solid selectively asymmetric reduction under can the high concentration of substrate of catalysis need not extra adding coenzyme or adds the minute quantity coenzyme in the reaction process, be with a wide range of applications undoubtedly.
Summary of the invention
Therefore; Technical problem to be solved by this invention is; To the biocatalysis asymmetric reduction reported preparation (R)-o-Chloromelic acid methyl esters, (R)-2-hydroxy-4-phenyl ethyl n-butyrate with (R)-need the enzyme of different sources in the reaction of 4-chloro-ethyl 3-hydroxybutanoate, enzymatic activity is on the low side, production concentration is low, optical purity is not high; And need problem such as the expensive coenzyme of extra interpolation; Provide that a kind of catalytic activity is high, enantioselectivity is strong, the carbonyl reductase of substrate better tolerance; The gene of this carbonyl reductase contains the recombinant expression vector of this gene, recombinant expressed transformant and high efficiency preparation method thereof, and the purposes of this carbonyl reductase in catalysis of carbonyl substrate asymmetric reduction.
The present invention through following technical proposals to solve the problems of the technologies described above:
One of technical scheme that the present invention takes is: a kind of isolating protein, and it is (a) or protein (b) as follows:
(a) by aminoacid sequence is formed shown in the SEQ ID No.2 in the sequence table protein;
(b) in the aminoacid sequence of (a) through replacing, lack or adding one or several amino acid and have a carbonyl reductase active by (a) deutero-and the protein identical with the aminoacid sequence of (a) at least 62%.
The protein source that aminoacid sequence shown in the SEQ ID No.2 is formed has the function of carbonyl reductase in Candida glabrata (Candida glabrata) CGMCC 2.234, is a kind of new carbonyl reductase.
The protein that aminoacid sequence shown in the described SEQ ID No.2 is formed can separate acquisition from Candida glabrata (Candida glabrata) CGMCC 2.234; Also can from recombinant expressed this protein expression transformant, separate acquisition, also can obtain by synthetic.
The homology (being amino acid sequence similarity) of the carbonyl reductase shown in the SEQ ID No.2 and other known carbonyl reductases is seen table 1.The homology of the aminoacid sequence of carbonyl reductase and the known carbonyl reductase of aminoacid sequence shown in SEQ ID No.2 is lower than 62% among the present invention, has the significance difference opposite sex.
The homology of table 1. novel carbonyl reductase of the present invention and other known carbonyl reductases
Protein (b) is through replacing, lack or adding one or several amino acid and have a carbonyl reductase active by (a) deutero-and the protein identical with the aminoacid sequence of (a) at least 62% in the aminoacid sequence of (a).Wherein said " several " are meant two to less than 100, and are better for 30.Such as the fusion rotein that adds an external secretion signal peptide, the present invention finds that such fusion rotein has the carbonyl reduction enzymic activity equally.For another example,, in protein (a) molecule of aminoacid sequence shown in the SEQ ID No.2, carry out the sudden change of 1～5 amino-acid residue, also can obtain above-mentioned protein (b), and still keep the carbonyl reduction enzymic activity according to the present invention.That is to say that as long as have the carbonyl reduction enzymic activity by (a) deutero-protein, and deriving mode is as stated, and with the homology of protein (a) more than 62%, can reach goal of the invention of the present invention.The homology of wherein said protein-protein (a) is more than 62%, and better is more than 90%, and best is more than 98.6%.
Obtain in screening on the basis of high reactivity and stereoselectivity enzyme; We have carried out the protein transformation to the enzyme of wild-type; The amino-acid residue in some sites in the hydrophobic channel in active site and guiding active site is sported other amino-acid residue; With the catalytic performance of further this enzyme of reinforcement, improve the expression of enzyme.Described active site is defined as near the approximately diameter of Spherical Volume of
of substrate binding site.
Preferably, (b) protein to be in the 85th, the 92nd, the 94th, the 99th of the aminoacid sequence of (a) and the 174th amino acids one or multidigit active by (a) deutero-protein through replacing an amino acid and having a carbonyl reductase.
Wherein preferred; (a) the 85th of described aminoacid sequence the Xie Ansuan is substituted by Isoleucine, get mutein CgKR1V85I, or the 174th glycocoll is substituted by L-Ala; Get mutein CgKR1G174A, their thermostability has obtained certain raising.
Wherein preferred; (a) the 92nd of described aminoacid sequence the phenylalanine(Phe) is substituted by nonpolar amino acids such as being selected from L-Ala, Xie Ansuan, leucine, Isoleucine and methionine(Met); More preferably be substituted by leucine; Get mutein CgKR1F92L, it still has certain carbonyl reduction enzymic activity.
Wherein preferred; (a) the 94th of described aminoacid sequence the phenylalanine(Phe) is substituted by nonpolar amino acids such as being selected from L-Ala, Xie Ansuan, leucine, Isoleucine or methionine(Met); Preferably be substituted by Xie Ansuan; Get mutein CgKR1F94V, its carbonyl reduction enzymic activity is brought up to original 7 times.
Wherein preferred; (a) the 99th of described aminoacid sequence the Isoleucine is substituted by and is selected from Serine, halfcystine, Stimulina, l-asparagine or tyrosine isopolarity amino acid; More preferably for being substituted by tyrosine; Obtain derived protein CgKR1I99Y, its solubility expression and thermostability have all obtained improving more significantly.
Wherein preferred; (a) the 92nd of described aminoacid sequence the phenylalanine(Phe) is substituted by leucine; Simultaneously the 94th phenylalanine(Phe) is substituted by Xie Ansuan; Get mutein CgKR1F92L/F94V, its carbonyl reduction enzymic activity is brought up to original 12 times, and its thermostability is greatly improved.
Wherein preferred; (a) the 92nd of described aminoacid sequence the phenylalanine(Phe) is substituted by leucine, and the 94th phenylalanine(Phe) is substituted by Xie Ansuan simultaneously, and the 99th Isoleucine is substituted by tyrosine simultaneously; The 174th glycocoll is substituted by L-Ala simultaneously; Get mutein CgKR1F92L/F94V/I99Y/G174A, its carbonyl reduction enzymic activity is brought up to original 12 times, and its thermostability is greatly improved.
