CN105925506A - Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate - Google Patents

Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate Download PDF

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CN105925506A
CN105925506A CN201610369158.5A CN201610369158A CN105925506A CN 105925506 A CN105925506 A CN 105925506A CN 201610369158 A CN201610369158 A CN 201610369158A CN 105925506 A CN105925506 A CN 105925506A
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buffer
zjph1504
pseudomonas aeruginosa
butyl
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王普
孙佳
黄金
沈琦
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses a pseudomonas aeruginosa ZJPH1504 and an application of pseudomonas aeruginosa ZJPH1504 in preparation of a sitagliptin chiral intermediate. The pseudomonas aeruginosa ZJPH1504 can be used for asymmetric reduction of a prochiral ketone compound (II) with high stereoselectivity to prepare a sitagliptin chiral intermediate (I-a) compound, the product prepared by using the pseudomonas aeruginosa ZJPH1504 is high in optical purity, and the e.e. (Errors Excepted) value is greater than 99.9%, 9g/L of a substrate is added in a phosphate buffer solution system of which the pH is 7.5, and reacted for 30 hours, and the yield of an S-reduction product is 60.2%. When an organic solvent is added in a reaction system, the catalytic efficiency can be effectively improved, the reaction yield can be increased, and the reaction time can be shortened, especially when di-n-butyl phthalate is added in the reaction system, the reaction yield can be increased to 75.6%, the reaction time can be shortened to 24 hours and the e.e. value is greater than 99.9%.

Description

Pseudomonas aeruginosa ZJPH1504 and prepare the application of sitagliptin chiral intermediate
(1) technical field
The present invention relates to the preparation of a kind of sitagliptin chiral intermediate, utilize new strains particularly to one--Pseudomonas aeruginosa The method of ZJPH1504 living things catalysis asymmetric synthesis sitagliptin chiral intermediate.
(2) background technology
3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) Butyl-1-ketone (I) (its molecular weight: 408.102) is to prepare formula III compound (sitagliptin, Sitagliptin, trade name) Key intermediate, wherein compound (I) S configuration, i.e. formula (I-a) are preparing in sitagliptin key chirality of addressing of the present invention Mesosome.The chemical name of formula III is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydrochysene-1,2,4-triazol [4,3-a] pyrazine -7-base]-4-(2,4,5-trifluorophenyl) butyl-1-ketone.
Formula (I-a) is (the S)-isomer of formula I, and formula (I-b) is (the R)-isomer of formula I
Sitagliptin is by the exploitation of Merck Sharp&Dohme Ltd. company, in Nikkei U.S. FDA October 17 in 2006 First dipeptidyl peptidase-4 (DPP-4) inhibitor of approval listing, can be used for treating type ii diabetes.Formula III is in patent WO Its concrete ingredient disclosed in 03004498.
WO 2010032264 discloses formula I compound, and electronation syntheti c route 1: under (non-) acid condition, In suitable solvent, utilize the suitably reducing agent Han borine, by formula II compound 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro -[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl) butyl-2-ketone or its metal salt back, it is thus achieved that formula I Raceme compound 3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5- Trifluorophenyl) butyl-1-ketone.
Syntheti c route 1:
This route there is trace metal thing to remain, simultaneously in production process owing to the use of metallic catalyst causes in reacting final product Use a large amount of organic solvent, it is thus achieved that product be raceme, for non-selective catalytic chemosynthesis.
WO 2012/046254 A2 discloses by building coexpression oxidoreductase and the engineering bacteria of glucose dehydrogenase, use Prepare the syntheti c route 2 of (I-a) in catalysis formula II asymmetric reduction, the response time is 25～30h, and productivity is 72%, e.e. Value > 99%.The transformation efficiency of this route is the most relatively low, and in reaction system, biocatalyzer dosage is big, unit thalline converted product Production capacity is on the low side.
WO 2015/071861 A2 discloses and utilizes penicillium Penicillium brevicompactum MTCC 5962 biological Converting the process program of preparation optical activity formula (I-a), react 48h, conversion ratio is 99%, e.e. value > 99%.This route will be anti- Answer process to carry out with thalline sweat simultaneously, add the separating difficulty of late-stage products.
Syntheti c route 2:
(3) summary of the invention
It is an object of the present invention to provide a strain and produce the microorganism new strains-Pseudomonas aeruginosa ZJPH1504 of carbonyl reductase, utilize complete Cell catalysis 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-1-(2,4,5-trifluoro-benzene Base) butyl-2-ketone asymmetric reduction prepares (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine -7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1-ketone.Compared with electronation route, the stereo selectivity of the method is high, catalysis Agent preparation cost is low, and conversion process environmental friendliness.And it is difficult effectively to solve substrate by building a kind of organic medium reaction system Dissolubility and the problem such as productivity is on the low side.
The technical solution used in the present invention is:
The present invention provides strain new strains Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504, in being preserved in State's Type Tissue Collection, deposit number: CCTCC NO:M 2016188, preservation date is: April 11 in 2016 Day, preservation address is Wuhan, China, Wuhan University, postcode 430072.
