CN105018440A - Aminotransferase and application thereof to synthesis of sitagliptin intermediate - Google Patents
Aminotransferase and application thereof to synthesis of sitagliptin intermediate Download PDFInfo
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Abstract
The invention provides a new aminotransferase, a gene of the new aminotransferase, a recombinant expression vector containing the gene, a recombinant expression transformant, a recombinase, a preparation method of the recombinase, and application of the aminotransferase to preparation of active chiral amine by performing asymmetrical transamination on a carbonyl compound. The aminotransferase is derived from mycobacterium (mycobacterium vanbaalenii) PYR-1, and is applied to preparation of (R)-3-amino-4-(2, 4, 5-trifluorophenyl)-methyl butyrate. Compared with other preparation methods, the preparation methods provided by the present invention have the advantages that catalyzed and prepared products are high in concentration, enantioselectivity is high, reaction conditions are mild, operations are simple and convenient, enlargement is easy and the like, so that the industrial application prospect in production of sitagliptin phosphate is excellent.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of transaminase, recombinant expression vector containing this enzyme coding gene and recombinant expressed transformant, the recombinase of expressing and the preparation method of this recombinase, and this transaminase is as the application of catalyzer in asymmetric synthesis sitagliptin intermediate.
Background technology
Diabetes are because insulin secretion changes, and cause insulin deficit and declines, or insulin activity reduces, or under both joint effects, the metabolic disease occurred take hyperglycemia as feature, and simultaneously with protein, sugar and fatty metabolism disorder.Diabetes and complication thereof, to the 3rd after the hazard rating intentions vascular disease of human health, tumour, become the important diseases of harm humans health.International Diabetes Federation estimates, to the year two thousand thirty, ill total number of persons will more than 4.35 hundred million people.And China has become one of the fastest country of world's diabetes prevalence rate of growth, about have 40,000,000 diabetic subjects at present, number has been only second to India, occupies second place of the world.In the Four types of diabetes, type ii diabetes accounts for more than 90%, is more common in the elderly of more than 30 years old, and the cause of disease is mainly because body is to insulin insensitivity.Treat clinically in the medicine of type ii diabetes, although the blood sugar reducing functions such as sulfonylurea insulin secernent, biguanides insulin sensitivity enhancer, alpha-glucosidase inhibitor, Study of Thiazolidinedione derivatives as Insulin Sensitizer, non-sulfonylurea insulin secretion stimulators are ideal, but all there is certain side effects patient to adhere to medication, do not reach good glycemic control effect.Sitagliptin phosphate (Sitagliptin phosphate) is first two peptidyl enzyme-IV (DPP-4) inhibitor of FDA approval listing in 2006, be used for the treatment of type ii diabetes, it is alone or share obvious hypoglycemic activity with N1,N1-Dimethylbiguanide, pioglitazone, and take safety, better tolerance, untoward reaction is few.It is mainly through suppressing DPP-4 to the degraded of glucagon-like-peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) to reach the object for the treatment of, slight increase GLP-1 content also weakens the antagonistic action of GLP-1 metabolite, so can not cause the side effects such as the nausea and vomiting caused because of GLP-1 too high levels while effectively playing blood sugar reducing function; Meanwhile, GIP promotes that insulin secretion has blood sugar dependency, therefore greatly can reduce the incidence of the hypoglycemia side effect that oral antidiabetic drug causes.Sitagliptin phosphate is developed by Merck & Co., Inc., in in August, 2006 and the medicine being approved as treatment type ii diabetes October respectively by the Ministry of Health of Mexico and U.S. FDA, commodity are called Jie Nuowei (Januvia), in the whole world, more than 60 state approval uses at present, 2012 annual sales amounts have reached 40.86 hundred million dollars, increase by 23% on a year-on-year basis.Therefore, Sitagliptin phosphate belongs to international up-to-date and added value is high " cookle ".
Committed step in sitagliptin synthesis is the structure of chiral amino intermediate.US Patent No. 6699871 discloses a kind of synthetic method of sitagliptin, adopts chiral source to induce the a-amino acid of chirality, then produces beta-amino acids by diazotization reaction and builds required chiral centre.Raw materials cost needed for this route is relatively high, and reaction comparatively bothers, and in industrialization process, technological process and quality product are all difficult to control.
International monopoly WO2004087650 discloses the synthetic route of Merck & Co., Inc. about sitagliptin, utilizes chiral ruthenium catalyst to carry out asymmetric hydrogenation to ketone and builds chiral alcohol, then chiral alcohol is changed into Chiral Amine.In this synthetic method, need the asymmetric hydrogenation using ruthenium catalysis, catalyzer is expensive, and total recovery only has 52%, uses high pressure hydrogen in technique, and stereoselectivity is not high yet.
International monopoly WO2005003135 discloses the synthetic method of sitagliptin of Merck & Co., Inc.'s exploitation, comes inducing catalysis hydrogenation and synthesis of chiral amine using S-benzene G-NH2 as chiral auxiliary(reagent).This route needs twice catalytic hydrogenation, and platinum catalyst used is for the first time expensive, needs with a large amount of Pd (OH) during second time deprotection
2-C catalyst, cost is higher, and ee value is 96%, needs further recrystallization.
International monopoly WO2007050485 discloses the synthetic method of Merck & Co., Inc. about sitagliptin, have employed the asymmetric hydrogenation of chirality rhodium catalyst to enamine and carry out synthesis of chiral amine, productive rate reaches 84%, ee value 94%, but the rhodium chiral catalyst that the method needs are expensive, removes and reclaims also comparatively difficulty.
US Patent No. 8293507 discloses Codexis company replaces in above-mentioned technique rhodium catalyst to the biological catalyst that the transaminase transformation that Arthrobacter is originated obtains, turns aminate ee value and reaches 99%, and substrate feeds intake 100g/L.But due to substrate poorly water-soluble, need to add the DMSO hydrotropy up to 50%, make the reprocessing loss of product comparatively large, the solvent residues of DMSO is higher, and reclaim difficulty, cost is higher.
Chinese patent CN102838511 discloses the production method of sea, Zhejiang Xiang medicine company about sitagliptin intermediate, the chiral epoxy chloropropane of grignard reagent is adopted to carry out nucleophilic substitution, then carry out replacement hydrolysis beta-hydroxy acid with prussiate, the method total recovery only 40%, and owing to adopting hypertoxic prussiate, apply limited.
Chinese patent CN102485718 discloses the route that sea, Zhejiang Xiang medicine company synthesize about sitagliptin, is synthesized as chiral source by employing methionine(Met), but productive rate only 14%, even if with 2,4,5-trifluorobromobenzene meter also only 55%.
Chinese patent CN103014081 discloses Suzhou Han Mei company and utilizes transaminase by 3-carbonyl-4-(2,4,5-trifluorophenyl)-methyl-butyrate is converted into R-3-amino-4-(2,4,5-trifluorophenyl)-methyl-butyrate, but sequence and the cloning process of concrete transaminase are not disclosed.
