CN101613672A - A kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate - Google Patents
A kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate Download PDFInfo
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Landscapes
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Abstract
The invention discloses a kind of recombination bacillus coli of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate, it is the intestinal bacteria that import carbonyl acyl reductase gene and glucose dehydrogenase gene.The invention also discloses the construction process of above-mentioned recombination bacillus coli.Utilize the method for above-mentioned recombination bacillus coli preparation (S)-4-chloro-ethyl 3-hydroxybutanoate: with the 4-chloroacetyl acetacetic ester is substrate, with glucose is cosubstrate, with oxidized form of nicotinamide-adenine dinucleotide I is cofactor, carries out conversion reaction by above-mentioned recombination bacillus coli and obtains (S)-4-chloro-ethyl 3-hydroxybutanoate.The PsCR enzyme is lived and is that 18U/mg, BmGDH enzyme live and is 9U/mg in the recombination bacillus coli that the present invention makes up, and has avoided adding expensive pure enzyme GDH and coenzyme NADP 11, and only needing to add coenzyme NAD P just can be so that the coenzyme NADP 11 highly efficient regeneration greatly reduces production cost.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of recombination bacillus coli and construction process and its application in asymmetric production (S)-4-chloro-ethyl 3-hydroxybutanoate of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate.
Background technology
(S)-4-chloro-3 3-hydroxyethyl butyrates (Ethyl 4-chloro-3-hydroxybutanoate, (S)-CHBE) be a kind of important organic intermediate, can be used for the synthetic of a lot of active medicines, as statins---hydroxymethyl glutaryl CoA (HMG-CoA) reductase inhibitor and 4-pyridone alkane ketone (4-hydroxypyrrolidone) etc.
[1]
With the latent chiral substrates of 4-chloroacetyl acetacetic ester (4-chloroacetoacetate Ethyl COBE) as reduction reaction, being easy to syntheticly and cheap, is that substrate carries out asymmetric reduction reaction to obtain (S)-CHBE be very cost-effective preparation approach with it.
Prepare the existing many relevant research reports of chirality CHBE about the COBE asymmetric reduction up to now.Summarize and get up to mainly contain chemical method and biological process.Chemical method synthetic COBE productive rate is low, and pollutes greatly, and biological process has the better development prospect by contrast.
Shimizu etc.
[2,3]Use the aldehyde radical reductase enzyme that relies on from the NADPH of Sporobolomyces salmonicolorAKU 4429 to prepare chirality CHBE at single aqueous phase system and water/organic solvent two-phase system catalytic reduction COBE respectively, because using the used enzyme of enzyme catalysis reduction reaction will obtain from microorganism cells in separation and purification, loaded down with trivial details and the easy inactivation of enzyme of process operation, compare with whole-cell catalytic, use less.Yasohara etc.
[4]Screening has obtained a strain Candida magnoliae from 400 saccharomycetes, in water/n-butyl acetate system, in adding glucose, NADP and Hexose phosphate dehydrogenase and reaction process, control under the condition of pH value, product (S)-CHBE can reach 90g/L at the accumulated concentrations of organic phase, and the optical purity enantiomeric excess value of product (enantiomeric excess e.e) reaches 96%.Owing to adopt and often to contain the multiple reductase enzyme of isomorphism type CHBE not of can catalysis COBE being in the wild yeast, therefore the optical activity of the product that catalysis obtained is often very low, uses the recombination bacillus coli asymmetric synthesis to have (the S)-CHBE of highly-solid selectively so recent research concentrates on emphatically.
