CN106801043B

CN106801043B - A kind of recombination transaminase and its preparation method and application

Info

Publication number: CN106801043B
Application number: CN201611239147.1A
Authority: CN
Inventors: 石利平; 陈峻青; 蔡进; 漆志文; 张维冰; 叶银梅; 龚仕荣; 徐春涛
Original assignee: JIANGSU ALPHA PHARMACEUTICAL CO Ltd
Current assignee: Jiangsu alpha Pharmaceutical Co.,Ltd.
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2019-08-06
Anticipated expiration: 2036-12-28
Also published as: CN106801043A

Abstract

The invention discloses a kind of recombination transaminases and its preparation method and application.A kind of recombination transaminase mutant of high activity, amino acid sequence is as shown in SEQ ID NO.2, and encoding gene is as shown in SEQ ID NO.1.A method of the recombination transaminase is prepared, the genetic engineering bacterium of above-mentioned encoding gene is contained including fermented and cultured, and collect and preparation and reorganization transaminase.Recombination transaminase of the present invention is applied to asymmetric syntheses chirality aminated compounds, especially for synthesizing sitagliptin intermediate (R) -3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate.Enzyme according to the present invention has excellent stereoselectivity, regioselectivity and catalysis activity, and the reaction of its catalysis is mild, and the yield of reaction conversion ratio and product is high, with good application prospect.

Description

A kind of recombination transaminase and its preparation method and application

Technical field

The invention belongs to technical field of bioengineering, and in particular to a kind of recombination transaminase and its preparation method and application.

Background technique

Diabetes are the chronic progressive diseases of a kind of whole body caused by human body own metabolism disorder, are by insulin secretion Caused by insufficient or insulin action obstacle.Diabetes are usually concurrent with coronary heart disease, cranial vascular disease, nephrosis, lower limbs necrosis etc. Disease is a kind of frequently-occurring disease of complexity.Whole world maturity-onset diabetes number of patients is up to 4.22 hundred million at present, it is contemplated that 2025, the whole world will There are more than 700,000,000 people to suffer from diabetes.

Diabetes are broadly divided into two kinds of I type and II type, and wherein type II diabetes is Non-Insulin Dependent Diabetes Mellitus, be by Insulin resistance or islet beta cell function, which are damaged, to be caused, and patient accounts for 90% of diabetic or more.II type for the treatment of sugar at present There are many drugs for urinating disease, as sulfonylurea, biguanides, thiazolidinediones, alpha-glucosidase restrainer, benzoic acid are derivative Species etc. influence patient due to there are side effect and adhere to medication, preferable blood glucose is not achieved although blood sugar reducing function is ideal Control purpose.

Dipeptidyl peptidase-IV (DPP-IV) can be cracked including glucagon-like-peptide-1 (GLP-1) and Gastric inhibitory polypeptide (GIP) A variety of peptide hormones inside, and the two and type II diabetes have close ties.DPP-IV inhibitor can pass through inhibition DPP-IV reduces the degradation of GLP-1, increases the plasma concentration of GLP-1, so as to improve postprandial blood sugar.In addition, DPP-IV inhibitor It may also suppress other peptides of the participations such as GIP, pituitary adenylyl cyclase activating polypeptide and gastrin releasing peptide adjusting blood glucose Degradation.

In October, 2006, Food and Drug Adminstration of the US (FDA) ratify sitagliptin phosphate monohydrate (Sitagliptin, trade name Januvia) is for treating type II diabetes.Sitagliptin is first for treating II type sugar Urinate disease dipeptidyl peptidase (DPP-IV) inhibitor class drug, can inhibit glucagon secretion and beta Cell of islet proliferation and It is horizontal to improve glucose-tolerant, blood sugar reducing function is relatively mild, does not cause patient's oedema and weight gain, leads to the wind of hypoglycemia Danger is smaller, and the side effects such as nothing is put on weight, Nausea and vomiting.Sitagliptin is used alone or shares with other antidiabetic drugs, It can reach the purpose of hypoglycemic.