Summary of the invention
For in the reaction of the asymmetric synthesis sitagliptin reported and intermediate thereof, productive rate is on the low side, stereoselectivity is not good, catalyzer is expensive, solvent is difficult to the problems such as recovery, the invention provides that a kind of catalytic activity is high, enantioselectivity is strong, substrate tolerance and the good transaminase of solvent tolerance carry out Enzyme catalyzed synthesis R-3-amino-4-(2,4,5-trifluorophenyl)-methyl-butyrate, and then synthesize the enzyme-chemically synthetic method of sitagliptin further.Additionally provide the gene of this transaminase, the recombinant expression vector containing this gene, recombinant expressed transformant and high efficiency preparation method thereof, and this transaminase is in the asymmetric purposes turned in ammonia synthesis sitagliptin intermediate of catalysis of carbonyl substrate.
The present invention by following technical proposals to solve the problems of the technologies described above:
A first aspect of the present invention provides a kind of protein of separation, and it is the protein of following (a) or (b):
A protein that () is made up of aminoacid sequence shown in SEQ ID No:2,4,6,8 or 10;
(b) in the aminoacid sequence of (a) through replacement, lack or add one or several amino-acid residue and have transaminase activity by (a) derivative and and the aminoacid sequence of (a) there is the protein of at least 90% identity, those particularly hereinafter mentioned aminoacid replacement.
The protein source of the aminoacid sequence composition shown in SEQ ID No:2 is in mycobacterium (Mycobacterium vanbaalenii) PYR-1, and having the function of transaminase, is a kind of new transaminase.
The protein of the aminoacid sequence composition shown in SEQ ID No:2 can be separated and obtain from mycobacterium PYR-1, also can be separated from this protein expression transformant recombinant expressed and obtain, and also can obtain by synthetic.
Article two, the identity between aminoacid sequence or two nucleotide sequences all obtains by the algorithm that this area is conventional, preferably adopts NCBI Blastp and Blastn software to calculate according to default parameters.
Amino acid sequence identity between the ATA117 in the transaminase shown in SEQ ID No:2 and Arthrobacter source is 52%.In the present invention, the identity of the aminoacid sequence of the transaminase of aminoacid sequence as shown in SEQ ID No:2 and known transaminase is lower than 70%, has the significance difference opposite sex.
Protein (b) be in the aminoacid sequence of (a) through replacement, lack or add one or several amino acid and have transaminase activity by (a) derivative and and the aminoacid sequence of (a) there is the protein of at least 90% identity.Wherein, described " several " refer to that 2 are more preferably less than 30 to being less than 100, are less than 10 best.Such as add the fusion rotein of an external secretion signal peptide, the present invention finds that such fusion rotein has transaminase activity equally.That is, as long as have transaminase activity by the protein that (a) is derivative, and deriving mode is described above, can reach goal of the invention of the present invention.According to the present invention, the sudden change carrying out 4,10,15,20 amino-acid residues in protein (a) molecule of such as aminoacid sequence shown in SEQ IDNo:2 obtains the protein of aminoacid sequence shown in SEQ ID No:4,6,8,10, also belongs to protein (b); Or SEQ ID No:2,4,6,8,10 is combined, or carry out the sudden change of 1-20 amino-acid residue, also can obtain above-mentioned protein (b), but still keep transaminase activity.
Obtain in screening on the basis of wild-type transaminase shown in SEQ ID No:2, contriver also transforms wild-type enzyme, the amino-acid residue in some sites, active centre is sported other amino-acid residue, to strengthen the catalytic performance of this enzyme further.Described active centre is defined as near substrate binding site about
diameter of Spherical Volume.
Preferably, (b) protein is one or more through replacing and having the protein derivative by (a) of transaminase activity in the 56th, 68,69,72,76,96,129,131,143,157,206,216,222,230,243,276,289,291,295,304 amino acids residues of aminoacid sequence at (a).
In a specific embodiment, the leucine of the 72nd of the aminoacid sequence described in SEQ ID No:2 is substituted by halfcystine, the α-amino-isovaleric acid of the 76th is substituted by L-Ala, the phenylalanine of the 129th is substituted by α-amino-isovaleric acid, and the L-Ala of the 291st be substituted by glycine gained SEQ ID No:4 shown in the protein of aminoacid sequence composition to substrate, there is certain activity.
In a specific embodiment, the Histidine of the 69th of the aminoacid sequence described in SEQ ID No:2 is substituted by leucine, the leucine of the 72nd is substituted by halfcystine, the α-amino-isovaleric acid of the 76th is substituted by L-Ala, the phenylalanine of the 129th is substituted by α-amino-isovaleric acid, the glycine of the 143rd is substituted by Isoleucine, the Serine of the 206th is substituted by α-amino-isovaleric acid, the Threonine of the 216th is substituted by Isoleucine, the proline(Pro) of the 230th is substituted by L-Ala, the Threonine of the 289th is substituted by L-Ala, and the L-Ala of the 291st be substituted by glycine gained SEQ ID No:6 shown in the activity of protein to substrate of aminoacid sequence composition be greatly improved.
In a specific embodiment, the L-glutamic acid of the 56th of the aminoacid sequence described in SEQ ID No:2 is substituted by Serine, the Histidine of the 69th is substituted by leucine, the leucine of the 72nd is substituted by halfcystine, the α-amino-isovaleric acid of the 76th is substituted by L-Ala, the phenylalanine of the 129th is substituted by α-amino-isovaleric acid, the glycine of the 143rd is substituted by Isoleucine, the Serine of the 206th is substituted by α-amino-isovaleric acid, the Threonine of the 216th is substituted by Isoleucine, the proline(Pro) of the 230th is substituted by L-Ala, the L-Ala of the 243rd is substituted by arginine, the Serine of the 276th is substituted by proline(Pro), the Threonine of the 289th is substituted by L-Ala, the L-Ala of the 291st is substituted by glycine, the Threonine of the 295th is substituted by tryptophane, and the proline(Pro) of the 304th be substituted by Threonine gained SEQ ID No:8 shown in the protein of aminoacid sequence composition to substrate, there is very high activity, thermostability there has also been very large raising.