The soil that Pseudomonas aeruginosa ZJPH1504 of the present invention gathers in being isolatable from Zhejiang Polytechnical University's (Zhejiang Hangzhou) campus Sample, as follows through following separation screening flow process: soil sample collection → enrichment culture → flat board primary dcreening operation → bacterial strain purification → liquid culture → work Property sieves again → and TLC/HPLC detects product → acquisition purpose bacterial strain.
The present invention also provides for a kind of described Pseudomonas aeruginosa ZJPH1504 application in preparing sitagliptin chiral intermediate, Concrete described application is with the fermented wet thallus obtained of cultivating of Pseudomonas aeruginosa ZJPH1504 for enzyme source, with 4-oxygen-4-[3-(three Methyl fluoride)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl) butyl-2-ketone is substrate, at least Be that reaction medium is constituted reaction system with the buffer (preferably pH 7～8) of pH 6～9,25～50 DEG C (preferably 40～45 DEG C), React under the conditions of 150～250rpm (preferably 200rpm), after reaction terminates, reactant liquor is isolated and purified, it is thus achieved that (S)-3- Hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1- Ketone.
Further, described substrate consumption is calculated as 2～21g/L (preferably 4～9g/L) with buffer volume, and wet thallus consumption is with buffering Liquid is long-pending is calculated as 50～350g/L (preferably 100～150g/L).
Further, described enzyme source is prepared as follows: (1) slant culture: be seeded to by Pseudomonas aeruginosa ZJPH1504 Slant medium, cultivates 36h for 25～30 DEG C, it is thus achieved that slant strains；Slant medium final concentration consists of: glucose 15g/L, Peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, agar powder 15～20g/L, solvent is water, pH 6.5；
(2) seed culture: slant strains is seeded in seed culture medium, 25～30 DEG C, 150～250rpm cultivation 12～24h, Obtain seed liquor；Seed culture medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, solvent is water, pH 6.5；
(3) fermentation culture: be 4 by seed liquor with volumetric concentration～the inoculum concentration of 10% (preferably 8%) is seeded to fermentation medium In, liquid amount is 90mL/250mL shaking flask, and 25～30 DEG C, 150～250rpm cultivate 24～28h, preferably 30 DEG C, 200rpm Cultivate after 28h fermentation ends, fermentation liquid is centrifuged, gained precipitation 0.1M, pH 8.0 buffer solution, collect wet thallus, It is enzyme source；Fermentation medium final concentration consists of: maltose 30.45g/L, peptone 17.31g/L, NaCl 0.98g/L, Solvent is water, pH 7.5.
Further, described buffer Application Range includes phosphate buffer, Tris-HCl buffer, and at 0.01M and 0.2M Ionic strength range in select.Phosphate buffer ionic strength range used is 0.01～0.2M, preferably 0.1M.
Further, for promoting regenerating coenzyme, improve reaction efficiency, described reaction system also needs to add cosubstrate, described auxiliary It is one of following for helping substrate: glucose, maltose, sucrose, methanol, ethanol, isopropanol, glycerol, ALANINE, L-paddy Propylhomoserin and Cys, described cosubstrate addition is calculated as 4～100g/L with buffer volume.Preferably, described reaction With the one in glucose, sucrose and glycerol as cosubstrate, most preferably glycerol (preferably 40～80g/L, most preferably 60g/L). React 24h under the same terms, be 14.7% (aqueous phase reforming, referring specifically to enforcement without the living things catalysis productivity of cosubstrate Example 12), add 60g/L glycerol make the living things catalysis productivity of cosubstrate can improve to 55.7% (aqueous phase reforming, referring specifically to Embodiment 19).
Present invention reaction is suppressed by metal ion, adds metal ion, including Co in reaction system2+(CoCl2·6H2O)、 Cu2+(CuCl2·2H2O)、Mg2+(MgSO4·7H2And Mn O)2+(MnCl2·4H2O), anti-to conversion of the present invention Inhibitory action (referring specifically to embodiment 23,24,25,26) should be there is；Add dithiothreitol, DTT (DTT) and ethylenediamine tetraacetic Acetic acid disodium (EDTA), there is also inhibitory action (referring specifically to embodiment 27,28) to conversion reaction of the present invention.
Further, reaction medium of the present invention is the mixed liquor of organic solvent and pH 6～9 (preferably pH 7.5) buffer, institute It is one of following for stating organic solvent: normal hexane, dichloromethane, isopropyl acetate, N,N-dimethylformamide (DMF), two Methyl sulfoxide (DMSO), glycerol and dibutyl phthalate, in described organic solvent, glycerol addition is with buffer volume Being calculated as 20g/L, the volume dosage of other organic solvents is calculated as 1～20% with buffer volume, and preferably 5%.Preferably glycerine or neighbour Phthalic acid dibutyl ester, living things catalysis efficiency increases.Most preferably dibutyl phthalate, can improve conversion yield, contracting The short conversion reaction time.