Sitagliptin Chinese is 7- [(3R) -3- amino -1- oxo -4- (2,4,5- trifluorophenyl) butyl] -5, 6,7,8- tetrahydro -3- (trifluoromethyl) -1,2,4- triazoles [4,3- α] pyrazoles phosphoric acid (1:1) monohydrate, molecular formula are C₁₆H₁₅F₆N₅O, relative molecular mass 407.31, No. CAS is 654671-77-9.

At present there are many synthetic methods of sitagliptin, wherein yield it is higher be United States Merck company synthetic method (WO2004085378, WO2005020920): asymmetric hydrogenation is carried out to unprotected enamine with chirality rhodium catalyst, is reacted Total recovery is higher, is 74.5%, and lower production costs.But the resulting product optical purity of the route is lower (ee 97%), needs Crystallization concentration can just obtain optically pure sitagliptin, and reaction needs high-pressure hydrogenation (250psi), to consersion unit require compared with It is high.

That there are reaction routes is long for chemical synthesis sitagliptin and its intermediate, need using toxic raw materials, product yield and The disadvantages of optical purity is low, severe reaction conditions；And Enzyme catalyzed synthesis sitagliptin intermediate is mild with reaction condition, reaction Specificity is strong, side reaction is few, the yield of product and optical purity are high, can be easily separated the advantages that purifying, before having preferable application Scape.The present invention develops the recombination transaminase that a kind of catalytic activity is high, enantioselectivity is good, substrate tolerance is good, can be used for urging It is combined to chiral amine compound, especially catalyzes and synthesizes sitagliptin intermediate (R) -3- amino -4- (2,4,5- trifluorophenyl) - Methyl butyrate.

Summary of the invention

The technical problem to be solved by the present invention is to the long, needs for current sitagliptin and its intermediate synthetic route The disadvantages of using toxic raw materials, product yield and chiral purity is low, severe reaction conditions, provides that a kind of catalytic activity is high, mapping The recombination transaminase that selectivity is good, substrate tolerance is good, the encoding gene of the transaminase, the recombinant expression containing the gene carry Body, recombinant expression transformants and preparation method thereof and the recombination transaminase mutant are prepared in chiral amine compound in catalysis Application.Particularly, which is used for sitagliptin intermediate (R) -3- amino -4- (2,4,5- trifluorophenyl) - The preparation of methyl butyrate.

The purpose of the present invention can be achieved through the following technical solutions:

A kind of recombination transaminase mutant of high activity, the Aspergillus terreus encoded with gene shown in SEQ ID NO.3 (Aspergillus terreus) NIH2624 wild type transaminase is the enzyme that sets out, will be in the enzyme active center and guiding activity It is higher that the amino acid residue in some sites in the hydrophobic channel of the heart sports the catalytic activity that other amino acid residues obtain Recombination transaminase mutant；The activated centre is defined as substrate binding site nearby aboutDiameter of Spherical Volume； Any one of described preferred SEQ ID NO.2 2 of recombination transaminase variant amino acid sequence.

Nucleic acid source shown in SEQ ID NO.3 is in Aspergillus terreus (Aspergillus terreus) in the present invention NIH2624.Nucleic acid shown in SEQ ID NO.3 of the present invention can be from Aspergillus terreus (Aspergillus terreus) It separates and obtains in the genome of NIH2624, it can also from the recombinant expression carrier containing nucleic acid shown in SEQ ID NO.3 or again It separates and obtains in group transformant, artificial synthesized can also obtain.

A kind of gene encoding recombination transaminase mutant of the present invention, nucleotide sequence are selected from SEQ ID NO.1。

A kind of recombinant expression carrier comprising recombination transaminase mutant code gene of the present invention.It can pass through this The nucleic acid sequence of recombination aminotransferase gene of the invention is connected to built-up on various carriers by field conventional method.Described Carrier can be the various carriers of this field routine, such as commercially available plasmid, bacteriophage or viral vectors, preferred plasmid pET21a。

A kind of recombinant expression comprising recombination transaminase mutant code gene or its recombinant expression carrier of the invention turn Change body, the preferred escherichia coli of the present invention (Escherichia coli) BL21 (DE3).Aforementioned recombinant expression plasmid is converted Into escherichia coli (Escherichia coli) BL21 (DE3), currently preferred genetic engineering bacterium can be obtained.