In a specific embodiment, the L-glutamic acid of the 56th of the aminoacid sequence described in SEQ ID No:2 is substituted by Serine, the glycine of the 68th is substituted by tyrosine, the Histidine of the 69th is substituted by leucine, the leucine of the 72nd is substituted by halfcystine, the α-amino-isovaleric acid of the 76th is substituted by L-Ala, the aspartic acid of the 96th is substituted by L-Ala, the phenylalanine of the 129th is substituted by α-amino-isovaleric acid, the l-asparagine of the 131st is substituted by Threonine, the glycine of the 143rd is substituted by Isoleucine, the tyrosine of the 157th is substituted by Threonine, the Serine of the 206th is substituted by α-amino-isovaleric acid, the Threonine of the 216th is substituted by Isoleucine, the L-Ala of the 222nd is substituted by halfcystine, the proline(Pro) of the 230th is substituted by L-Ala, the L-Ala of the 243rd is substituted by arginine, the Serine of the 276th is substituted by proline(Pro), the Threonine of the 289th is substituted by L-Ala, the L-Ala of the 291st is substituted by glycine, the Threonine of the 295th is substituted by tryptophane, and the proline(Pro) of the 304th be substituted by Threonine gained SEQ ID No:10 shown in the protein of aminoacid sequence composition to substrate, there is very high activity, thermostability and solvent stability are all greatly improved.
A second aspect of the present invention provides a kind of nucleic acid of separation, and it is the nucleic acid of following (1) or (2):
(1) nucleic acid be made up of nucleotide sequence shown in SEQ ID No:1,3,5,7 or 9;
(2) to encode the nucleic acid of following protein (a) or (b):
A protein that () is made up of aminoacid sequence shown in SEQ ID No:2,4,6,8 or 10;
(b) in the aminoacid sequence of (a) through replacement, lack or add one or several amino-acid residue and have transaminase activity by (a) derivative and and the aminoacid sequence of (a) there is the protein of at least 90% identity.
The nucleic acid source of the nucleotide sequence composition shown in SEQ ID No:1 is in mycobacterium PYR-1, it can be separated and obtain from mycobacterium PYR-1 genome, also can obtain from containing being separated the recombinant expression vector of this nucleic acid or in recombinant conversion body, also can obtain by full genome synthetic.
In the present invention, SEQ ID No:1,3,5, the unnamed gene shown in 7,9 is MvAT, total length 1014bp.Wherein, its encoding sequence (CDS) stops to the 1011st base from the 1st base, and initiator codon is ATG, and terminator codon is TGA.This sequence intronless, the aminoacid sequence of the protein of its coding is respectively as shown in SEQ ID No:2,4,6,8,10.
As is known to the person skilled in the art, due to the degeneracy of codon, coding SEQ ID No:2,4,6,8, the nucleotide sequence of the aminoacid sequence of 10 is not only confined to SEQ ID No:1,3,5,7,9.The nucleotide sequence of aminotransferase gene of the present invention also can be other any nucleotide sequences of aminoacid sequence shown in SEQ ID No:2 in polynucleotide, 4,6,8,10.In addition, can also by suitably introducing replacement, disappearance or inserting the homologue providing a polynucleotide.In the present invention polynucleotide homologue can by nucleic acid sequence SEQ ID No:1,3,5,7, one or more bases of 9 replace, lack or add and obtain within the scope of maintenance enzymic activity.
SEQ ID No:1,3,5,7, the homologue of 9 also refers to promoter variants.Promotor before described nucleotide sequence or signal sequence change by the replacement of one or more nucleic acid, insertion or disappearance, but these changes do not have negative impact to the function of promotor.And pass through the sequence of change promotor or even replace completely by the more effective promotor from difference kind organism, the expression level of target protein can be improved.
SEQ ID No:1,3,5,7, the homologue of 9 also refer to a kind of have can carry out the Polynucleotide of the base sequence of hybridizing with the Polynucleotide of sequence shown in SEQID No:1,3,5,7,9 at the standard conditions.Carry out hybridization at the standard conditions to carry out according to the mode described in such as " Molecular Cloning: A Laboratory guide ": Cold Spring Harbor Laboratory Press, the general scheme (Current Protocolsin Molecular Biology) in molecular biology.Specifically, hybridization can be carried out in accordance with the following steps, and the film and the label probe that one are loaded with transcribed DNA to be measured or RNA molecule are hybridized in hybridization buffer.Hybridization buffer consist of 0.1wt%SDS, 5wt% dextran glucosides, the dilution inhibitor of a box 1/20 and 2 ~ 8 × SSC.20 × SSC is the solution of the citric acid composition of 3M sodium-chlor and 0.3M.Hybridization temperature is 50 ~ 70 DEG C.Cultivation several hours or after spending the night, clean film with cleaning buffer solution.Cleaning temperature is room temperature, is more preferably hybridization temperature.Cleaning buffer solution consist of 6 × SSC+0.1wt%SDS solution, be more preferably 5 × SSC+0.1wt%SDS.After with this cleaning buffer solution cleaning film, just can identify this DNA or RNA molecule by the mark on the probe of hybridizing with DNA or RNA molecule.
A third aspect of the present invention provides a kind of recombinant expression vector comprising nucleotide sequence of the present invention.Aminotransferase gene of the present invention is connected to structure on various expression vector by this area ordinary method and forms by it.Described expression vector can be the various carriers of this area routine, as commercially available plasmid, clay, phage or virus vector etc., and preferred plasmid pET21a.Preferably, recombinant expression vector of the present invention is obtained: use restriction enzyme Nde I and EcoR I double digestion respectively by by the nucleic acid product of pcr amplification gained and expression vector pET21a or its mutant by following method, form complementary sticky end, connect through T4DNA ligase enzyme, form the recombinant expression plasmid pET21a-MvAT expression plasmid containing aminotransferase gene of the present invention.
A fourth aspect of the present invention provides a kind of recombinant expressed transformant comprising recombinant expression vector of the present invention.Obtained by recombinant expression vector of the present invention being converted in host cell.Described host cell can be the host cell of this area routine, copy voluntarily, and entrained aminotransferase gene of the present invention can by effective expression as long as can meet recombinant expression vector Absorbable organic halogens ground.The preferred intestinal bacteria of the present invention (E.coli), more preferably E.coli BL21 (DE3).Aforementioned recombinant expression plasmid pET21a-MvAT is converted in E.coli BL21 (DE3), the preferred engineering strain of the present invention can be obtained, be i.e. E.coliBL21 (DE3)/pET21a-MvAT.Method for transformation can select this area ordinary method, and as electric robin, heat shock method etc., preferably select heat shock method to carry out transforming, hot shock condition is preferably: 45 DEG C, thermal shock 90 seconds.
A fifth aspect of the present invention provides a kind of preparation method of transaminase of recombinating, comprising following steps: cultivate recombinant expressed transformant of the present invention, obtain restructuring transaminase from culture.