Additionally, the mixed liquor that another type reaction medium of the present invention is ionic liquid and pH 6～9 buffer.Described ion Liquid mainly has: 1. conventional ion liquid, including 1-octyl group-3-Methylimidazole. hexafluorophosphate, tetramethyl-ammonium hexafluorophosphate, 1-octyl group-3-methyl imidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium nitrate, N-butyl-pyridinium tetrafluoroborate, tetramethyl Ammonium tetrafluoroborate.2. amino acid ion liquid, including tetramethyl-ammonium glutamic acid ([N1,1,1,1]+[Glu]-), tetraethyl ammonium paddy Propylhomoserin ([N2,2,2,2]+[Glu]-), preparation method sees Chinese patent CN 103695480 A；Tetramethyl-ammonium cysteine ([N1,1,1,1]+[Cys]-), preparation method sees document (Bioresource Technology 175 (2015) 42-50).The lowest common Molten class ionic liquid, including [choline chloride]+[glycine]-, preparation method sees document (Journal of Molecular Catalysis B:Enzymatic 122(2015)188-198)；[choline chloride]+[glutathion]-, preparation method sees Chinese patent CN 103709230 A.Each ionic liquid anion compound is 1:1 with the mol ratio of cationic compound.Described ionic liquid body constitution Amount addition is calculated as 0.1～10g/L with buffer volume, preferably 5g/L.Especially, when adding [choline chloride]+[glycine]-Time, Living things catalysis productivity can be improved to 70.1% (being specifically shown in embodiment 37) by 60.2% (being specifically shown in embodiment 31) of buffer solution system.
Compared with prior art, the beneficial effects are mainly as follows: the present invention provides a strain to can be used for highly-solid selectively Asymmetric reduction prochiral ketone compound (II) prepares (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] Pyrazine-7-(8H)-yl] the microorganism new strains-Pseudomonas aeruginosa of-4-(2,4,5-trifluorophenyl) butyl-1-ketone (formula (I-a) compound) (Pseudomonas aeruginosa) ZJPH1504, utilizes this bacterial strain to prepare the optical purity height of products therefrom, e.e. value > 99.9%. In the phosphoric acid buffer liquid system of pH 7.5, adding the substrate of 9g/L, the productivity of reaction 30h, S-type reduzate is 60.2% (being specifically shown in embodiment 31).Catalytic efficiency can be effectively improved when reaction system is added organic solvent, improve reaction yield, And shorten the response time, reaction system is especially added dibutyl phthalate, reaction yield can be made to improve to 75.6%, Response time foreshortens to 24h, e.e. value more than 99.9% (being specifically shown in embodiment 32).
(4) accompanying drawing explanation
Fig. 1 is the colonial morphology of Pseudomonas aeruginosa ZJPH1504.
Fig. 2 is Pseudomonas aeruginosa ZJPH1504 cellular morphology (10 × 100 times, oil mirror) under an optical microscope.
Fig. 3 is that HPLC chromatogram detects collection of illustrative plates；A: formula (I-a) compound, formula (I-b) compound, formula II compound are mixed Compound；B: embodiment 1 Pseudomonas aeruginosa ZJPH1504 bacterial strain conversional solution sample.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that:
Embodiment 1: be used for being catalyzed reduction 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] microorganism strains filtration of-1-(2,4,5-trifluorophenyl) butyl-2-ketone
Enrichment culture: 1g fresh soil sample (soil sample gathered in Zhejiang Polytechnical University's (Zhejiang Hangzhou) campus) is joined dress Have in the 250mL shaking flask of 50mL enrichment medium, 30 DEG C, 200rpm, cultivate 5d, stand, take 1mL culture fluid and turn It is connected in fresh enrichment medium, continues to cultivate 5d, so repeat to be enriched with 3 times.Enrichment medium consists of: (NH4)2SO4 2g/L, KH2PO41g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, formula II compound (2g/L) is sole carbon Source, solvent is water, 6.5,120 DEG C of sterilizing 20min of pH.
Flat board primary dcreening operation: enrichment culture liquid normal saline dilution 104-106Plate screening culture medium, 30 DEG C of cultivations are coated after Bei 2d.Picking list bacterium colony carries out plate streaking again, and juxtaposition 30 DEG C cultivates 2d.Purification bacterium is obtained after 2 line separation and Culture Strain ZJPH1504, and it is preserved in solid slant culture base.Plating medium final concentration consists of: (NH4)2SO42g/L, KH2PO4 1g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, 20g/L agar powder, solvent is water, pH 6.5.Slant medium Final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO4 1.5 G/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, agar powder 20g/L, solvent is water, pH 6.5.120 DEG C of sterilizings 20min。
Microbial cells is cultivated and is collected: picking purification seed is seeded to seed culture medium, 30 DEG C, and 200rpm cultivates 12～24h, Being forwarded in Preliminary fermentation culture medium with the inoculum concentration of 10% (v/v), 30 DEG C, 200rpm cultivates 24～48h.By fermentation liquid In 4 DEG C, centrifugal 10min, supernatant discarded under the conditions of 9000rpm, and wash thalline twice with pH 8.0 phosphate buffer, then Secondary centrifugation obtains resting cell.Seed culture medium and Preliminary fermentation culture medium final concentration composition are: glucose 15g/L, Peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, solvent is water, pH 6.5.