Two of the technical scheme that the present invention takes is: a kind of isolating nucleic acid, and it is the nucleic acid of (1) or (2) as follows:
(1) by nucleotide sequence is formed shown in the SEQ ID No.1 in the sequence table nucleic acid;
(2) encode following protein (a) or nucleic acid (b):
(a) by aminoacid sequence is formed shown in the SEQ ID No.2 in the sequence table protein;
(b) in the aminoacid sequence of (a) through replacing, lack or adding one or several amino acid and have a carbonyl reductase active by (a) deutero-and the protein identical with the aminoacid sequence of (a) at least 62%.
Nucleic acid shown in the SEQ ID No.1 according to the invention derives from Candida glabrata (Candida glabrata) CGMCC 2.234.Nucleic acid shown in the SEQ ID No.1 according to the invention can separate acquisition from Candida glabrata (Candida glabrata) CGMCC 2.234 genomes; Also can separate acquisition from the recombinant expression vector that contains nucleic acid shown in this SEQ ID No.1 or in the recombinant conversion body, also the gene synthetic obtains entirely.
Among the present invention, the gene of nucleotide sequence shown in SEQ ID No.1 in the sequence table, called after CgKR1, total length 1059bp.Wherein, its encoding sequence (CDS) ends from 1056 bases of the 1st base to the, and initiator codon is ATG, and terminator codon is TAA.This sequence intronless, the aminoacid sequence of its encoded protein matter is shown in SEQ ID No.2 in the sequence table.
As is known to the person skilled in the art, because the degeneracy of codon, the nucleotide sequence of the aminoacid sequence of coding SEQ ID No.2 not only is confined to SEQ ID No.1.The nucleotide sequence of carbonyl reductase gene of the present invention also can be other any nucleotide sequences of aminoacid sequence shown in the SEQ ID No.2 in the code sequence tabulation.In addition, can also or insert the homologue that a polynucleotide is provided through suitable introducing replacement, disappearance.The homologue of polynucleotide can make through one or more bases of nucleic acid sequence SEQ ID No.1 are replaced, lacked or increase in keeping the enzymic activity scope among the present invention.
The homologue of SEQ ID No.1 also refers to promoter variants.Promotor or signal sequence before described nucleotide sequence can change through replacement, insertion or the disappearance of one or more nucleic acid, but these functions that change promotor do not have negative impact.And the sequence through changing promotor or even use more effective promotor wholly replace from difference kind organism, can improve the expression level of target protein.
The homologue of SEQ ID No.1 also refers to a kind of Polynucleotide that has under standard conditions the base sequence that can hybridize with the Polynucleotide of sequence shown in the SEQ ID No.1.Under standard conditions, hybridize and to carry out according to the mode of describing in " molecular cloning ": Cold Spring Harbor Laboratory Press, the general scheme in the molecular biology (Current Protocols in Molecular Biology).Specifically, hybridization can be carried out according to following steps, is loaded with the DNA to be measured that transcribed or film and label probe of RNA molecule hybridized with one in hybridization buffer.The dilution suppressor factor and 2～8 * SSC that consist of 0.1wt%SDS, 5wt% sulfuric acid DEXTRAN 500.000, a box 1/20 of hybridization buffer.20 * SSC is the solution that the Hydrocerol A of 3M sodium-chlor and 0.3M is formed.Hybridization temperature is 50～70 ℃.After cultivating several hrs or spending the night, clean film with cleaning buffer solution.Cleaning temperature is room temperature, more preferably hybridization temperature.Cleaning buffer solution consist of 6 * SSC+0.1wt%SDS solution, more preferably 5 * SSC+0.1wt%SDS.After having cleaned film, just can discern DNA or RNA molecule through the mark on the probe of being hybridized at DNA or RNA intramolecularly with this cleaning buffer solution.
Three of the technical scheme that the present invention takes is: a kind of recombinant expression vector that comprises nucleotide sequence of the present invention.It can be connected in the nucleic acid of described carbonyl reductase gene of the present invention or its two mutants to make up on the various expression vectors and form through this area ordinary method.Described expression vector can be the conventional various carriers in this area, like commercially available plasmid, clay, phage or virus vector etc., preferred plasmid pET28a.Preferable; Can make recombinant expression vector of the present invention through following method: will use restriction enzyme Nde I and BamH I double digestion respectively through the nucleic acid product and the expression vector pET28a of pcr amplification gained; Form the complementary sticky end; Connect through the T4 dna ligase, form the recombinant expression plasmid pET28a-CgKR1 or its two mutants expression plasmid that contain carbonyl reductase gene of the present invention.
Four of the technical scheme that the present invention takes is: a kind of recombinant expressed transformant that comprises recombinant expression vector of the present invention.Can make through recombinant expression vector of the present invention is converted in the host cell.Described host cell can be the conventional host cell in this area, can stably duplicate voluntarily as long as can satisfy recombinant expression vector, and entrained reductase gene of the present invention can be got final product by effective expression.The preferred intestinal bacteria of the present invention, more preferably intestinal bacteria (E.coli) BL21 (DE3) or intestinal bacteria (E.coli) DH5 α.Aforementioned recombinant expression plasmid pET28a-CgKR1 or its two mutants are converted among ETEC (E.coli) BL21 (DE3); Can obtain the preferred engineering strain of the present invention, i.e. intestinal bacteria (E.coli) BL21 (DE3)/pET28a-CgKR1 or its two mutants.Method for transformation can be selected this area ordinary method, changes method like electricity, and thermal shock method etc. are preferably selected the thermal shock method to transform and got final product, the thermal shock condition preferably: 45 ℃, thermal shock 90 seconds.
Five of the technical scheme that the present invention takes is: a kind of preparation method of the carbonyl reductase of recombinating, and comprising following steps: cultivate recombinant expressed transformant of the present invention, from culture, obtain the reorganization carbonyl reductase.