Wherein, the described same foregoing description of recombinant expressed transformant, obtains by recombinant expression vector of the present invention is converted into host cell.Cultivating recombinant expressed transformant substratum used can be that this area routine any makes transformants grew and produce the substratum of transaminase of the present invention, for coli strain, preferred LB substratum (peptone 10g/L, yeast extract paste 5g/L, NaCl10g/L, pH7.0).Cultural method and culture condition do not have special restriction, can carry out appropriate selection according to the difference of the factor such as host type and cultural method by this area general knowledge, as long as can make transformants grew and produce transaminase of the present invention.Other concrete operations of cultivating transformant all can be undertaken by this area routine operation.For coli strain, shake-flask culture fermentative production enzyme preferably selects following method: be seeded to by the recombination bacillus coli that the present invention relates to (preferred E.coliBL21 (DE3)/pET21a-MvAT) in the LB substratum containing penbritin and cultivate, as the optical density(OD) OD of nutrient solution
600when reaching 0.5 ~ 0.7 (being more preferably 0.6), adding final concentration is that the isopropyl-beta D-thio galactopyranoside (IPTG) of 0.05 ~ 1.0mmol/L (being more preferably 0.2mmol/L) is induced, inducing temperature 10 ~ 40 DEG C (being more preferably 35 DEG C), can high expression restructuring transaminase of the present invention.
In the present invention, catalysis prochiral carbonyl compounds carries out the catalyzer that asymmetric transamination reaction forms optical activity chirality amine, can be the culture of the transformant of the restructuring transaminase of above-mentioned generation, also can be the transformant cell by obtaining after substratum centrifugation or the goods with its processing.Here " goods of processing " refer to the extract obtained by transformant cell, by to the product separation that the transaminase in extract is separated and/or purifying obtains, or by the product separation of immobilization transformant cell and extract or extract and the immobilizing product obtained.
A sixth aspect of the present invention provides a kind of protein of the present invention and carries out asymmetric transamination reaction at catalysis prochiral carbonyl compounds and form application in Chiral Amine.
In above-mentioned application, each condition of described asymmetric transamination reaction can be selected by the normal condition of this type of reaction of this area, is preferably as follows:
The preferred transaminase of the present invention of described protein or restructuring transaminase.Described prochiral carbonyl compounds is preferably 3-carbonyl-4-(2,4,5-trifluorophenyl)-bungeana acid esters compound, is namely the compound shown in formula I:
Wherein,
R is alkyl or benzyl.
Preferably,
R to be carbon chain lengths be 1 ~ 8 alkyl or benzyl.
More preferably,
R is-CH
3,-CH
2cH
3,-CH (CH
3)
2,-CH
2cH
2cH
3,-C (CH
3)
3or-CH
2c
6h
5.
Best, R is-CH
3, namely formula I is 3-carbonyl-4-(2,4,5-trifluorophenyl)-methyl-butyrate.
Each condition of asymmetric transamination 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: in the aqueous ethanolic solution of pH7.0 ~ 10.0, under the existence of Isopropylamine and pyridoxal phosphate (PLP), transaminase of the present invention or restructuring transaminase catalysis under, prochiral carbonyl compounds carries out asymmetric transamination reaction, forms optical activity chirality amine.
The preferred concentration of wherein said prochiral carbonyl compounds in reaction solution is 1 ~ 800mmol/L.Transaminase consumption of the present invention is catalytically effective amount, is preferably 0.1 ~ 50g/L.The consumption of Isopropylamine is preferably 1 ~ 60g/L.The consumption of the PLP additionally added is preferably 0 ~ 1.0mmol/L.The described aqueous solution can be this area conventional buffers, as long as its pH scope is 7.0 ~ 10.0, 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 alcohol concn is preferably 5% ~ 50%.Described asymmetric transamination reaction is preferably and carries out under vibration or agitation condition.The temperature of described asymmetric transamination reaction is preferably 20 ~ 55 DEG C.The time of described asymmetric transamination reaction, the time that production concentration no longer continues to improve was as the criterion preferably with in reaction process.After asymmetric transamination reaction terminates, Chiral Amine product can be extracted by this area ordinary method from reaction solution.
In the present invention, use crude enzyme liquid to make catalyzer, preferably should add PLP coenzyme.If make catalyzer with resting cell, do not need to add PLP coenzyme, only need utilize PLP coenzyme contained in cell.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: in the reaction of the asymmetric synthesis sitagliptin reported and intermediate thereof, productive rate is on the low side, stereoselectivity is not good, catalyzer is expensive, solvent is difficult to the problems such as recovery, provide that a kind of catalytic activity is high, enantioselectivity is strong, substrate tolerance and the good new transaminase of solvent tolerance carry out Enzyme catalyzed synthesis R-3-amino-4-(2,4,5-trifluorophenyl) method of-methyl-butyrate.When catalytic level is up to 0.8mol/L (200g/L) substrate, the optical purity of product is still up to more than 99%.Relative to other preparation method, the production concentration that use the inventive method prepares gained is high, and product optical purity is high, and solvent easily reclaims, and reaction conditions is gentle, environmentally friendly, easy and simple to handle, is easy to industry and amplifies, therefore have good prospects for commercial application.
Accompanying drawing explanation
The agarose gel electrophoresis figure of Fig. 1 mycobacterium PYR-1 aminotransferase gene PCR primer.
The bacterium colony PCR of the recombinant expressed transformant of Fig. 2 mycobacterium PYR-1 transaminase schemes.M is molecular weight standard, and A swimming lane is E.coli DH5 α/pET21a – MvAT, and B swimming lane is E.coli BL21 (DE3)/pET21a – MvAT.
The polyacrylamide gel electrophoresis figure of the mycobacterium PYR-1 transaminase crude enzyme liquid that Fig. 3 is recombinant expressed.
M is molecular weight standard, and A swimming lane is for before induction, and B swimming lane is for after induction.
The agarose gel electrophoresis figure of Fig. 4 transaminase mutant library 1.M is molecular weight standard, and A swimming lane is the agarose gel electrophoresis figure of the PCR fragment product that rite-directed mutagenesis obtains.B swimming lane is the agarose gel electrophoresis figure that mutant fragments uses the gene clone primer product of PCR again.
The agarose gel electrophoresis figure of Fig. 5 transaminase mutant library 2.M is molecular weight standard, and A swimming lane is the agarose gel electrophoresis figure of the PCR fragment product that rite-directed mutagenesis obtains.B swimming lane is the agarose gel electrophoresis figure that mutant fragments uses the gene clone primer product of PCR again.
Embodiment
Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
Material source in the following example is:
The genomic dna of mycobacterium (Mycobacterium vanbaalenii) PYR-1 is provided by Hui doctor Wu of the School of Stomatology of Alabama State University of the U.S..
Expression plasmid pET21a, E.coli DH5 α and E.coli BL21 (DE3) competent cell, 2 × Taq PCRMasterMix, sepharose DNA reclaim test kit all purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
The clone of embodiment 1 mycobacterium PYR-1 aminotransferase gene
Be foundation according to the gene order (NCBI accession number: YP_955297.1) being predicted as mycobacterium PYR-1 transaminase that Genbank includes, design PCR primer is as gene clone upstream and downstream primer in table 1.Wherein, upstream primer underscore part is Nde I restriction enzyme site, and downstream primer underscore part is EcoR I restriction enzyme site.