Strain sieves again: prepare with microorganism resting cell for enzyme source catalytic substrate (formula II compound) Stereoselective reduction (S)-type product [formula I-a compound], reaction system is: 1g resting cell (in terms of weight in wet base) is resuspended in 10mL phosphoric acid and delays Rushing in liquid (0.1M, pH 8.0), add 2g/L substrate, cosubstrate made by 60g/L glycerol, 37 DEG C, and 200rpm reacts 24h.After reaction terminates, reactant liquor is extracted with ethyl acetate, and analyzes detection converted product (ginseng through thin plate chromatograph (TLC) According to method detection described in patent application CN 104893989 A), compare racemic modification reduzate formula I compound, observe raw Whether thing catalyzed samples occurs formula I reduzate speckle.Formula II compound can be reduced to formula I by bacterial strain ZJPH1504 Compound.Chirality HPLC method is used to detect the enantiomeric excess value (e.e. value) of this bacterial strain reduzate, after testing, e.e. Value > 99.9%.
Thin plate chromatography (TLC) qualitative analysis: developing solvent is ethyl acetate: petroleum ether: glacial acetic acid=80:20:0.1 (v/v)；Thin Laminate is GF254；Develop the color under the ultraviolet that wavelength is 254nm, formula I compound RfValue is 0.44, formula II chemical combination Thing RfValue is 0.64.
Liquid chromatography Qualitative and quantitative analysis: detection conversion reaction afterproduct and the content of remaining substrate, and calculate related substances Concentration, productivity (Yield) and e.e. value.
C in formula (1)i、C0The molar concentration of substrate when the molar concentration of product initiates with reaction at the end of being respectively reaction.
The optical purity of product is characterized by enantiomeric excess value (enantiomeric excess, e.e.).
In formula (2): CSAnd CRIt is respectively (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrrole Piperazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1-ketone and (R)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl] molar concentration of-4-(2,4,5-trifluorophenyl) butyl-1-ketone.
Pseudomonas aeruginosa (Pseudomonas aeruginosa) morphological characteristic of ZJPH1504 bacterial strain, physio-biochemical characteristics and 16S RDNA sequence:
1. morphological observation is on nutrient agar panel, observe the bacterium colony mode of appearance of bacterial strain ZJPH1504, quality, color and luster, The features such as the speed of growth, and somatic cells is carried out Gram’s staining film-making, by observation by light microscope cellular morphology.30 DEG C, Cultivate 36h, nutrient agar panel is formed the relatively petite that color is shallow, flat, moistening, sees Fig. 1；Under optical microscope considerable Observe bacterial strain ZJPH1504 cell in dispersion, straight or micro-bend shaft-like, in Gram-negative, see Fig. 2.
2. physio-biochemical characteristics utilize Biolog (GEN III) automatic microbe identification systems to analyze bacterial strain ZJPH1504 to carbon source Utilization power.This bacterial strain can utilize more by force and include dextrin, maltose, D-trehalose, D-cellobiose, gentiobiose, sugarcane Sugar, D-turanose, stachyose, D-Raffinose, α-D-lactose, D-6-(.alpha.-D-galactosido)-D-glucose., β-formyl-D-glucoside, D-salicin, N-acetyl-GLUCOSAMINE, D-Glucose, D-MANNOSE, D-Fructose, L-fucose, D-glucitol, PEARLITOL 25C, D-Ah NSC-11905, D-Fructose-6-phosphoric acid, D-Asp, D-Ser, e glycyl-L-PROLINE, ALANINE, L-paddy ammonia Acid, D-galacturonic acid, L-galacturonic acid acid lactone, methyl pyruvate, D-ALPHA-Hydroxypropionic acid methyl ester, polysorbate40, gamma-amino-butanoic acid and Alpha-hydroxy-butanoic acid is in 34 kinds of interior carbon sources；Meanwhile, sensitive to triacetyloleandomycin.
3. feature 16S rDNA sequential extraction procedures cell STb gene and with as template, utilize universal primer to carry out feature 16S RDNA sequence amplification, forward primer PI (27F) 5'-3':AGTTTGATCCTGGCTCAG, reverse primer P2 (1492R) 5'-3':GGTTACCTTGTTA CGACTT a, it is thus achieved that size is the DNA fragmentation of 1343bp.Through order-checking (Shanghai Sheng Gong biological engineering company limited), it is thus achieved that this bacterial strain 16S rDNA sequence (SEQ ID NO:1).
The 16S rDNA sequence of bacterial strain ZJPH1504 is carried out on NCBI website (http://www.ncbi.nlm.nih.gov) Sequence analysis (BLAST).Result shows: bacterial strain ZJPH1504 and the portion of Rhodopseudomonas (Pseudomonas sp.) Point strain sequence homology is higher, and reaches with the sequence homology of strain Pseudomonas aeruginosa N83 (JQ900543.1) To 98.6%.Therefore by named for bacterial strain ZJPH1504 Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504.