A method of it preparing the recombination transaminase, including the fermented and cultured genetic engineering bacterium, and collects and make Standby recombination transaminase.

The step that the method includes under centainly production tank fermentation condition, carrying out recombinating transaminase described in preparation of industrialization Suddenly；The production tank fermentation condition is preferred: 35% or more DO, air mass flow 1:1.5vvm.

Recombination transaminase of the present invention is applied to asymmetric syntheses chirality aminated compounds, especially for synthesizing west Ta Lieting intermediate (R) -3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate.

The transaminase-catalyzed reaction of present invention recombination is as follows:

It is preferred that: in the reaction solution of pH 6.5~7.5, in the presence of pyridoxime 5-phosphate, in recombination of the present invention Under the effect of transaminase mutant, reduction is catalyzed as substrate enantioselective enzyme using compound A and prepares optics chiral amine compound

Wherein, R is alkyl or substituted heteroaryl, preferably C_nH_2n+1And benzyl, wherein n is selected from 1~8 integer.

A kind of synthetic method of sitagliptin, reaction route are as follows:

The utility model has the advantages that

The present invention by chemistry, molecular biology, bioinformatics and high-throughput screening method, exploitation to it is a kind of with compared with The recombination transaminase mutant of high specific (enantiomeric excess value > 99.5%) and preferably catalytic activity, for catalyzing and synthesizing hand Property amine compounds.The co-factor of recombination transaminase of the invention is pyridoxime 5-phosphate PLP.

Enzyme according to the present invention have excellent stereoselectivity, regioselectivity and catalysis activity, and its catalysis Reaction is mild, and the yield of reaction conversion ratio and product is high, with good application prospect.

Specific embodiment

The foundation of 1 genetic engineering bacterium of embodiment

The gene order of Aspergillus terreus (the Aspergillus terreus NIH2624) transaminase included according to Genbank (NCBI accession number: XM_001209325.1), the artificial synthesized genetic fragment extend segment (the segment two sides by PCR amplification Add I incision enzyme gene segment of Nde I and BamH), nucleotide sequence is as shown in SEQ ID NO.3.And utilize Nde I and BamH I Gene is inserted into pET21a plasmid by restriction enzyme site, and the carrier after connection is transferred to foundation in e. coli bl21 (DE3) and is turned Adnosine deaminase genetic engineering bacterium.The wherein primer of PCR amplification aminotransferase gene are as follows: forward primer F1:GGGGCCATATGGCCTCCATG GACAAAGTCTTT (SEQ ID NO.4), reverse primer R1:GGGCCGGATCCCGTTATAATCAATCTCGAAGC (SEQ ID NO.5)。

The acquisition of 2 transaminase mutant gene of embodiment

This research and utilization fallibility PCR method has carried out protein engineering transformation to transaminase.Fallibility PCR is using DNA When polymerase carries out target gene amplification, by adjusting reaction condition, magnesium ion concentration is such as improved, manganese ion is added, changes body Four kinds of dNTPs concentration or utilization low fidelity archaeal dna polymerase etc. in system, to change the frequency of mutation in amplification procedure, thus It is randomly incorporated into mutation into target gene with certain frequency, obtains the random mutant of protein molecule.This research is using guarantor It really spends lower polymerase to be easy to mix the principle of random mutation into amplified production under certain measures, while utilizing Mn²⁺ Substitute natural confactor Mg²⁺Increase fallibility probability.

50 μ l PCR reaction systems are as follows: 10 × amplification buffer 5 μ l, 4 kinds of each 4 μ l of dNTP mixture (2.5mmol/L) draw Each 50pmol of object, 1.5 μ g, Taq archaeal dna polymerase of template DNA 0.5 μ L, Mg²⁺2mmol/L adds distilled water to 50 μ l.