Wherein, described recombinant expressed transformant is ditto given an account of and is continued, and can obtain through recombinant expression vector of the present invention is converted into host cell.Used substratum can be the conventional any substratum that makes the transformant growth and produce carbonyl reductase of the present invention in this area in the recombinant expressed transformant of described cultivation; For coli strain; Preferred LB substratum: peptone 10g/L; Yeast extract paste 5g/L, NaCl 10g/L, pH 7.0.Cultural method and culture condition do not have special limitation, can carry out appropriate selection by this area general knowledge according to the different of factor such as host type and cultural methods, as long as can make the transformant growth and produce carbonyl reductase of the present invention.Other culture transformation body concrete operations all can be undertaken by this area routine operation.For coli strain; The shake-flask culture enzymatic production is preferably selected following method for use: the recombination bacillus coli that the present invention relates to (preferred E.coli BL21 (DE3)/pET28a-CgKR1 or its two mutants) is seeded in the LB substratum that contains kantlex and cultivates, as the optical density(OD) OD of nutrient solution
600When reaching 0.5～0.7 (more preferably being 0.6); The adding final concentration is that the sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) of 0.05～1.0mmol/L (more preferably being 0.1mmol/L) is induced; 10～30 ℃ of inducing temperatures (more preferably being 16 ℃) can efficiently express reorganization carbonyl reductase according to the invention.
Catalysis prochirality carbonyl compound carries out the catalyzer that asymmetric reduction reaction forms optical activity chirality alcohol among the present invention; Can be the culture of transformant of the reorganization carbonyl reductase of above-mentioned generation, also can be through with the transformant cell that obtains after the substratum spinning or with the goods of its processing.Here " goods of processing " are meant the extract that obtained by transformant or through to the separating prod that the carbonyl reductase in the extract separates and/or purifying obtains, perhaps the immobilizing product that obtains of the separating prod through immobilization transformant cell or extract or transformant.
Six of the technical scheme that the present invention takes is: a kind of protein of the present invention carries out asymmetric reduction reaction at catalysis prochirality carbonyl compound and forms the application in the chiral alcohol.
In the above-mentioned application, each condition of described asymmetric reduction reaction can be selected by the normal condition of this type of reaction of this area, preferably as follows:
The preferred carbonyl reductase of the present invention of described protein or the carbonyl reductase of recombinating.What described prochirality carbonyl compound was preferable is α-ketone ester, 'beta '-ketoester or aryl ketones compounds, is respectively the compound shown in formula 1,2 or 3:
Wherein,
R
1For alkyl, phenyl or have substituent phenyl, the substituting group of phenyl is halogen or alkyl;
R
2Be alkyl;
R
3Be alkyl or haloalkyl;
R
4Be haloalkyl.
Preferably,
R
1For carbon chain lengths is 1～8 alkyl, phenyl or has substituent phenyl, the substituting group of phenyl is that halogen or carbon chain lengths are 1～2 alkyl;
R
2It for carbon chain lengths 1～2 alkyl;
R
3For carbon chain lengths is 1～2 alkyl or haloalkyl, more preferably, the halogen of said haloalkyl is Cl, Br or F; Best, R
3Be one to three Cl, Br or the substituted methyl of F atom.
R
4Be haloalkyl.Preferably, the halogen of described haloalkyl is Cl or F.
More preferably be,
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
2CH
3,-CH
2Cl ,-CH
2Br or-CF
3
R
4For-CH
2Cl or-CF
3
Best, R
1For-C
6H
4-o-Cl, R
2For-CH
3, promptly formula 1 is adjacent chlorobenzoyl methyl-formiate.
Best, R
1For-(CH
2)
2C
6H
5, R
2For-CH
2CH
3, promptly formula 1 is 2-carbonyl-4-phenylbutyrate.
Best, R
3For-CH
2Cl, promptly formula 2 is 4-chloro-3-carbonyl ethyl n-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, and is preferable, and described application comprises the steps: in the aqueous solution of pH 5.5～7.0, at Hexose phosphate dehydrogenase, glucose and NADP
+Existence under, under the catalysis of carbonyl reductase of the present invention or reorganization carbonyl reductase, the prochirality carbonyl compound carries out asymmetric reduction reaction, forms optical activity chirality alcohol.
Preferable, said protein consumption is 5～60kU/L, and the consumption of Hexose phosphate dehydrogenase is 5～60kU/L, and the consumption of glucose is 5～600g/L, NADP
+Consumption be 0～0.5mmol/L, the concentration of said prochirality carbonyl compound is 10mmol/L～2.0mol/L, the aqueous solution of said pH 5.5～7.0 is phosphate buffered saline buffer, temperature of reaction is 20～35 ℃.
The preferred concentration of wherein said prochirality carbonyl compound (except that adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate and 4-chloro-3-carbonyl ethyl n-butyrate) in reaction solution is 10～100mmol/L.Carbonyl reduction enzyme dosage of the present invention is a catalytically effective amount, and that preferable is 5～60kU/L.That the consumption of Hexose phosphate dehydrogenase is preferable is 5～60kU/L.That the consumption of glucose is preferable is 5～600g/L.The NADP of extra adding
+Consumption preferable be 0～1.0mmol/L.The described aqueous solution can be the conventional damping fluid in this area, needs only its pH scope 5.5～7.0, and the preferably phosphoric acid salt buffer is like phosphoric acid-sodium phosphate buffer.That the concentration of phosphate buffered saline buffer is preferable is 0.05～0.1mol/L, and described concentration is meant the total concn of conjugate acid and base in the buffered soln.Described asymmetric reduction reaction preferably carries out under vibration or agitation condition.What the temperature of described asymmetric reduction reaction was preferable is 20～35 ℃.The time of described asymmetric reduction reaction preferable with in the reaction process, the time that production concentration no longer continue to improve is as the criterion.