With mycobacterium PYR-1 genomic dna for template, carry out pcr amplification.PCR system is: 10 × KOD-Plus PCR buffer2 μ L, 25mM MgSO
41.2 μ L, 2mM dNTP2 μ L, KOD-PlusPCR high-fidelity enzyme 0.3 μ L, DNA profiling 0.5 μ L (containing DNA profiling 0.1 μ g), ddH
2o13 μ L, carries out pcr amplification with gene clone upstream primer in table 1 and each 0.5 μ L (10mmol/L) of gene clone downstream primer (SEQ ID No:11 and 12).Pcr amplification step is: (1) 95 DEG C, denaturation 3min; (2) 98 DEG C, sex change 15s; (3) 55 DEG C of annealing 30s; (4) 72 DEG C extend 1min; Step (2) ~ (4) are repeated 30 times; (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.PCR primer is through agarose gel electrophoresis purifying, sepharose DNA is utilized to reclaim the target stripe (see Fig. 1) in test kit recovery 900 ~ 1200bp interval, obtain a complete mycobacterium PYR-1 transaminase full-length gene order, through DNA sequencing, total length 1014bp, called after MvAT, the aminoacid sequence of its nucleotide sequence and coding is respectively as shown in SEQ ID No:1 and 2 in sequence table.
Table 1PCR primer table
The structure of embodiment 2 recombinant expression vector
By the aminotransferase gene DNA fragmentation of gained in embodiment 1 at 37 DEG C with restriction enzyme NdeI and EcoRI double digestion 8h, 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 pET21a after NdeI and EcoRI enzyme is cut, at 16 DEG C, connection is spent the night and is obtained recombinant expression plasmid pET21a-MvAT.
The preparation of the recombinant expressed transformant of embodiment 3
Recombinant expression plasmid is transformed in colon bacillus (E.coli) DH5 α competent cell, conversion condition 45 DEG C, thermal shock 90 seconds, containing in the resistant panel of penbritin, positive recombinants is being screened, picking mono-clonal, bacterium colony PCR verifies positive colony (the A swimming lane see in Fig. 2).Cultivate recombinant bacterium, plasmid is extracted after plasmid amplification, again be converted in E.coli BL21 (DE3) competent cell, conversion fluid is applied on the LB flat board containing penbritin, be inverted overnight incubation for 37 DEG C, namely obtain positive recombinant conversion body E.coli BL21 (DE3)/pET21a – MvAT, bacterium colony PCR and verify positive colony (the B swimming lane see in Fig. 2).
Embodiment 4 is recombinated the expression of transaminase
By the restructuring E.coli BL21 (DE3) of embodiment 3 gained, be seeded to LB substratum (the peptone 10g/L containing penbritin, yeast extract paste 5g/L, NaCl10g/L, pH7.0) in, 37 DEG C of shaking culture are spent the night, be equipped with in the 500ml triangular flask of 100ml LB substratum by the inoculum size access of 1% (v/v), put 37 DEG C, the jolting of 180rpm shaking table is cultivated, when the OD600 of nutrient solution reaches 0.6, adding final concentration is that the IPTG of 0.2mmol/L is as inductor, after 35 DEG C of induction 12h, by medium centrifugal, collecting cell, and with brine twice, obtain resting cell.The resting cell of gained is suspended in the damping fluid of pH8.5, ultrasonication in ice bath, collected by centrifugation supernatant liquor, is the crude enzyme liquid of restructuring transaminase.Protein concentration is measured by Bradford method.Crude enzyme liquid is through Polyacrylamide Gel Electrophoresis (see Fig. 3) together with precipitation, and recombinant protein exists with partly soluble form.Crude enzyme liquid is used freeze drier freeze-drying, be the thick enzyme powder of freeze-drying.
Embodiment 5 the asymmetric of transaminase-catalyzed carbonyl substrates of recombinating turns ammonia
At 50ml sodium phosphate-Isopropylamine damping fluid (100mmol/L, pH7.5) crude enzyme liquid to the final protein concentration adding MvAT prepared by embodiment 4 in is 10g/L, add the 3-carbonyl-4-(2 that final concentration is 10mmol/L, 4,5-trifluorophenyl)-methyl-butyrate and final concentration be the pyridoxal phosphate of 1mmol/L, add 2.5ml ethanol, insert bottom at 20 DEG C with syringe needle and carry out nitrogen purging reaction.Reaction extracted with equal-volume ethyl acetate after 48 hours, extracting twice, combining extraction liquid, added the anhydrous sodium sulfate drying post analysis that spends the night and measured substrate conversion efficiency and turn the ee value of ammonia product.Find no and turn the generation of ammonia product.
Transformation efficiency measuring method is: reaction solution dilution in acetonitrile 100 times, get 20 μ L after centrifugal and analyze transformation efficiency on Agilent1200HPLC, analytical column is Agilent Eclipse XAD-C18 reverse phase silica gel post, moving phase is water: acetonitrile=40:60, separately add 10mmol/L ammonium formiate, flow velocity is per minute 1mL, and determined wavelength is 210nm and 260nm, the retention time turning ammonia product is 4.6min, and the retention time of substrate is 7.4min.
The ee values determination method turning ammonia product is: the optical purity e.e value detecting product on Agilent1200HPLC, Daicel Chiralpak AD-H chiral chromatographic column (4.6 × 150mm) is adopted to analyze, moving phase is ethanol: normal heptane: diethylamine: water=60:40:0.1:0.1, flow velocity is per minute 1mL, column temperature is 35 DEG C, retention time is respectively: substrate 5.6min, (S)-product: 7.9min; (R)-product: 9.7min.Determined wavelength is 210nm and 260nm.
The structure of embodiment 6 transaminase mutated library 1
With plasmid pET21a-MvAT constructed in embodiment 3 for template, carry out pcr amplification.PCR system is: 10 × KOD-Plus PCR buffer2 μ L, 25mM MgSO
41.2 μ L, 2mM dNTP2 μ L, KOD-Plus PCR high-fidelity enzyme 0.3 μ L, DNA profiling 0.5 μ L (containing DNA profiling 0.1 μ g), ddH
2o13 μ L, carries out pcr amplification with A291 downstream primer (SEQID No:18), A291 upstream primer (SEQ ID No:17) with each 0.5 μ L (10mmol/L) of gene clone downstream primer (SEQ ID No:12) with F129 downstream primer (SEQ ID No:16), F129 upstream primer (SEQ ID No:15) with gene clone upstream primer in table 1 (SEQ ID No:11) and L72+V76 downstream primer (SEQ ID No:14), L72+V76 upstream primer (SEQ ID No:13) respectively.Pcr amplification step is: (1) 95 DEG C, denaturation 3min; (2) 98 DEG C, sex change 15s; (3) 55 DEG C of annealing 30s; (4) 72 DEG C extend 1min; Step (2) ~ (4) are repeated 30 times; (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.PCR primer, through agarose gel electrophoresis purifying, utilizes sepharose DNA to reclaim test kit and reclaims 250,150,500, about 180 target stripe (the A swimming lane see in Fig. 4).With these PCR primer for template, carry out pcr amplification by embodiment 1 and reclaim the PCR primer (the B swimming lane see in Fig. 4) of about 1100bp, after building plasmid by embodiment 2, namely obtain transaminase mutated library 1 (L72FSCVAG/V76CSAG/F129LMV/A291AG) by embodiment 3 Transformed E .coli BL21 (DE3) competent cell.