PCR amplification program are as follows: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 45s, 55 DEG C of denaturation 30s, 72 DEG C of denaturation 120s, into Row 30 circulations；Continue to extend 10min at 72 DEG C, is cooled to 4 DEG C.The genetic fragment of amplification is after detecting with 1% agarose Gel electrophoresis recycling, and amplified production is purified, remove extra primer.

Experiment flow

PCR amplification aminotransferase gene and gene is inserted using Nde I and I restriction enzyme site of BamH according to the method for embodiment 1 Enter into pET21a plasmid, as gene mutation template；

The gene of fallibility PCR amplification transaminase, genetic fragment links to pET21a carrier after amplification, by the carrier after connection It is transferred in e. coli bl21 (DE3) and establishes aminotransferase gene mutant library；It is host using e. coli bl21 (DE3), PET21a plasmid is carrier, expression extension transaminase, high flux screening high activity mutant strain；To high activity transaminase base after mutation Because being identified.The nucleic acid sequence of the high activity transaminase mutant gene filtered out is as shown in SEQ ID NO.1.

Aminotransferase gene primer are as follows: forward primer F1:GGGGCCATATGGCCTCCATGGACAAAGTCTTT (SEQ ID NO.4), reverse primer R1:GGGCCGGATCCCGTTATAATCAATCTCGAAGC (SEQ ID NO.5).

The genetic engineering bacterium for expressing the transaminase mutant is constructed by 1 the method for embodiment.

After obtaining mutant sequence, I He of Nde is utilized after the gene order can also be synthesized by chemical synthesis mode Gene is inserted into pET21a plasmid by I restriction enzyme site of BamH, then converts Escherichia coli building genetic engineering bacterium.

It is prepared by the shaking flask of 3 transaminase of embodiment

Embodiment 1, embodiment 2 are constructed into resulting recombination bacillus coli and are seeded to 50mL respectively containing ampicillin (100 μ g/mL) LB culture medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH7.2) in, in 37 DEG C, 200rpm's is shaken Shaken cultivation 16 hours or more in bed.2mL bacterial culture fluid of transferring is cultivated in LB of the 50mL containing chloramphenicol (or ampicillin) In base, it is placed in shaken cultivation under similarity condition, light absorption value of the Timing measurement bacterium solution at 600nm is to monitor thalli growth density. When 600 value of OD of bacterium solution is in 0.6-0.8, inducer IPTG to final concentration of 0.2mmol/L, 30 DEG C of inducing expressions 12 are added Hour or more.Thallus is collected by centrifugation (5000rpm, 15min, 4 DEG C) after expression, and with phosphate buffer (pH7.2,50mM) Cleaning twice, is scattered in the buffer being equally pre-chilled, ultrasonication is carried out in ice-water bath.It is centrifuged (8000rpm, 30min, 4 DEG C) supernatant is collected, concentration, freeze-drying obtain recombination transaminase or recombinate the crude powder of transaminase mutant.

The measurement of the recombination aminotransferase activity of embodiment 4

With 3- carbonyl -4- (2,4,5- trifluorophenyl) methyl butyrate for substrate, HPLC detects recombination prepared by embodiment 3 and turns Adnosine deaminase activity, so that the genetic engineering bacterium with highest transaminase enzyme activity can be expressed by filtering out.It is efficient using Shimadzu LC-20AT Liquid chromatograph is equipped with C18 column (150mm*4.6mm, 5um), 0.1% trifluoroacetic acid aqueous solution of mobile phase-methanol (40:60), Detection wavelength 254nm, 30 DEG C of column temperature, flow velocity 1.0mLmin^-1。

Testing conditions are as follows: reaction system total volume 2mL, wherein including 0.5-50 μm of ol/L substrate, 200 μ L 1mol/L are different Propylamin hydrochloride, 100 μm of ol pyridoxime 5-phosphate, 5%-25% (v/v) DMSO, 500 μ L enzyme solutions, 45 DEG C of reaction 30min are surveyed Determine the consumption and product (R) -3- amino -4- (2,4,5- trifluoro of substrate 3- carbonyl -4- (2,4,5- trifluorophenyl) methyl butyrate Phenyl)-methyl butyrate production quantity.