Embodiment 2: the chiral configuration of bacterial strain ZJPH1504 reduzate determines
With reference to there being the separation method of related substance described in patent 201510289729.X (publication number CN 104893989 A), will be real Execute the bacterial strain ZJPH1504 bioconversion broth equal-volume ethyl acetate obtained in example 1 to extract 2 times, solvent be evaporated off through rotation, By preparative thin layer chromatography, (developing solvent is ethyl acetate: petroleum ether: glacial acetic acid=80:20:0.1 (v/v/v) again；Lamellae is GF254；Develop the color under the ultraviolet of wavelength 254nm) separate, collect RfValue is the product of 0.44, obtains reduzate formula (I) compound.This material, corresponding to one of them peak in the HPLC chromatogram analysis chart of raceme formula I compound, is shown in Fig. 3. The specific rotatory power (SOR) recording this reduzate by standard method is: [α]D 25=21.68 (CHCl3, c=1), with patent WO After material specific rotatory power comparison disclosed in 2012/046254 A2, determine that this product is (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6- Dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1-ketone (formula (I-a) compound).
Embodiment 3: the acquisition in resting cell enzyme source under shake flask culture conditions
(1) slant culture: be seeded in slant medium by Pseudomonas aeruginosa ZJPH1504, cultivates 36h for 30 DEG C, it is thus achieved that Cultivate ripe slant strains.Slant medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6 G/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, agar powder 20g/L, Solvent is water, pH 6.5.
(2) seed culture: slant strains is seeded in seed culture medium, 30 DEG C, 200rpm cultivates and obtain seed after 12h Liquid.Seed culture medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO4 3 G/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, solvent is water, pH 6.5.
(3) fermentation culture: seed liquor is seeded in fermentation medium with the inoculum concentration that volumetric concentration is 8%, liquid amount 90 ML/250mL shaking flask, 30 DEG C, 200rpm cultivate 28h.After fermentation ends, fermentation liquid is centrifuged, gained precipitation phosphoric acid Buffer (0.1M, pH 8.0) washs 2 times, and every liter of fermentation liquid obtains wet thallus 3.6g (giving money as a gift heavily for 2.7g).Fermentation Culture medium final concentration consists of: maltose 30.45g/L, peptone 17.31g/L, NaCl 0.98g/L, and solvent is water, pH 7.5.
Embodiment 4:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 8.0) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L).Add 4g/L substrate 4-oxygen-4-[3-(trifluoromethyl)-5,6- Dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl) butyl-2-ketone (i.e. 0.04g formula II chemical combination Thing), add 60g/L (i.e. 0.6g) glycerol as cosubstrate, under different temperatures (25～50 DEG C, be shown in Table 1), The shaking table of 200rpm converts 24h.Add isopyknic ethyl acetate terminate reaction and extract 30min, centrifugal, take supernatant Organic facies carries out liquid-phase chromatographic analysis quantitatively (with embodiment 1) after treatment.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6- Dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone (formula I-a compound) It is shown in Table 1 with e.e. value.Preferable reaction temperature is 40～45 DEG C, and the productivity of formula I-a compound is about 92%, e.e. value > 99.9%.
The concentration of table 1 product [formula I-a compound] and ee value
Embodiment 5:
The K of 10mL 0.1M, pH 6.0 will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffering Liquid, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add 8g/L substrate (i.e. 0.08g formula II chemical combination Thing), add 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, the shaking table of 200rpm converts 24h.With Equal-volume ethyl acetate extracts, and uses liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] Triazol [4,3-a] pyrazine-7-(8H)-yl] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 19.3%, e.e. value > 99.9%.
Embodiment 6:
Buffer in embodiment 5 is replaced with the K of 10mL 0.1M, pH 7.02HPO4-KH2PO4Buffer, other Part is with embodiment 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 52.5%, e.e. value > 99.9%.
Embodiment 7:
Buffer in embodiment 5 is replaced with the K of 10mL 0.1M, pH 7.52HPO4-KH2PO4Buffer, other Part is with embodiment 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 55.7%, e.e. value > 99.9%.
Embodiment 8:
Buffer in embodiment 5 is replaced with the K of 10mL 0.1M, pH 8.02HPO4-KH2PO4Buffer, other Part is with embodiment 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 50.7%, e.e. value > 99.9%.
Embodiment 9:
Buffer in embodiment 5 replaces with the Tris-HCl buffer of 10mL 0.1M, pH 9.0, and other conditions are with real Execute example 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5- Trifluorophenyl) productivity of butyl-1-ketone is 34.8%, e.e. value > 99.9%.
Embodiment 10:
Buffer in embodiment 5 is changed into the K of 10mL 0.01M, pH 7.52HPO4-KH2PO4Buffer, other Part is with embodiment 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 15.7%, e.e. value > 99.9%.
Embodiment 11:
Change buffer in embodiment 5 K of 10mL 0.2M, pH 7.5 into2HPO4-KH2PO4Buffer, other conditions are same Embodiment 5.Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 35.5%, e.e. value > 99.9%.