The screening of embodiment 7 transaminase mutated library
Choose sudden change bacterium colony from mutated library, be seeded in 200 μ L2 × YT substratum (penbritins containing 100 μ g/m L) of microtiter plate, cultivate 24 hours for 37 DEG C.Get the above-mentioned culture of 100 μ L be seeded to containing 500 μ L express substratum (2 × YT, 100 μ g/ml penbritins, 1mM IPTG) deep-well plates in, 25 DEG C cultivate 24 hours.3500rpm carries out centrifugal 15 minutes, and add 400 μ L lysis buffers (20mM phosphoric acid buffer, pH7.5, containing 1mg/mL N,O-Diacetylmuramidase) and make its resuspension, multigelation makes cytoclasis.Centrifugal 15 minutes of 4000rpm, gets 200 μ L supernatants to new microtiter plate in each hole.Add reaction solution (200mM sodium phosphate, 200mM Isopropylamine, 20mM3-carbonyl-4-(2,4,5-trifluorophenyl)-methyl-butyrate, 2mM pyridoxal phosphate, 10% ~ 80% (v/v) ethanol, pH7.5 ~ 8.5), measure transformation efficiency by embodiment 5 after 20 DEG C ~ 50 DEG C shaking tables vibrate 24 hours.
Embodiment 8 is screened mutated library 1 and is obtained transaminase mutant 1
The mutated library 1 that embodiment 6 builds is screened by embodiment 7, screening conditions: 10% ethanol, pH7.5,20 DEG C, obtain a plant mutant strain transformation efficiency and reach 11% (mutant 1).Check order to this mutant, it is L72C/V76A/F129V/A291G mutant, and nucleotide sequence and aminoacid sequence are respectively as shown in SEQ ID No:3 and 4 in sequence table.
The structure of embodiment 9 transaminase mutated library 2
With plasmid constructed in embodiment 3 for template, carry out pcr amplification.PCR system is: 10 × KOD-PlusPCR buffer2 μ L, 25mM MgSO
41.2 μ L, 2mM dNTP2 μ L, KOD-Plus PCR high-fidelity enzyme 0.3 μ L, DNA profiling 0.5 μ L (containing DNA profiling 0.1 μ g), ddH
2o13 μ L, respectively with gene clone upstream primer in table 1 (SEQ ID No:11) and H69 downstream primer (SEQ ID No:20), H69 upstream primer (SEQ ID No:19) and G143 downstream primer (SEQ ID No:22), G143 upstream primer (SEQ ID No:21) and S206 downstream primer (SEQ ID No:24), S206 upstream primer (SEQ ID No:23) and T216 downstream primer (SEQ ID No:26), T216 upstream primer (SEQID No:25) and P230 downstream primer (SEQ ID No:28), P230 upstream primer (SEQ ID No:27) and each 0.5 μ L (10mmol/L) of gene clone downstream primer (SEQ ID No:12) carry out pcr amplification.Pcr amplification step is: (1) 95 DEG C, denaturation 3min; (2) 98 DEG C, sex change 15s; (3) 55 DEG C of annealing 30s; (4) 72 DEG C extend 1min; Step (2) ~ (4) are repeated 30 times; (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.PCR primer, through agarose gel electrophoresis purifying, utilizes sepharose DNA to reclaim test kit and reclaims 200,200,200,50,80, about 300 target stripe (the A swimming lane see in Fig. 5).With these PCR primer for template, carry out pcr amplification by embodiment 1 and reclaim the PCR primer (the B swimming lane see in Fig. 5) of about 1100bp, after building plasmid by embodiment 2, namely obtain transaminase mutated library 2 (H69FLV/G143ATIV/S206ATIV/T216IV/P230LPAV) by embodiment 3 Transformed E .coli BL21 (DE3) competent cell.
Embodiment 10 is screened mutated library 2 and is obtained transaminase mutant 2
Screen by embodiment 7 mutated library 2 that embodiment 9 builds, screening conditions: 10% ethanol, pH7.5, obtains a plant mutant strain transformation efficiency and reaches 99% (mutant 2) by 20 DEG C.Check order to this mutant, it is H69L/L72C/V76A/F129V/G143I/S206V/T216I/P230A/T289A/A291G mutant, and nucleotide sequence and aminoacid sequence are respectively as shown in SEQ ID No:5 and 6 in sequence table.
The foundation of embodiment 11 Random mutagenesis libraries
With transaminase mutant gene for template, with gene clone upstream primer and gene clone downstream primer for primer, Random Mutagenesis Kit is used to carry out PCR sudden change.Pcr amplification step is: (1) 95 DEG C, denaturation 3min; (2) 94 DEG C, sex change 30s; (3) 68 DEG C extend 1min; Step (2) ~ (3) are repeated 25 times; (5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.After building plasmid by embodiment 2, namely obtain transaminase random mutagenesis mutated library by embodiment 3 Transformed E .coli BL21 (DE3) competent cell.
Embodiment 12 is screened Random mutagenesis libraries and is obtained mutant 3
With embodiment 10 gained transaminase mutant 2 gene for template, build random library by embodiment 11, screen by embodiment 7, screening conditions: 10% ethanol, pH7.5,50 DEG C, obtain a plant mutant strain transformation efficiency and reach 99% (mutant 3).This mutant is checked order, it is E56S/H69L/L72C/V76A/F129V/G143I/S206V/T216I/P230A/A243R/ S276P/T289A/A291G/T295W/P304T mutant, and nucleotide sequence and aminoacid sequence are respectively as shown in SEQ IDNo:7 and 8 in sequence table.
Embodiment 13 is screened Random mutagenesis libraries and is obtained mutant 4
With embodiment 12 gained transaminase mutant 3 gene for template, build random library by embodiment 11, screen by embodiment 7, screening conditions: 50% ethanol, pH8.5,50 DEG C, obtain a plant mutant strain transformation efficiency and reach 99% (mutant 4).This mutant is checked order, it is E56S/G68Y/H69L/L72C/V76A/D96A/F129V/N131T/G143I/Y157T/S2 06V/T216I/A222C/P230A/A243R/S276P/T289A/A291G/T295W/P304 T mutant, and nucleotide sequence and aminoacid sequence are respectively as shown in SEQ ID No:9 and 10 in sequence table.