Enzyme activity unit (U) is defined as: under the above-described reaction conditions, catalysis generates 1 μm of ol (R) -3- amino-per minute Enzyme amount needed for 4- (2,4,5- trifluorophenyl)-methyl butyrate or per minute 1 μm of ol substrate 3- carbonyl -4- (2,4,5- tri- of consumption Fluorophenyl) enzyme amount needed for methyl butyrate.

The enzyme activity for measuring recombination transaminase mutant is 926.2U/L, than original enzyme activity (NCBI accession number: XM_ 001209325.1) 1.1 times are improved.

Embodiment 5 recombinates the fermentation preparation of transaminase

Fermentation medium components are as follows: ammonium sulfate 0.76g/L, ironic citrate 0.06mg/L, NaCl 10.5g/L, phosphoric acid hydrogen two Potassium 12.2g/L, potassium dihydrogen phosphate 6.1g/L, yeast extract 11.5g/L, peptone 7.1g/L, glycerol 17.3g/L, ammonium molybdate Tetrahydrate 0.01g/L.Fermentation liquid maintains pH 7.0 by the way that ammonium hydroxide is added, and 37 DEG C of tank temperature, speed of agitator 300-1300rpm, sends out 35% or more DO, air mass flow 1:1.5vvm are controlled during ferment.Access 1205 seed liquor of E.Coli BL21 (DE3) ST OD600 is 0.68, and access amount is the 10% of fermentating liquid volume, and IPTG to final concentration 1mmol/ is added when fermentation liquid OD600 is up to 0.8 Hereafter L continues fermentation 15 hours, 25 DEG C of tank temperature to induce the expression of transaminase.By the way that 460g/ containing glycerol is added in fermentation process L, yeast extract 150g/L, ammonium chloride 11.5g/L, ammonium molybdate tetrahydrate 0.04mg/L, ironic citrate 0.06mg/L it is molten The growth of liquid maintenance culture.Culture is cooled to 4 DEG C of preservations after fermentation.

Fermentation liquid is centrifuged the conventional treatments such as (8000rpm, 10min), clasmatosis, freeze-drying, is prepared and turns Adnosine deaminase polypeptide freeze-dried powder is simultaneously saved in -80 DEG C.

Embodiment 6 recombinates transaminase-catalyzed synthesis (R) -3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate (XT-4)

32.6kg substrate 3- carbonyl -4- (2,4,5- trifluorophenyl)-methyl butyrate, 132kg are added into 2000L reaction kettle Isopropylamine hydrochloride, 0.55kg pyridoxime 5-phosphate (PLP), 550L DMSO, then to being added in kettle according to 5 method of embodiment The recombination transaminase freeze-dried powder 29.5kg of preparation is warming up to 45 DEG C under nitrogen protection, and 4mol/L isopropylamine solution is added The pH for adjusting reaction solution is 8.5.The pH for controlling reaction solution in reaction process by the way that 4mol/L isopropylamine solution is added is 8.5- 9.0, it is stirred to react 22 hours or so, until substrate content terminates reaction when being down to 3% or less.

It is operated after reaction terminating through centrifugation, extraction, decoloration etc., obtains product (R) -3- amino -4- (2,4,5- trifluoro-benzenes Base)-methyl butyrate:¹H NMR(CDCl₃)d7.08(m,1H),6.94(m,1H),3.85(s,2H),3.77(s,3H),3.55(s, 2H)；¹³C NMR(CDCl₃)d 197.9,167.1,156.9,154.5,150.4,147.9,145.5,119.4,117.0, 105.5,52.4,48.3,41.9.It is analyzed through HPLC, determines the substrate transformation rate 97.6%, product ee 99.5%.

The concrete analysis condition of product ee value are as follows: Chiralpak AD-H chromatographic column (150mm × 4.6mm, 5um), flowing Phase n-hexane-isopropanol (80:20, v/v)；Detection wavelength 254nm, flow velocity 1mL/min, 30 DEG C of column temperature.