Embodiment 12～22:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add substrate (the i.e. 0.08g formula II of 8g/L Compound), then it is separately added into glucose, maltose, sucrose, methanol, ethanol, isopropanol, glycerol, ALANINE, L-paddy Propylhomoserin, Cys as cosubstrate, with without any cosubstrate for comparison, 40 DEG C, in the shaking table of 200rpm Convert 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(fluoroform Base)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone and e.e. value be shown in Table 2.In preferred embodiments, when the glycerol of 60g/L is as cosubstrate, products collection efficiency is 54.7%, e.e. value > 99.9%.
Living things catalysis productivity when table 2 adds different cosubstrate and ee value
Embodiment 23:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add the substrate (i.e. formula II compound 0.08) of 8g/L, Add 60g/L (i.e. 0.6g) glycerol and, as cosubstrate, add CoCl2·6H2O makes Co2+Final concentration of 1mmol/L, 40 DEG C, the shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) fourth The productivity of-1-ketone is 45.1%, e.e. value > 99.9%.
Embodiment 24:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add substrate (the i.e. 0.08g formula II of 8g/L Compound), add 60g/L (i.e. 0.6g) glycerol and, as cosubstrate, add CuCl2·2H2O makes Cu2+Final concentration of 1mmol/L, converts 24h by 40 DEG C in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method inspection Survey, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-tri- Fluorophenyl) productivity of butyl-1-ketone is 1.7%, e.e. value > 99.9%.
Embodiment 25:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add 8g/L substrate (i.e. 0.08g formula II chemical combination Thing), add 60g/L (i.e. 0.6g) glycerol and, as cosubstrate, add MgSO4·7H2O makes Mg2+Final concentration of 1mmol/L, converts 24h by 40 DEG C in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method Detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5- Trifluorophenyl) productivity of butyl-1-ketone is 52%, e.e. value > 99.9%.
Embodiment 26:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add 8g/L substrate (i.e. 0.08g formula II chemical combination Thing), add 60g/L (i.e. 0.6g) glycerol and, as cosubstrate, add MnSO4·7H2O makes Mn2+Final concentration of 1mmol/L, converts 24h by 40 DEG C in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method Detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5- Trifluorophenyl) productivity of butyl-1-ketone is 45.2%, e.e. value > 99.9%.
Embodiment 27:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add 8g/L substrate (i.e. 0.08g formula II chemical combination Thing), addition 60g/L (i.e. 0.6g) glycerol, as cosubstrate, adds dithiothreitol, DTT (DTT) and makes final concentration of 1mmol/L, 40 DEG C, the shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) fourth The productivity of-1-ketone is 40.6%, e.e. value > 99.9%.
Embodiment 28:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add 8g/L substrate (i.e. 0.08g formula II chemical combination Thing), addition 60g/L (i.e. 0.6g) glycerol, as cosubstrate, adds disodiumedetate (EDTA) and makes end Concentration is 1mmol/L, 40 DEG C, converts 24h in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid phase color Spectral method detects, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)- Base] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 46.9%, e.e. value > 99.9%.
Embodiment 29:
10mL K will be suspended according to the wet thallus 1g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 100g/L of wet thallus (give money as a gift weight 74.6g/L), add final concentration be respectively 4,6,8,10,12,16, The substrate (i.e. formula II compound is respectively 0.04g, 0.06g, 0.08g, 0.12g, 0.16g, 0.20g) of 20g/L, Add 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, the shaking table of 200rpm converts 24h.With equal-volume Ethyl acetate extracts, and uses liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazole And [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) concentration of butyl-1-ketone, productivity, e.e. value be shown in Table 3.At preferred tranquillization When cell/initial substrate mass ratio is 1:0.06, converting 24h, production concentration reaches 10.3mM, and productivity is 68.4%, e.e. Value > 99.9%.
The bioconversion result of the different initial substrate concentration of table 3
Embodiment 30:
10mL will be suspended according to wet thallus 1g, 1.5g, 2g, 2.5g, 3g, 3.5g of embodiment 3 gained K2HPO4-KH2PO4In buffer (0.1M, pH 7.5), wet thallus final concentration is respectively 100,150,200,250,300, 350g/L, corresponding add final concentration of 6,9,12,15,18, (i.e. the dosage of formula II compound divides the substrate of 21g/L Wei 0.06g, 0.09g, 0.12g, 0.15g, 0.18g, 0.21g), add 60g/L (i.e. 0.6g) glycerol as auxiliary Substrate, converts 24h by 40 DEG C in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluoro-benzene Base) concentration of butyl-1-ketone, productivity be shown in Table 4.Consider productivity and products therefrom concentration, preferably resting cell concentration and the initial end When substrate concentration is respectively 150g/L, 9g/L, the productivity of product formula I-a compound is 55.5%.