Recombinate transaminase and the asymmetric of mutant catalysis of carbonyl substrate thereof of embodiment 14 turns ammonia
At 50ml sodium phosphate-Isopropylamine damping fluid (100mmol/L, pH7.0 ~ 8.5) in add according to embodiment 4 prepare MvAT or its mutant crude enzyme liquid to final protein concentration be 10g/L, add the 3-carbonyl-4-(2 that final concentration is 10mmol/L, 4,5-trifluorophenyl)-methyl-butyrate and final concentration be the pyridoxal phosphate of 1mmol/L, add 2.5 ~ 50ml ethanol, insert bottom at 20 ~ 50 DEG C with syringe needle and carry out nitrogen purging reaction.Reaction extracted by equal-volume ethyl acetate after 48 hours, extracting twice, combining extraction liquid, added after anhydrous sodium sulfate drying spends the night and measured substrate conversion efficiency and the ee value turning ammonia product by embodiment 5.The results are shown in Table 2.
The result of table 2MvAT and the asymmetric transamination reaction of mutant catalytic substrate thereof
| MvAT enzyme | SEQ ID NO | pH | Temperature of reaction (%) | Alcohol concn (%) | Transformation efficiency (%) | Ee value (%) |
| Wild-type | 2 | 7.5 | 25 | 5 | <0.1 | Do not survey |
| Mutant 1 | 4 | 7.5 | 25 | 5 | 16 | 99 |
| Mutant 2 | 6 | 7.5 | 25 | 5 | 99 | 99 |
| Mutant 2 | 6 | 7.5 | 50 | 5 | 7 | 99 |
| Mutant 3 | 8 | 7.5 | 50 | 5 | 99 | 99 |
| Mutant 3 | 8 | 8.5 | 50 | 50 | 13 | 99 |
| Mutant 4 | 10 | 8.5 | 50 | 50 | 99 | 99 |
The asymmetric of embodiment 15 transaminase mutant 4 catalysis of carbonyl compound turns ammonia
At 50ml sodium phosphate-Isopropylamine damping fluid (100mmol/L, pH8.5) crude enzyme liquid to the final protein concentration adding the transaminase mutant 4 prepared according to embodiment 4 in is 10g/L, add the 3-carbonyl-4-(2 that final concentration is 100mmol/L, 4,5-trifluorophenyl)-butyric ester substrate and final concentration be the pyridoxal phosphate of 1mmol/L, add 50mL ethanol, insert bottom at 50 DEG C with syringe needle and carry out nitrogen purging reaction.Measure transformation efficiency to 99% in reaction process or react stopped reaction after 24 hours.Reaction terminates rear 100mL ethyl acetate and extracts, extracting twice, combining extraction liquid, adds after anhydrous sodium sulfate drying spends the night and measures substrate conversion efficiency and the ee value turning ammonia product by embodiment 5.The results are shown in Table 3.
The result of the asymmetric transamination reaction of table 3 transaminase mutant 4 catalysis of carbonyl compound
The asymmetric of embodiment 16 transaminase mutant 4 catalysis methyl esters substrate turns ammonia
At 100mL sodium phosphate-Isopropylamine damping fluid (100mmol/L, pH8.5) crude enzyme liquid to the final protein concentration adding the transaminase mutant 4 prepared according to embodiment 4 in is 10g/L, add the 3-carbonyl-4-(2 that final concentration is 800mmol/L, 4,5-trifluorophenyl)-methyl-butyrate (19.7g) and final concentration be the pyridoxal phosphate of 1mmol/L, add 100mL ethanol, insert bottom at 50 DEG C with syringe needle and carry out nitrogen purging reaction.The rear stopped reaction of transformation efficiency to 99% is measured in reaction process.Reaction terminates rear 80 DEG C of rotary evaporations removing ethanol, cross and filter protein, adjust pH to be 11.0, extract by 100mL ethyl acetate, extracting twice, combining extraction liquid, add anhydrous sodium sulfate drying spend the night after rotary evaporation except desolventizing, underpressure distillation obtains 18.1g (R)-3-amino-4-(2,4,5-trifluorophenyl)-methyl-butyrate, yield 92%, the ee value of product is 99%.
The Boc protection of embodiment 17 turns of ammonia products
Take (R)-3-amino-4-(2 of 12.3 grams of embodiment 16 gained, 4,5-trifluorophenyl)-methyl-butyrate (0.05mol), be dissolved in the solution of 50mL water and 50mL tetrahydrofuran (THF), add 4g NaOH (0.10mol), under ice bath, add 12.0g (Boc)
2o (0.055mol), rises to room temperature reaction 12 hours.Adding sodium carbonate regulates pH to be 12, and use 50mL dichloromethane extraction, give up organic phase, aqueous phase 1N salt acid for adjusting pH is 2,50mL dichloromethane extraction merges organic phase, anhydrous sodium sulfate drying, and rotary evaporation is except desolventizing, recrystallizing methanol, obtains 14.8g white solid, productive rate 89%.
The sitagliptin turning ammonia Product formation Boc protection of embodiment 18Boc protection
Take the product (0.01mol) and 2.28g trifluoromethyl triazole piperazine hydrochloride (0.01mol) that obtain in 3.32g embodiment 17 in 20mL methylene dichloride, 1.62g1-hydroxy benzo triazole (0.012mol) and 2.29g1-ethyl-3-(3-dimethylamino-propyl) carbimide hydrochloride (0.012mol) is added under cryosel bath, drip 3g triethylamine (0.03mol), stir lower room temperature reaction 24 hours, reaction solution 20mL water washing 3 times, organic phase anhydrous magnesium sulfate drying, rotary evaporation removes desolventizing and obtains 4.72g solid, productive rate 93%.
The deprotection of the sitagliptin of embodiment 19Boc protection
The sitagliptin (0.01mol) taking the Boc protection obtained in 5.07g embodiment 18, in 50mL methyl alcohol, adds 50mL concentrated hydrochloric acid: the solution of methyl alcohol=1:5, stirring at room temperature 3 hours, and rotary evaporation removing methyl alcohol, uses Na
2cO
3neutralization, with 50mL extraction into ethyl acetate 3 times, merge organic phase, anhydrous magnesium sulfate drying, desolventizes to obtain oily matter.Add 60mL ethanol and 10mL water, be heated to 80 DEG C, add 1.5g strong phosphoric acid, stir and within 2 hours, be down to stirring at room temperature 12 hours, separate out 4.39g solid and Sitagliptin phosphate, productive rate 87%.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (24)
1. the protein be separated, it is the protein of following (a) or (b):
A protein that () is made up of aminoacid sequence shown in SEQ ID No:2,4,6,8 or 10;
(b) in the aminoacid sequence of (a) through replacement, lack or add one or several amino-acid residue and have transaminase activity by (a) derivative and and the aminoacid sequence of (a) there is the protein of at least 90% identity.
2. protein according to claim 1, wherein the protein of (b) is one or more sites in the 56th, 68,69,72,76,96,129,131,143,157,206,216,222,230,243,276,289,291,295,304 amino acids residues of aminoacid sequence at (a) through replacing and having the protein derivative by (a) of transaminase activity.