Embodiment 7 (R) -3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate (XT-4)

The addition 90ml dimethyl sulfoxide in reactor, 3- carbonyl -4- (2,4,5- trifluorophenyl)-methyl butyrate of 15g, The isopropyl amine salt aqueous acid 90ml of pH=9.00 is added in stirring and dissolving, and crude enzyme liquid 120g and 0.08g phosphoric acid pyrrole is added and trembles Aldehyde, 35 DEG C are stirred to react for 24 hours, are during which maintained between pH8.00-8.60 with isopropylamine solution, and acid adding terminates reaction, then 35 DEG C Insulated and stirred 1h, is filtered to remove albumen, and water phase sodium hydrate aqueous solution tune pH to 10-11 is extracted with dichloromethane three times, closes And the dry 2h of anhydrous sodium sulfate is added in methylene chloride, obtains product 14g, 99% or more purity after recycling methylene chloride.

Embodiment 8 (R) -3- t-butoxycarbonyl amino -4- (2,4,5- trifluorophenyl)-methyl butyrate (XT-5)

14.8g (R) -3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate (0.06mol) is weighed, 50ml bis- is dissolved in In chloromethanes, 1ml triethylamine is added, 5 DEG C or less addition 14.4g di-tert-butyl dicarbonates (0.066mol) are warming up to room temperature Reaction 15 hours is added 30ml water quenching and goes out, collects organic phase, and water phase is extracted with dichloromethane 2 times again, merges organic phase, is added Anhydrous sodium sulfate is dry, rotates recycling design, obtains 18.7g white solid, yield 90%.

Embodiment 9 (R) -3- t-butoxycarbonyl amino -4- (2,4,5- trifluorophenyl)-butyric acid (XT-6)

By 17.4g (R) -3- t-butoxycarbonyl amino -4- (2,4,5- trifluorophenyl)-methyl butyrate (0.05mol), dissolution In the in the mixed solvent of 120ml methanol and 60ml water, 6g sodium hydroxide (0.15mol) is added, is stirred to react at 40-45 DEG C 1.5 hours, revolving removed partial solvent, and it is 2-3 that 1N hydrochloric acid tune pH is added under ice bath, is extracted with ethyl acetate 3 times, is associated with Machine phase, organic to be added to the dry 2h of anhydrous sodium sulfate with brine It 1 time, filtering rotates recycling design and obtains white solid 15.7g, yield 94%.

10 7- of embodiment [(3R) -3- t-butoxycarbonyl amino -1- oxo -4- (2,4,5- trifluorophenyl) butyl] -5,6, 7,8- tetrahydro -3- Trifluoromethyl-1,2,4- triazol [4,3-a] pyrazine (XT-7)

Under nitrogen protection, by 8.6g (R) -3- t-butoxycarbonyl amino -4- (2,4,5- trifluorophenyl)-butyric acid Simultaneously piperazine hydrochloride (0.045mol) is dissolved in 50ml methylene chloride for (0.045mol), 10.28g trifluoromethyl triazole, ice Bath is lower to be added 7.27g 1- hydroxy benzo triazole (0.054mol) and 10.3g 1- ethyl -3- (3- dimethylamino-propyl) phosphinylidyne 13.5g triethylamine is added dropwise in inferior amine salt hydrochlorate (0.054mol), and lower reaction 24 hours, reaction solution 50ml water washing 3 is stirred at room temperature Secondary, organic anhydrous sodium sulfate that is added to is 2 hours dry, and recycling design obtains 20.9g, yield 91%.

11 Sitagliptin phosphate of embodiment

It weighs 10.14g XT-7 (0.02mol) and is dissolved in 100ml concentrated hydrochloric acid: the in the mixed solvent of ethyl alcohol (1:5), room temperature It is stirred to react 4 hours, revolving removes ethyl alcohol, is neutralized, is extracted with ethyl acetate 3 times with sodium bicarbonate, merges organic phase, and nothing is added Aqueous sodium persulfate is 2 hours dry, filtering, and after recycling design, under nitrogen protection, 80ml isopropanol and 10ml water is added, and 85 DEG C are stirred, 85% phosphoric acid of 3g is added, 85 DEG C are stirred to react 2 hours, are cooled to room temperature and are stirred for 10 hours, filtering drying obtains white Xi Talie Spit of fland phosphate 9.09g, yield 90%.