Production concentration under different resting cell, initial substrate concentration and productivity in table 4 living things catalysis system
Embodiment 31:
10mL K will be suspended according to the wet thallus 1.5g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus (give money as a gift weight 111.9g/L), add the substrate (i.e. 0.09g) of 9g/L, add 60 G/L (i.e. 0.6g) glycerol, as cosubstrate, 40 DEG C, converts 30h in the shaking table of 200rpm.Extract with equal-volume ethyl acetate Take, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrrole Piperazine-7-(8H)-yl] productivity of-4-(2,4,5-trifluorophenyl) butyl-1-ketone is 60.2%, e.e. value > 99.9%.
Embodiment 32:
9.5mL K will be suspended according to the wet thallus 1.5g of embodiment 3 gained2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, system adds 0.5mL normal hexane, or 0.5mL dichloromethane, or 0.5mL isopropyl acetate, or 0.5mL is adjacent Phthalic acid dibutyl ester, or 0.5mL DMF, or 0.5mL dimethyl sulfoxide, or 2g glycerol (is used as Solvent and cosubstrate), add the substrate (i.e. adding 0.09g formula II compound) of final concentration of 9g/L, additionally, Add 0.6g glycerol (sequence number group 1 no longer adds this, i.e. the group using glycerol as solvent) as cosubstrate, 40 DEG C, 200rpm Shaking table in convert 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(three Methyl fluoride)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) productivity of butyl-1-ketone, e.e. Value is shown in Table 5.The preferably buffer solution system added with glycerol and the buffer solution system added with dibutyl phthalate, its catalytic reaction The productivity of product formula (I-a) compound is respectively 58.5%, 75.6%, and e.e. value is all higher than 99.9%.
Table 5 buffer adds the different organic solvents impact on bioconversion
Note: " nd " expression is not detected by
Embodiment 33:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds imidazoles, pyrrolidine, quaternary ammonium salts ionic liquid, including 1-octyl group-3- Methylimidazole. hexafluorophosphate, tetramethyl-ammonium hexafluorophosphate, 1-octyl group-3-methyl imidazolium tetrafluoroborate, 1-butyl-3-methyl Imidazole nitrate, N-butyl-pyridinium tetrafluoroborate, tetramethyl-ammonium tetrafluoroborate, ionic liquid final concentration is 5g/L. Add the substrate (i.e. formula II compound 0.09g) of final concentration of 9g/L, add 60g/L (i.e. 0.6g) glycerol as auxiliary Help substrate, 40 DEG C, the shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method inspection Survey, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-tri- Fluorophenyl) productivity of butyl-1-ketone and e.e. value be shown in Table 6.
Table 6 conventional ion liquid-buffer solution system adds the impact on bioconversion of the variety classes ionic liquid
Embodiment 34:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds the tetramethyl-ammonium glutamic acid of final concentration of 5g/L, adds final concentration of The substrate (i.e. formula II compound 0.09g) of 9g/L, add 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, The shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl Base-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1-ketone Productivity is 62.1%, e.e. value > 99.9%.
Embodiment 35:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds the tetraethyl ammonium glutamic acid of final concentration of 5g/L, adds final concentration of The substrate (i.e. formula II compound 0.09g) of 9g/L, add 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, The shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl Base-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) butyl-1-ketone Productivity is 61.5%, e.e. value > 99.9%.
Embodiment 36:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds the tetramethyl-ammonium cysteine of final concentration of 5g/L, adds final concentration For the substrate (i.e. formula II compound 0.09g) of 9g/L, add 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, the shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) fourth The productivity of-1-ketone is 5.3%, e.e. value > 99.9%.Embodiment 37:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds [choline chloride] of final concentration of 5g/L+[glycine]-, add the denseest Degree is the substrate (i.e. formula II compound 0.09g) of 9g/L, adds 60g/L (i.e. 0.6g) glycerol as cosubstrate, 40 DEG C, the shaking table of 200rpm converts 24h.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) fourth The productivity of-1-ketone is 70.1%, e.e. value > 99.9%.
Embodiment 38:
10mL K will be suspended according to embodiment 3 gained wet thallus 1.5g2HPO4-KH2PO4Buffer (0.1M, pH 7.5) In, the final concentration of 150g/L of wet thallus, system adds [choline chloride] of final concentration of 5g/L+[glutathion]-, add eventually Concentration is the substrate (i.e. formula II compound 0.09g) of 9g/L, adds 60g/L (i.e. 0.6g) glycerol as the auxiliary end Thing, converts 24h by 40 DEG C in the shaking table of 200rpm.Extract with equal-volume ethyl acetate, use liquid-phase chromatography method detection, Product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluoro-benzene Base) productivity of butyl-1-ketone is 40.1%, e.e. value > 99.9%.

Claims (10)

1. Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504, is preserved in China typical culture collection The heart, deposit number: CCTCC NO:M 2016188, preservation date is: on April 11st, 2016, preservation address is Wuhan, China, Wuhan University, postcode 430072.
2. the answering in preparing sitagliptin chiral intermediate of Pseudomonas aeruginosa ZJPH1504 described in a claim 1 With.