Asymmetric reduction reaction can extract the chiral alcohol product by this area ordinary method after finishing from reaction solution.For adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate and 4-chloro-3-carbonyl ethyl n-butyrate, its concentration of substrate best result can not reach 1.5mol/L, 2.0mol/L and 2.0mol/L.Reductase enzyme consumption of the present invention is a catalytically effective amount, and what the amount ratio of carbonyl reductase and adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate or 4-chloro-3-carbonyl ethyl n-butyrate was preferable is 10～100kU carbonyl reductase/mol substrate.What the amount ratio of glucose and adjacent chlorobenzoyl methyl-formiate, 2-carbonyl-4-phenylbutyrate or 4-chloro-3-carbonyl ethyl n-butyrate was preferable is 200～300g glucose/mol substrate.What the consumption of Hexose phosphate dehydrogenase was preferable is 10～100kU Hexose phosphate dehydrogenase/mol substrate, the NADP of extra adding
+Consumption preferable be 0～1.0mmol/L.Described damping fluid can be the conventional damping fluid in this area, is 6.0 phosphate buffered saline buffer as long as its pH scope, is preferably selected pH 5.5～7.0, like phosphoric acid-sodium phosphate buffer.That the concentration of phosphate buffered saline buffer is preferable is 0.05～0.1mol/L, and described concentration is meant the total concn of conjugate acid and base in the buffered soln.Described asymmetric reduction reaction preferably carries out under agitation condition.What the temperature of described asymmetric reduction reaction was preferable is 20～35 ℃, more preferably is 25 ℃.The time of described asymmetric reduction reaction preferable with in the reaction process, the time that production concentration no longer improves is as the criterion.Asymmetric reduction reaction can extract the chiral alcohol product by this area ordinary method after finishing from reaction solution.
Among the present invention, carry out if use pure carbonyl reductase, then need adding coenzyme NAD P in the reaction of asymmetric reduction reaction formation chiral alcohol at described carbonyl reduction enzyme catalysis prochirality carbonyl compound
+, if use is carbonyl reductase crude enzyme liquid that from microorganism cells, extracts or the resting cell of expressing carbonyl reductase, then need not add coenzyme, only need utilize the coenzyme that is contained in the cell to get final product.
On the basis that meets this area general knowledge, above-mentioned each optimum condition, but arbitrary combination promptly get each preferred embodiments of the present invention.
Agents useful for same of the present invention and raw material are all commercially available to be got.
Positive progressive effect of the present invention is: the present invention is directed in the research of biocatalysis asymmetric synthesis (R)-2-hydroxy-4-phenyl ethyl n-butyrate of having reported and exist production concentration not high; Need problems such as the expensive coenzyme of extra interpolation; A kind of new carbonyl reductase is provided and has utilized the reorganization reductase enzyme to combine regenerating coenzyme asymmetric reduction 2-carbonyl-4-phenylbutyrate, prepared the method for optical purity (R)-2-hydroxy-4-phenyl ethyl n-butyrate.In catalysis concentration during up to the substrate of 2mol/L (412g/L), the optical purity of product is still up to more than 99%, and do not need the expensive coenzyme of extra interpolation.With respect to other asymmetric reduction preparation method, the production concentration that use the inventive method prepares gained is high, needn't the expensive coenzyme NAD P of extra interpolation
+, and it is high to have the product optical purity, and reaction conditions is gentle, and is environmentally friendly, easy and simple to handle, is easy to the advantage that industry is amplified, and therefore has better industrial application prospect.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 is the pcr amplification electrophoretogram of gene C gKR1, wherein, and 1.DNA Marker (Marker II, sky, Beijing root biochemical technology ltd); The pcr amplification product of 2～3. gene C gKR1.
Fig. 2 is the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) DH5 α/pET28a-CgKR1, wherein, and 1.DNA Marker (Marker II, sky, Beijing root biochemical technology ltd); The bacterium liquid pcr amplification electrophorogram of 2～3. ETEC (E.coli) DH5 α/pET-CgKR1.
Fig. 3 is the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET28a-CgKR1, wherein, and 1.DNA Marker (Marker II, sky, Beijing root biochemical technology ltd); The bacterium liquid pcr amplification electrophorogram of 2～3. ETEC (E.coli) BL21 (DE3)/pET28a-CgKR1.
Fig. 4 is the bacterium liquid pcr amplification electrophorogram of intestinal bacteria (E.coli) BL21 (DE3)/pET28a-CgKR1 F92L/F94V/I99Y/G174A, wherein, and 1.DNA Marker (Marker II, sky, Beijing root biochemical technology ltd); The bacterium liquid pcr amplification electrophorogram of 2～3. ETEC (E.coli) BL21 (DE3)/pET28a-CgKR1 F92L/F94V/I99Y/G174A.
Fig. 5 is the structure synoptic diagram of recombinant expression plasmid pET28a-CgKR1.
Fig. 6 is the polyacrylate hydrogel electrophorogram of reorganization carbonyl reductase CgKR1.
Fig. 7 is the polyacrylate hydrogel electrophorogram of reorganization carbonyl reductase CgKR1F92L/F94V/I99Y/G174A.
Embodiment
The methods analyst that the inventor excavates through genome database some genome sequences, the candidate a collection of sequence that on some information biology, is predicted to be the carbonyl reductase gene.Described method for digging is specially; And have the good biological catalytic performance, make probe from the aminoacid sequence of the carbonyl reductase Gre2p (or being called YOL151w) of Saccharomyces cerevisiae; In ncbi database, carry out the pBLAST search, the carbonyl reductase gene order of selected a collection of prediction.Then these candidate genes are carried out clonal expression respectively, make up recombinant Bacillus coli cells.Through measuring these carbonyl reductases to the activity of the adjacent chlorobenzoyl methyl-formiate of substrate, 2-carbonyl-4-phenylbutyrate or 4-chloro-3-carbonyl ethyl n-butyrate and stereoselectivity etc.; Enzyme to institute's clonal expression carries out comparing repeatedly and screening; The final carbonyl reductase CgKR1 that obtains catalytic performance the best, thus the present invention accomplished.
The present invention obtains on the basis of high reactivity and stereoselectivity enzyme in screening; Enzyme to wild-type has carried out the protein transformation; The amino-acid residue in some sites in the hydrophobic channel in active site and guiding active site is sported other amino-acid residue; With the catalytic performance of further this enzyme of reinforcement, improve the expression of enzyme.