12 Sitagliptin phosphate pilot scale of embodiment preparation

It weighs 5.1kg XT-7 (1mol) and is dissolved in 50L concentrated hydrochloric acid: the in the mixed solvent of ethyl alcohol (1:5), be stirred at room temperature anti- It answers 4 hours, revolving removes ethyl alcohol, is neutralized, is extracted with ethyl acetate 3 times with sodium bicarbonate, merges organic phase, and anhydrous slufuric acid is added Sodium is 2 hours dry, filtering, and after recycling design, under nitrogen protection, 45L isopropanol is added: the mixed solvent of water (8:1), 85 DEG C are stirred It mixes, 85% phosphoric acid of 1.5kg is added, continue 85 DEG C and be stirred to react 2 hours, be cooled to room temperature and be stirred for 10 hours, filtering drying obtains White Sitagliptin phosphate 4.71kg, yield 93%.

<110>Jiangsu Alpha Pharmaceutical Co., Ltd.

<120>a kind of recombination transaminase and its preparation method and application

<160> 5

<210> 1

<211> 978

<212> DNA

<213>artificial sequence

<220>

<223>encoding gene of transaminase mutant is recombinated

<400> 1

atggcctcca tggacaaagt ctttgccggc tacgccgccc gccaagcgat cctcgaatca 60

accgagacca ccaacccctt tgcgaagggt atcgcctggg tagaaggcga gctggtgccc 120

ctggcagagg cacgcattcc actgctcgac cagggcttca tgaaaagcga tctcacctac 180

gacgtgccct ccgtctggga cggccgcttc ttccggctag acgaccacat cacgcggctc 240

gaagccagct gcaccaagct ccggctgcga ctgccactcc cgcgcgacaa agtcaagcag 300

attctcgtcg agatggtgcg caagagcggc atccgcgacg cctttgtcga gctgatcgtg 360

acgcgcgggc tgaagggcgt gcgggggaca cgccccgagg acatcgtcaa caatctgtac 420

atgtttgtgc agccgtacgt gtgggtgatg gagccggata tgcagcgtgt cggcggcagc 480

gcggtcgtcg cccgcaccgt gcgccgggtg cccccgggtg ccatcgaccc aaccgtcaag 540

aacctgcaat ggggcgatct cgtgcgcggc atgttcgagg ctgcggatcg cggtgcaact 600

tatccgttct tgacggacgg agatgcccat ctcaccgaag gctctgggtt caatattgtg 660

ctcgtcaagg acggcgtgct gtacacacca gaccgtggtg tgctgcaggg cgtgacacga 720

aagagtgtta tcaatgcggc ggaagccttc gggattgaag tccgcgttga gtttgtgccg 780

gttgagctgg cgtaccgttg tgatgagatc tttatgtgta ccaccgctgg cggcatcatg 840

cctatcacta cgctggatgg gatgcccgtg aatggaggac agatcggtcc tattacgaag 900

aagatttggg atggatattg ggctatgcat tatgatgcgg cttacagctt cgagattgat 960

tataacgaga ggaactga 978

<210> 2

<211> 325

<212> PRT

<213>artificial sequence

<220>

<223>transaminase mutant is recombinated

<400> 2

Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala

5 10 15

Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala

20 25 30

Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu

35 40 45

Leu Asp Gln Gly Phe Met Lys Ser Asp Leu Thr Tyr Asp Val Pro Ser

50 55 60

Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu

65 70 75 80

Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp

85 90 95

Lys Val Lys Gln Ile Leu Val Glu Met Val Arg Lys Ser Gly Ile Arg

100 105 110

Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg

115 120 125

Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln

130 135 140

Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser

145 150 155 160

Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp

165 170 175

Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe

180 185 190

Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp

195 200 205

Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp

210 215 220

Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg

225 230 235 240

Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val

245 250 255

Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met

260 265 270

Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met

275 280 285

Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp

290 295 300

Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp

305 310 315 320

Tyr Asn Glu Arg Asn

325

<210> 3

<211> 978

<212> DNA

<213>Aspergillus terreus (Aspergillus terreus) NIH2624

<220>

<223>encoding gene of transaminase

<400> 3

atggcctcca tggacaaagt ctttgccggc tacgccgccc gccaagcgat cctcgaatca 60

accgagacca ccaacccctt tgcgaagggt atcgcctggg tagaaggcga gctggtgccc 120

ctggcagagg cacgcattcc actgctcgac cagggcttca tgcacagcga tctcacctac 180

gacgtgccct ccgtctggga cggccgcttc ttccggctag acgaccacat cacgcggctc 240

gaagccagct gcaccaagct ccggctgcga ctgccactcc cgcgcgacca ggtcaagcag 300

attctcgtcg agatggtggc caagagcggc atccgcgacg cctttgtcga gctgatcgtg 360

acgcgcgggc tgaagggcgt gcgggggaca cgccccgagg acatcgtcaa caatctgtac 420

atgtttgtgc agccgtacgt gtgggtgatg gagccggata tgcagcgtgt cggcggcagc 480

gcggtcgtcg cccgcaccgt gcgccgggtg cccccgggtg ccatcgaccc aaccgtcaag 540

aacctgcaat ggggcgatct cgtgcgcggc atgttcgagg ctgcggatcg cggtgcaact 600

tatccgttct tgacggacgg agatgcccat ctcaccgaag gctctgggtt caatattgtg 660

ctcgtcaagg acggcgtgct gtacacacca gaccgtggtg tgctgcaggg cgtgacacga 720

aagagtgtta tcaatgcggc ggaagccttc gggattgaag tccgcgttga gtttgtgccg 780

gttgagctgg cgtaccgttg tgatgagatc tttatgtgta ccaccgctgg cggcatcatg 840

cctatcacta cgctggatgg gatgcccgtg aatggaggac agatcggtcc tattacgaag 900

aagatttggg atggatattg ggctatgcat tatgatgcgg cttacagctt cgagattgat 960

tataacgaga ggaactga 978

<210> 4

<211> 978

<212> DNA

<213>artificial sequence

<220>

<223>primers F 1

<400> 4

ggggccatat ggcctccatg gacaaagtct tt 32

<210> 5

<211> 978

<212> DNA

<213>artificial sequence

<220>

<223>primer R1

<400> 5

gggccggatc ccgttataat caatctcgaa gc 32

Claims

1. the recombination transaminase mutant that a kind of pyridoxime 5-phosphate relies on, it is characterised in that amino acid sequence such as SEQ ID Shown in NO.2.

2. a kind of gene for encoding recombination transaminase mutant described in claim 1, it is characterised in that nucleotide sequence such as SEQ Shown in ID NO.1.

3. a kind of expression vector containing gene as claimed in claim 2.

4. expression vector according to claim 3, it is characterised in that expression vector system pET21a.

5. a kind of for producing the genetic engineering bacterium of recombination transaminase mutant described in claim 1, it is characterised in that described Contain gene as claimed in claim 2 in genetic engineering bacterium.

6. genetic engineering bacterium according to claim 5, it is characterised in that the host cell of the genetic engineering bacterium is large intestine Escherichia (Escherichia coli) BL21 (DE3).

7. a kind of preparation method of recombination transaminase mutant described in claim 1, it is characterised in that include the following steps: to train Genetic engineering bacterium described in any one of claim 5~6 is supported, the recombination transaminase mutant of recombinant expression is obtained.

8. according to the method described in claim 7, it is characterized in that carrying out work the method includes in the case where producing tank fermentation condition Industryization prepares the step of recombination transaminase mutant；The production tank fermentation condition are as follows: DO35% or more, air mass flow 1:1.5vvm。

9. recombination transaminase mutant described in claim 1 is as catalyst in preparation (R) -3- amino -4- (2,4,5- trifluoro Phenyl) application in-methyl butyrate.

10. a kind of synthetic method of sitagliptin, it is characterised in that reaction route is as follows:

Wherein the enzyme is recombination transaminase mutant described in claim 1.