Apply the most as claimed in claim 2, it is characterised in that described application is with Pseudomonas aeruginosa ZJPH1504 warp The wet thallus that fermentation culture obtains is enzyme source, with 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine -7-(8H)-yl]-1-(2,4,5-trifluorophenyl) butyl-2-ketone is substrate, at least buffer with pH 6～9 is constituted instead for reaction medium Answer system, convert under the conditions of 25～50 DEG C, 150～250rpm, after reaction terminates, reactant liquor is isolated and purified, Obtain (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazol [4,3-a] pyrazine-7-(8H)-yl]-4-(2,4,5-trifluoro Phenyl) butyl-1-ketone.
Apply the most as claimed in claim 3, it is characterised in that described substrate consumption is calculated as 2～21g/L with buffer volume, Wet thallus consumption is calculated as 50～350g/L with buffer volume.
Apply the most as claimed in claim 3, it is characterised in that described reaction system has been also added with cosubstrate, described Cosubstrate is one of following: glucose, maltose, sucrose, methanol, ethanol, isopropanol, glycerol, ALANINE, Pidolidone and Cys, described cosubstrate addition is calculated as 4～100g/L with buffer volume.
Apply the most as claimed in claim 3, it is characterised in that described enzyme source is prepared as follows: (1) slant culture: Pseudomonas aeruginosa ZJPH1504 is seeded to slant medium, cultivates 36h for 25～30 DEG C, it is thus achieved that slant strains；Inclined-plane Culture medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, agar powder 15～20g/L, solvent is water, pH 6.5；
(2) seed culture: slant strains is seeded to seed culture medium, 25～30 DEG C, cultivate 12～24 under 150-250rpm H, it is thus achieved that seed liquor；Seed culture medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract 6g/L, (NH4)2SO43g/L, KH2PO41.5g/L, NaCl 0.75g/L, MgSO4·7H2O 0.75g/L, solvent is water, pH 6.5；
(3) fermentation culture: seed liquor is seeded to fermentation medium, liquid amount 90 with the inoculum concentration of volumetric concentration 4～10% ML/250mL shaking flask, cultivates 24～28h, after fermentation ends, is centrifuged by fermentation liquid under 25～30 DEG C, 150～250rpm, Gained precipitation uses 0.1M, pH 8.0 buffer solution twice, collects wet thallus, is enzyme source；Fermentation medium final concentration Consisting of: maltose 30.45g/L, peptone 17.31g/L, NaCl 0.98g/L, solvent is water, pH 7.5.
Apply the most as claimed in claim 3, it is characterised in that described reaction medium is organic solvent and pH 6～9 buffer Mixed liquor, described organic solvent is one of following: normal hexane, dichloromethane, isopropyl acetate, N, N-dimethyl formyl Amine, dimethyl sulfoxide, glycerol and dibutyl phthalate.
Apply the most as claimed in claim 7, it is characterised in that in organic solvent, glycerol consumption is calculated as 20 with buffer volume G/L, remaining organic solvent volume consumption is calculated as 1～20% with buffer volume.
Apply the most as claimed in claim 3, it is characterised in that described reaction medium is ionic liquid and pH 6～9 buffer Mixed liquor, described ionic liquid is one of following: 1-octyl group-3-Methylimidazole. hexafluorophosphate, tetramethyl-ammonium hexafluorophosphoric acid Salt, 1-octyl group-3-methyl imidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium nitrate, N-butyl-pyridinium tetrafluoroborate, Tetramethyl-ammonium tetrafluoroborate, tetramethyl-ammonium glutamic acid, tetraethyl ammonium glutamic acid, tetramethyl-ammonium cysteine, [chlorination gallbladder Alkali]+[glycine]-, [choline chloride]+[glutathion]-。
Apply the most as claimed in claim 9, it is characterised in that described ionic liquid quality addition is with buffer stereometer It is 0.1～10g/L.
CN201610369158.5A 2016-05-27 2016-05-27 Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate Pending CN105925506A (en)

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Cited By (5)

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CN107189970A (en) * 2017-07-27 2017-09-22 盐城工学院 A kind of cloudy city pseudomonad YC1612 and its application and preparation method for producing halide alcohol dehalogenase
CN107189970B (en) * 2017-07-27 2020-03-06 盐城工学院 Pseudomonas adynaudiana YC1612 for producing halohydrin dehalogenase and application and preparation method thereof
CN107950655A (en) * 2017-12-25 2018-04-24 光明乳业股份有限公司 A kind of acidified milk for preparing the method for acidified milk using Paenibacillus polymyxa and preparing
CN108362690A (en) * 2018-01-10 2018-08-03 广西大学 A kind of method of quick discriminating red sandalwood and dyestuff red sandalwood
CN109207535A (en) * 2018-09-20 2019-01-15 新乡拓新药业股份有限公司 Utilize the method for pseudomonas aeruginosa synthesis uracil
CN109207535B (en) * 2018-09-20 2021-06-25 新乡拓新药业股份有限公司 Method for synthesizing uracil by using pseudomonas aeruginosa
CN110283733A (en) * 2019-06-20 2019-09-27 浙江工业大学 Saturn wheel head yeast ZJPH1807 and its application

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