3. protein according to claim 2, wherein the protein of (b) is one or more sites in the 56th, 68,69,72,76,96,129,131,143,157,206,216,222,230,243,276,289,291,295,304 amino acids residues of aminoacid sequence shown in SEQ ID No:2 through replacing and having the protein of transaminase activity.
4. protein according to claim 3, wherein said shown in SEQ ID No:2 aminoacid sequence the 56th, 68, 69, 72, 76, 96, 129, 131, 143, 157, 206, 216, 222, 230, 243, 276, 289, 291, 295, the L-glutamic acid that the replacement of 304 amino acids residues is respectively the 56th is substituted by Serine, the glycine of the 68th is substituted by tyrosine, the Histidine of the 69th is substituted by leucine, the leucine of the 72nd is substituted by halfcystine, the α-amino-isovaleric acid of the 76th is substituted by L-Ala, the aspartic acid of the 96th is substituted by L-Ala, the phenylalanine of the 129th is substituted by α-amino-isovaleric acid, the l-asparagine of the 131st is substituted by Threonine, the glycine of the 143rd is substituted by Isoleucine, the tyrosine of the 157th is substituted by Threonine, the Serine of the 206th is substituted by α-amino-isovaleric acid, the Threonine of the 216th is substituted by Isoleucine, the L-Ala of the 222nd is substituted by halfcystine, the proline(Pro) of the 230th is substituted by L-Ala, the L-Ala of the 243rd is substituted by arginine, the Serine of the 276th is substituted by proline(Pro), the Threonine of the 289th is substituted by L-Ala, the L-Ala of the 291st is substituted by glycine, the Threonine of the 295th is substituted by tryptophane, the proline(Pro) of the 304th is substituted by Threonine.
5. the nucleic acid be separated, it is the nucleic acid of following (1) or (2):
(1) nucleic acid be made up of nucleotide sequence shown in SEQ ID No:1,3,5,7 or 9;
(2) to encode the nucleic acid of following protein (a) or (b):
A protein that () is made up of aminoacid sequence shown in SEQ ID No:2,4,6,8 or 10;
(b) in the aminoacid sequence of (a) through replacement, lack or add one or several amino-acid residue and have transaminase activity by (a) derivative and and the aminoacid sequence of (a) there is the protein of at least 90% identity.
6. one kind comprises the recombinant expression vector of nucleic acid according to claim 5.
7. recombinant expression vector according to claim 6, it is selected from plasmid, clay, phage and virus vector.
8. recombinant expression vector according to claim 7, it is pET21a.
9. one kind comprises the recombinant expressed transformant of the recombinant expression vector described in any one of claim 6-8.
10. recombinant expressed transformant according to claim 9, it is intestinal bacteria.
11. recombinant expressed transformant according to claim 10, it is E.coli BL21 (DE3).
The preparation method of 12. 1 kinds of transaminases of recombinating, it comprises the steps: to cultivate the recombinant expressed transformant described in any one of claim 9-11, and from culture, obtain restructuring transaminase.
13. 1 kinds of catalysis prochiral carbonyl compounds carry out the catalyzer that asymmetric transamination reaction forms optical activity chirality amine, it is selected from the culture of the recombinant expressed transformant described in any one of claim 6-8, by the transformant cell will obtained after the substratum centrifugation in described culture, or with the goods that described transformant cell is processed; Wherein, the goods of described processing are the extracts obtained by described transformant cell, by to the product separation that the transaminase in described extract is separated and/or purifying obtains, or by the product separation of immobilization transformant cell and extract or extract and the immobilizing product obtained.
Protein described in 14. any one of claim 1-4, the restructuring transaminase that method according to claim 12 obtains, or catalyzer according to claim 13 carries out the application in asymmetric transamination reaction formation optical activity chirality amine at catalysis prochiral carbonyl compounds.
15. application according to claim 14, wherein said prochiral carbonyl compounds is selected from following compound:
Wherein, R is alkyl or benzyl.
16. application according to claim 15, wherein R to be carbon chain lengths be 1 ~ 8 alkyl or benzyl.
17. application according to claim 15, wherein R is-CH
3,-CH
2cH
3,-CH (CH
3)
2,-CH
2cH
2cH
3,-C (CH
3)
3or-CH
2c
6h
5.
18. application according to claim 15, wherein R is-CH
3.
19. application according to any one of claim 15-18, wherein said asymmetric transamination reaction carries out in the aqueous ethanolic solution of pH7.0 ~ 10.0 containing Isopropylamine, optionally, also containing pyridoxal phosphate in described aqueous ethanolic solution.
20. application according to claim 19, wherein, described in reaction solution, the concentration of prochiral carbonyl compounds is 1 ~ 800mmol/L, the concentration of Isopropylamine is 1 ~ 60g/L, the concentration of pyridoxal phosphate is 0 ~ 1.0mmol/L, the phosphate buffered saline buffer of the described aqueous solution to be conjugate acid and base total concn be 0.05 ~ 0.1mol/L, and alcohol concn is 5% (v/v) ~ 50% (v/v), react and carry out under vibration or agitation condition, temperature of reaction is 20 ~ 55 DEG C.
21. 1 kinds of synthesis (R)-3-amino-4-(2,4,5-trifluorophenyl) method of-methyl-butyrate, comprise the protein used described in any one of claim 1-4, the restructuring transaminase that method according to claim 12 obtains, or catalyst 3-carbonyl-4-(2,4,5-trifluorophenyl)-methyl-butyrate according to claim 13 carries out the step of asymmetric transamination reaction.
22. methods according to claim 21, wherein, 3-carbonyl-4-(2 containing 10 ~ 800mmol/L in the reaction solution of described transamination reaction, 4,5-trifluorophenyl)-methyl-butyrate, the sodium phosphate of 50 ~ 100mmol/L, the Isopropylamine of 50 ~ 100mmol/L, the pyridoxal phosphate of 0 ~ 1mmol/L, the ethanol of 5% (v/v) ~ 50% (v/v), reaction pH is 7.5 ~ 8.5, and temperature of reaction is 25 ~ 50 DEG C.
23. methods according to claim 22, wherein, the protein catalysis that described transamination reaction is made up of the aminoacid sequence shown in SEQ ID NO:10,3-carbonyl-4-(2 containing 800mmol/L in reaction solution, 4,5-trifluorophenyl)-methyl-butyrate, the sodium phosphate of 50mmol/L, the Isopropylamine of 50mmol/L, the pyridoxal phosphate of 1mmol/L, the ethanol of 50% (v/v), reaction pH be 8.5, temperature of reaction is 50 DEG C.
24. 1 kinds of methods of synthesizing sitagliptin, comprise method synthesis of chiral amino intermediate (R)-3-amino-4-(2,4,5-the trifluorophenyl)-methyl-butyrate using any one of claim 21-23.
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