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Material source in the following example is:
Candida glabrata (Candida glabrata) CGMCC 2.234.
Expression plasmid pET28a is available from Shanghai Novagen company.
E.coli DH5 α and E.coli BL21 (DE3) competent cell, 2 * Taq PCR MasterMix, sepharose DNA reclaim test kit available from sky, Beijing root biochemical technology ltd.
The clone of embodiment 1 carbonyl reductase gene
The gene order that is predicted as Candida glabrata (Candida glabrata) carbonyl reductase of including according to Genbank (the NCBI accession number: CAG58832) be foundation, design PCR primer is following:
CgKR1f：5’-
CATATGGCTTCTGATAACAGCAAC-3’；
CgKR1r：5’-
GGATCCTTAATTAGAGTTCTTCTCGGC-3’。
Wherein, the upstream primer underscore partly is a Nde I restriction enzyme site, and the downstream primer underscore partly is a BamH I restriction enzyme site.
Genomic dna with Candida glabrata (Candida glabrata) CGMCC 2.234 is a template, carries out pcr amplification.The PCR system is: 2 * Taq PCR MasterMix, 10 μ l, each 1 μ l (0.3 μ mol/L) of upstream primer and downstream primer, dna profiling 1 μ l (0.1 μ g) and ddH
2O 7 μ l.The pcr amplification step is: (1) 95 ℃, and preparatory sex change 3min; (2) 94 ℃, sex change 1min; (3) 55 ℃ of annealing 30s; (4) 72 ℃ are extended 1min; Repeat 30 times step (2)～(4); (5) 72 ℃ are continued to extend 10min, are cooled to 4 ℃.The PCR product utilizes sepharose DNA to reclaim test kit and reclaims the interval target stripe (Fig. 1) of 900～1200bp through the agarose gel electrophoresis purifying.Obtain the carbonyl reductase full-length gene order of a complete Candida glabrata (Candida glabrata) CGMCC 2.234, through dna sequencing, total length 1059bp, called after CgKR1, SEQ ID No.1 in its base sequence such as the sequence table.
The rite-directed mutagenesis of embodiment 2 CgKR1 genes
The carbonyl reductase full-length gene order (SEQ ID No.1) of the Candida glabrata CGMCC 2.234 of embodiment 1 gained is carried out base mutation.
Design PCR primer is following:
Sudden change V85I:
Upstream primer: 5 '-CAAGGTTATCTTACACACCGCCTCTCC-3 '
Downstream primer: 5 '-CGGTGTGTAAGATAACCTTGATATCCTTGCCATG-3 '
Sudden change F92L:
Upstream primer: 5 '-CCGCCTCTCCACTGCACTTCAACACCACTGACATT-3 '
Downstream primer: 5 '-GTGCAGTGGAGAGGCGGTGTGTAAG-3 '
Sudden change F94V:
Upstream primer: 5 '-CACACCGCCTCTCCATTCCACGTTAACACCACTGA
CATTGAA-3’
Downstream primer: 5 '-TGGAGAGGCGGTGTG-3 '
Sudden change I99Y:
Upstream primer: 5 '-CCACTGACTATGAAAAGGATCTATTGATCCC-3 '
Downstream primer: 5 '-GATCCTTTTCATAGTCAGTGGTGTTGAAGTGG-3 '
Sudden change G174A:
Upstream primer: 5 '-CCAATCAGAGCTTACTGTGGTTCAAAGAAGTTTG-3 '
Downstream primer: 5 '-CCACAGTAAGCTCTGATTGGATCGGATTGAC-3 '
Combinatorial mutagenesis F92L/F94V:
Upstream primer: 5 '-CTCCACTGCACGTGAACACCACTGACATTGAAAAG-3 '
Downstream primer: 5 '-GTGTTCACGTGCAGTGGAGAGGCGGTGTGTAAG-3 '
Carbonyl reductase full-length gene order (SEQ ID No.1) with the Candida glabrata CGMCC 2.234 of embodiment 1 gained is a template, carries out pcr amplification.The PCR system is: 10 * KOD-Plus PCR buffer, 2 μ l, 25mM MgSO
41.2 μ l, 2mM dNTP 2 μ l, KOD-Plus PCR high-fidelity enzyme 0.3 μ l, dna profiling 0.5 μ l (0.1 μ g), ddH
2O 13 μ l pass through pcr amplification first dna fragmentation with each 0.5 μ l (10mmol/L) of downstream primer respectively with CgKR1f (seeing embodiment 1), pass through pcr amplification second dna fragmentation with CgKR1r and each 0.5 μ l (10mmol/L) of upstream primer.The pcr amplification step is: (1) 95 ℃, and preparatory sex change 3min; (2) 98 ℃, sex change 15s; (3) 55 ℃ of annealing 30s; (4) 72 ℃ are extended 1min; Repeat 30 times step (2)～(4); (5) 72 ℃ are continued to extend 10min, are cooled to 4 ℃.The PCR product utilizes sepharose DNA to reclaim test kit and reclaims target stripe through the agarose gel electrophoresis purifying.
Be template then, carry out pcr amplification with above-mentioned gained first dna fragmentation and second dna fragmentation.The PCR system is: 10 * KOD-Plus PCR buffer, 5 μ l, 25mM MgSO
43 μ l, 2mM dNTP 5 μ l, KOD-Plus PCR high-fidelity enzyme 1 μ l is as first dna fragmentation and each 1 μ l (0.1 μ g) of second dna fragmentation of dna profiling, ddH
2O 31 μ l, each 1.5 μ l (10mmol/L) of CgKR1f and CgKR1r.The pcr amplification step is: (1) 95 ℃, and preparatory sex change 3min; (2) 98 ℃, sex change 15s; (3) 55 ℃ of annealing 30s; (4) 72 ℃ are extended 1min; Repeat 30 times step (2)～(4); (5) 72 ℃ are continued to extend 10min, are cooled to 4 ℃.The PCR product utilizes sepharose DNA to reclaim test kit and reclaims the target stripe about 1.0～1.2kbp through the agarose gel electrophoresis purifying.
Wherein said F92L/F94V/I99Y/G174A sudden change building process is: be template with SEQ ID No.1 earlier; PCR primer with combinatorial mutagenesis F92L/F94V makes up F92L/F94V; Then sport template with this; PCR primer with sudden change I99Y makes up F92L/F94V/I99Y, sports template with this at last, uses the PCR primer of sudden change G174A to be built into F92L/F94V/I99Y/G174A.
Correct through dna sequencing, the gained target stripe is the gene of mutant protein of the present invention, and its title is respectively:
CgKR1 V85I, the Xie Ansuan that aminoacid sequence SEQ ID No.2 is the 85th is substituted by Isoleucine;
CgKR1 F92L, the phenylalanine(Phe) that aminoacid sequence SEQ ID No.2 is the 92nd is substituted by leucine;
CgKR1 F94V, the phenylalanine(Phe) that aminoacid sequence SEQ ID No.2 is the 94th is substituted by Xie Ansuan;
CgKR1 F92L/F94V, the phenylalanine(Phe) that aminoacid sequence SEQ ID No.2 is the 92nd is substituted by leucine, and the 94th phenylalanine(Phe) is substituted by Xie Ansuan simultaneously;
CgKR1 I99Y, the Isoleucine that aminoacid sequence SEQ ID No.2 is the 99th is substituted by tyrosine;
CgKR1 G174A, the glycocoll that aminoacid sequence SEQ ID No.2 is the 174th is substituted by L-Ala;
CgKR1 F92L/F94V/I99Y/G174A; The phenylalanine(Phe) that aminoacid sequence SEQ ID No.2 is the 92nd is substituted by leucine; The 94th phenylalanine(Phe) is substituted by Xie Ansuan, and the 99th for Isoleucine is substituted by tyrosine, and the 174th glycocoll is substituted by L-Ala.
The structure of embodiment 3 recombinant expression vectors (plasmid) and the preparation of recombinant expressed transformant
With the carbonyl reductase gene DNA fragment of gained in embodiment 1 or 2 37 ℃ with restriction enzyme NdeI and BamHI double digestion 12h, through the agarose gel electrophoresis purifying, utilize sepharose DNA to reclaim test kit and reclaim target fragment.With target fragment under the effect of T4DNA ligase enzyme, with the same plasmid pET28a after NdeI cuts with the BamHI enzyme, 4 ℃ down connection spend the night and obtain recombinant expression plasmid pET28a-CgKR1 and two mutants thereof, the plasmid construction collection of illustrative plates is as shown in Figure 5.
Above-mentioned recombinant expression plasmid is transformed in ETEC (E.coli) the DH5 α competent cell; 45 ℃ of conversion conditions, thermal shock 90 seconds screens positive recombinant chou containing on the resistant panel of kantlex; The picking mono-clonal, bacterium colony PCR verifies positive colony (Fig. 2).Cultivate the reorganization bacterium; Treat to extract plasmid behind the plasmid amplification; Again be converted in ETEC (E.coli) BL21 (DE3) competent cell; Conversion fluid is applied on the LB flat board that contains kantlex, is inverted overnight cultures, promptly obtains positive recombinant conversion body ETEC (E.coli) BL21 (DE3)/pET28a-CgKR1 and two mutants thereof for 37 ℃; Bacterium colony PCR verifies positive colony (Fig. 3), the bacterium liquid pcr amplification electrophorogram (Fig. 4) of BL21 (DE3)/pET28a-CgKR1 F92L/F94V/I99Y/G174A.
The expression of embodiment 4 reorganization carbonyl reductases and enzymic activity and stability are measured
With the recombination bacillus coli of embodiment 3 gained, be seeded to the LB substratum (peptone 10g/L, the yeast extract paste 5g/L that contain kantlex; NaCl 10g/L; PH 7.0) in, 37 ℃ of shaking culture are spent the night, and are equipped with in the 500ml triangular flask of 100ml LB substratum by the inoculum size access of 1% (v/v); Put 37 ℃, 180rpm shaking table jolting cultivation, as the OD of nutrient solution
600Reach at 0.6 o'clock, the IPTG that adds final concentration and be 0.5mmol/L is as inductor, 25 ℃ induce 12h after, with medium centrifugal, collecting cell, and use the saline water washed twice must resting cell.The resting cell of gained is suspended in the damping fluid of pH 7.0, ultrasonication in ice bath, centrifugal collection supernatant, be the reorganization carbonyl reductase crude enzyme liquid.Through the polyacrylamide gel electrophoresis map analysis, wherein the polyacrylate hydrogel electrophorogram of CgKR1 is shown in Figure 6 to crude enzyme liquid with deposition, and the polyacrylate hydrogel electrophorogram of reorganization carbonyl reductase CgKR1 F92L/F94V/I99Y/G174A is as shown in Figure 7.
Recombinant protein exists with partly soluble form.The soluble protein content method of calculation: the reorganization carbonyl reductase in supernatant among the polyacrylamide gel electrophoresis figure and the deposition carries out integration, and the per-cent of shared both summations of reorganization carbonyl reductase in the supernatant is soluble protein content.The solubility of reorganization carbonyl reductase CgKR1 and CgKR1 F92L/F94V/I99Y/G174A is seen table 2.Crude enzyme liquid with freezing Freeze Drying Equipment freeze-drying, is the thick enzyme powder of freeze-drying.
The vitality test of carbonyl reductase: the mode through detecting the light absorption value variation of 340nm place is measured.Concrete grammar is following: in 1ml reaction system (100mmol/L sodium phosphate buffer; PH 6.0) in; Adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 2mmol/L substrate, and final concentration is the NADPH of 0.1mmol/L, and 30 ℃ of insulations add the crude enzyme liquid after an amount of cytoclasis after 2 minutes; Mixing detects the variation of 340nm place light absorption value rapidly.The calculation formula of enzyme activity is: enzyme activity (U)=EW * V * 10
3/ (6220 * l); In the formula, EW is the changing value of 340nm place absorbancy in the 1min; V is the volume of reaction solution, Unit; 6220 is the molar extinction coefficient of NADPH, the L/ of unit (molcm); L is an optical path length, the cm of unit.Being defined as under these conditions of per unit (U) carbonyl reductase, the required enzyme amount of PM catalysis 1 μ mol NADPH oxidation.The enzyme activity of reorganization carbonyl reductase CgKR1 and mutant strain thereof is as shown in table 2.
The carbonyl reductase 4stability determination: according to document Angew.Chem.Int.Ed., 2006,27,7745-7751 measures the T of carbonyl reductase enzyme
50Value.The T of reorganization carbonyl reductase CgKR1 and mutant strain thereof
50Be worth as shown in table 2.
The Hexose phosphate dehydrogenase vigour-testing method is following: in 1ml reaction system (100mmol/L sodium phosphate buffer, pH 7.0), add 10mmol/L glucose, 1mmol/L NADP
+, 30 ℃ of insulations add an amount of enzyme liquid after 2 minutes, and mixing detects the variation of 340nm place light absorption value rapidly.Being defined as under these conditions of per unit (U) Hexose phosphate dehydrogenase, PM catalysis 1 μ mol NADP
+Reduce required enzyme amount.
Table 2.CgKR1 and mutant strain character thereof
The asymmetric reduction of embodiment 5～17 reorganization reductase enzyme CgKR1 catalysis of carbonyl compounds
At 0.4ml sodium phosphate buffer (100mmol/L; PH 7.0) in add the crude enzyme liquid CgKR1 of 2U embodiment 4 preparations and the Hexose phosphate dehydrogenase crude enzyme liquid of 2U (preparation method be referring to Journal of Industrial Microbiology and Biotechnology 2011; 38; 633-641), add ketone ester or aryl ketones (embodiment 5～17) that final concentration is 10mmol/L respectively, and final concentration is the NADP of 0.5mmol/L
+Glucose with 5g/L.At 30 ℃, the 1100rpm oscillatory reaction, the reaction times is 24 hours, this moment, the concentration of reaction product all no longer continued to increase.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE, extracted 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 result sees table 3.
The concrete analysis condition of product yield and ee value is following:
Embodiment 5 and 10～17 uses gas chromatograph to analyze transformation efficiency and ee value, and chromatographic column is chiral capillary column CP-Chirasil-DEX CB, is carrier gas with nitrogen, 280 ℃ of injector temperatures, and 280 ℃ of detector temperatures, other conditions are following:
Embodiment 5: 80 ℃ of column temperatures;
Embodiment 10: after 90 ℃ of initial column temperatures are kept 2min, be warming up to 120 ℃ with the speed of 1 ℃/min, keep 5min;
Embodiment 11 and 12: analyze after the reaction product acetylize, 110 ℃ of initial column temperatures, keep 2min after, rise to 126 ℃ with the speed of 2 ℃/min, keep 2min;
Embodiment 13: 120 ℃ of column temperatures;
Embodiment 14: 130 ℃ of column temperatures;
Embodiment 15 and 16: 140 ℃ of column temperatures;
Embodiment 17: 160 ℃ of column temperatures;
Embodiment 6～9 uses gas chromatograph to analyze transformation efficiency, and chromatographic column is chiral capillary column CP-Chirasil-DEX CB, is carrier gas with nitrogen, 280 ℃ of injector temperatures, 280 ℃ of detector temperatures, 180 ℃ of column temperatures;
Embodiment 6 and 7 uses liquid-phase chromatographic analysis ee value, chirality OD-H post, moving phase: normal hexane/Virahol/trifluoroacetic acid=94/6/0.2, flow velocity 1ml/min, detector wavelength 228nm.
Embodiment 8 and 9 uses liquid-phase chromatographic analysis ee value, chirality OD-H post, moving phase: normal hexane/Virahol=97/3, flow velocity 1ml/min, detector wavelength 254nm.
The result of table 3.CgKR1 catalysis of carbonyl compound asymmetric reduction reaction
The asymmetric reduction of the adjacent chlorobenzoyl methyl-formiate of embodiment 18 CgKR1 catalysis
At 100ml sodium phosphate buffer (100mmol/L; PH 5.5) in add the CgKR1 crude enzyme liquid and the Hexose phosphate dehydrogenase crude enzyme liquid of 4000U of embodiment 4 preparations of 4000U, the adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 0.75mol/L and the glucose of 220g/L.Be reflected under 20 ℃ and carry out, reaction solution pH is controlled to be 5.5, and reaction is till react completely, and promptly the concentration of reaction product no longer continues to increase, and this reaction time is 12 hours.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE, extracted twice, and combining extraction liquid adds anhydrous sodium sulfate drying and spends the night, and rotary evaporation removes and desolvates, and underpressure distillation obtains 1.16g (R)-o-Chloromelic acid methyl esters, yield 76%, the ee value of product is 98.0%.
The asymmetric reduction of the adjacent chlorobenzoyl methyl-formiate of embodiment 19 CgKR1 catalysis
At 100ml sodium phosphate buffer (100mmol/L; PH 6.0) in add the CgKR1 crude enzyme liquid and the Hexose phosphate dehydrogenase crude enzyme liquid of 6000U of embodiment 4 preparations of 6000U, the adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 1.5mol/L and the glucose of 450g/L.Be reflected under 25 ℃ and carry out, reaction solution pH is controlled to be 6.0, and reaction is till react completely, and promptly the concentration of reaction product no longer continues to increase, and this reaction time is 6 hours.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE, extracted twice, and combining extraction liquid adds anhydrous sodium sulfate drying and spends the night, and rotary evaporation removes and desolvates, and underpressure distillation obtains 2.61g (R)-o-Chloromelic acid methyl esters, yield 87%, the ee value of product is 98.5%.
The asymmetric reduction of embodiment 20 CgKR1 catalysis 2-carbonyl-4-phenylbutyrates
At 100ml sodium phosphate buffer (100mmol/L; PH 6.0) in add the CgKR1 crude enzyme liquid and the Hexose phosphate dehydrogenase crude enzyme liquid of 6000U of embodiment 4 preparations of 6000U, the adding final concentration is the 2-carbonyl-4-phenylbutyrate of 2.0mol/L and the glucose of 600g/L.Be reflected under 25 ℃ and carry out, reaction solution pH is controlled to be 6.0, and reaction is till react completely, and promptly the concentration of reaction product no longer continues to increase, and this reaction time is 12 hours.Reaction finishes the back and extracts extracted twice, combining extraction liquid with equal-volume ETHYLE ACETATE; Add anhydrous sodium sulfate drying and spend the night, rotary evaporation removes and desolvates, and underpressure distillation obtains 3.67g (R)-2-hydroxy-4-phenyl ethyl n-butyrate; Yield 89%, the ee value of product is 98%.
The asymmetric reduction of embodiment 21 CgKR1 catalysis 4-chloro-3-carbonyl ethyl n-butyrates
At 100ml sodium phosphate buffer (100mmol/L; PH 6.0) in add the CgKR1 crude enzyme liquid and the Hexose phosphate dehydrogenase crude enzyme liquid of 6000U of embodiment 4 preparations of 6000U, the adding final concentration is the 4-chloro-3-carbonyl ethyl n-butyrate of 2.0mol/L and the glucose of 600g/L.Be reflected under 25 ℃ and carry out, reaction solution pH is controlled to be 6.0, and reaction is till react completely, and promptly the concentration of reaction product no longer continues to increase, and this reaction time is 4 hours.Reaction finishes the back and extracts extracted twice, combining extraction liquid with equal-volume ETHYLE ACETATE; Add anhydrous sodium sulfate drying and spend the night, rotary evaporation removes and desolvates, and underpressure distillation obtains 3.0g (R)-4-chloro-ethyl 3-hydroxybutanoate; Yield 91%, the ee value of product is 97%.
The asymmetric reduction of the adjacent chlorobenzoyl methyl-formiate of embodiment 22～31 CgKR1 and two mutants catalysis thereof
According to the mutant strain of embodiment 2 structure CgKR1, according to embodiment 3 preparation recombinant expression vector (plasmid) and recombinant expressed transformant, according to the thick enzyme powder of embodiment 4 preparations.
At 100ml sodium phosphate buffer (100mmol/L; PH 6.0) in add CgKR1 and the thick enzyme powder and the thick enzyme powder of 4000U Hexose phosphate dehydrogenase of mutant strain thereof of the foregoing description 4 preparation gained of 4000U; Adding final concentration is the adjacent chlorobenzoyl methyl-formiate of 100g/L, the glucose of 150g/L and the NADP of 0.1mmol/L
+Be reflected under 25～35 ℃ and carry out, reaction solution pH is controlled to be 6.0, reacts to 24h, and this moment, the concentration of embodiment 21～31 reaction product all no longer continued to increase.According to embodiment 8 assaying reaction transformation efficiencys and ee value, the result is as shown in table 4.
The asymmetric reduction result of the adjacent chlorobenzoyl methyl-formiate of table 4.CgKR1 and two mutants catalysis thereof
Should be understood that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. isolating protein is characterized in that: it is (a) or protein (b) as follows:
(a) by aminoacid sequence is formed shown in the SEQ ID No.2 in the sequence table protein;
(b) in the aminoacid sequence of (a) through replacing, lack or adding one or several amino acid and have a carbonyl reductase active by (a) deutero-and the protein identical with the aminoacid sequence of (a) at least 62%.
2. protein as claimed in claim 1; It is characterized in that it is active by (a) deutero-protein through replacing an amino acid and having a carbonyl reductase that described (b) protein is in the 85th, the 92nd, the 94th, the 99th of the aminoacid sequence of (a) and the 174th amino acids one or multidigit.
3. isolating nucleic acid is characterized in that: it is the nucleic acid of (1) or (2) as follows:
(1) by nucleotide sequence is formed shown in the SEQ ID No.1 in the sequence table nucleic acid;
(2) encode following protein (a) or gene (b):
(a) protein of forming by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table;
(b) in the aminoacid sequence of (a) through replacing, lack or adding one or several amino acid and have a carbonyl reductase active by (a) deutero-and the protein identical with the aminoacid sequence of (a) at least 62%.
4. recombinant expression vector that comprises nucleic acid as claimed in claim 3.
5. recombinant expressed transformant that comprises recombinant expression vector as claimed in claim 4.
6. the preparation method of the carbonyl reductase of recombinating is characterized in that, comprises the steps: to cultivate recombinant expressed transformant as claimed in claim 5, from culture, obtains the reorganization carbonyl reductase.
7. one kind is carried out asymmetric reduction reaction like each described protein of claim 1～2 at catalysis prochirality carbonyl compound and forms the application in the chiral alcohol.
8. application as claimed in claim 7 is characterized in that, described application comprises following method: in the aqueous solution of pH 5.5～7.0, at Hexose phosphate dehydrogenase, glucose and NADP
+Existence under, under like each described proteinic catalysis of claim 1～2, the prochirality carbonyl compound carries out asymmetric reduction reaction, forms optical activity chirality alcohol.
9. application as claimed in claim 8 is characterized in that, said protein consumption is 5～60kU/L, and the consumption of Hexose phosphate dehydrogenase is 5～60kU/L, and the consumption of glucose is 5～600g/L, NADP
+Consumption be 0～0.5mmol/L, the concentration of said prochirality carbonyl compound is 10mmol/L～2.0mol/L, the aqueous solution of said pH 5.5～7.0 is phosphate buffered saline buffer, temperature of reaction is 20～35 ℃.
10. like each described application of claim 7～8, it is characterized in that described prochirality carbonyl compound is suc as formula the compound shown in 1,2 or 3:
Wherein, R
1For alkyl, phenyl or have substituent phenyl, the substituting group of phenyl is halogen or alkyl;
R
2Be alkyl;
R
3Be alkyl or haloalkyl;
R
4Be haloalkyl.
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| CN113528588A (en) * | 2021-06-15 | 2021-10-22 | 海南卓科制药有限公司 | Preparation method of levocarnitine |
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