The inventor has done a large amount of research in microbial method production (S)-4-chloro-3 3-hydroxyethyl butyrate fields; now mode by genome mine locating has obtained a new reductase gene (PsCR) that can catalytic substrate COBE from pichia spp; and the high reactivity expression is carried out in success in intestinal bacteria (pET-22b-PsCR); by fermentation with catalyzed reaction research; confirmed that this carbonyl acyl reductase can generate (S)-CHBE by catalysis COBE; and have high catalytic activity and a strict chiral selectivity; its enzyme work is up to 32U/mg; be the twice (the S1 enzyme is lived and is 13.7U/mg in the Japanese Patent) of bibliographical information, the optical purity in the water catalyst system surpasses 99%
[5,6]By ammonium sulfate precipitation and DEAE-Sephadex ion exchange resin this enzyme is carried out separation and purification, and its biochemical property systematically studied, the result shows: the PsCR that derives from pichia spp belongs to the short-chain dehydrogenase enzyme family in the oxydo-reductase, only depends on DPNH I (NADPH).Its Michaelis-Menton constant is 4.9mg/mL, and corresponding maximum speed of reaction is 337 μ mol/mg protein/min, is higher than to report and derive from other zymic reductase enzyme.Simultaneously, PsCR has advantages of high catalytic activity to ketone and aldehyde, the methyl aceto acetate or the α of chlorine replacing group is particularly arranged, beta-diketon etc. in 2 or 4 position
[7]
Yet, reductase enzyme with reorganization carries out catalyzed reaction, must press chemical dose and add a large amount of expensive coenzyme, reaction can continue to carry out, therefore, in order to obtain high yield, highly purified (S)-CHBE, need clone's dehydrogenase gene to realize the efficient in-situ regeneration of coenzyme, so that regenerating coenzyme circulating system efficiently to be provided.
[reference]:
[1]Karanewsky?DS，Badia?MC，Ciosek?CP?Jr，Robl?JF，Sofia?MJ，Simpkins?LM，DeLange?B，Harrity?TW，Biller?SA，Gorden?EM(1990)Phosphorus-containing?inhibitors?of?HMG-CoAreductase.1.4-[2-arylethyl]-hydroxyphosphinyl-3-hydroxybutanoic?acids：a?new?class?ofcell-selective?inhibitors?of?cholesterol?biosynthesis.Journal?of?Medicinal?Chemistry33：2925-2956.
[2]Shimizu?S，Kataoka?M，Morishita?A?Katoh?M，Morikawa?T，Miyoshi?T，Yamada?H(1990a)Microbial?asymmetric?reduction?of?ethyl?4-chloro-3-oxobutaoate?to?optically?active?ethyl4-4-chloro-3-hydroxybutanoate.Biotechnol?lett?12：593-596.
[3]Shimizu?S，Kataoka?M，Katoh?M，Morikawa?T，Miyoshi?T，Yamada?H(1990b)Stereoselective?reduction?of?ethyl?4-chloro-3-oxobutaoate?by?a?microbial?aldehyde?reductasein?an?organic?solvent-water?diphasic?system.Appl?Environ?Microbiol?56：2374-2377。
[4]Yasohara?Y，Kizaki?N，Hasegawa?J，Takahashi?S，Wada?M，Kataoka?M，Shimizu?S(1999)Synthesis?of?optically?activeethyl?4-chloro-3-hydroxybutanoate?by?microbial?reduction.ApplMicrobiol?Biotechnol?51：847-851.
[5] Ying Hanjie, strict and impartial, leaf is neat, Xu Lin, bear is strong, Bai Jianxin, Chen Yong, Li Zhenjiang.The application of a kind of carbonyl acyl reductase in producing (S)-4-chloro-3 3-hydroxyethyl butyrates.CN?101372699A.
[6]Qi?Ye，Ming?Yan，Lin?Xu，Hou?Cao.A?novel?carbonyl?reductase?from?Pichia?stipitis?for?theproduction?of?ethyl(S)-4-chloro-3-hydroxybutanoate.[J].Biotechnology?Letters，2009，(31)：537-542.
[7]Qi?Ye，Ming?Yan，Lin?Xu，Hou?Cao.A?new?member?of?the?short-chaindehydrogenases/reductases?superfamily：purification，characterization?and?substratespecificity?of?a?recombinant?carbonyl?reductase?from?Pichia?stipitis.[J].BioresourceTechnology，2009(DOI：10.1016/j.biortech.2009.06.014).
Summary of the invention
Technical problem to be solved by this invention provides the recombination bacillus coli that a kind of asymmetric conversion prepares (S)-4-chloro-ethyl 3-hydroxybutanoate, and this bacterial strain does not need to add coenzyme in producing (S)-4-chloro-ethyl 3-hydroxybutanoate process.
The technical problem that the present invention also will solve provides the construction process of above-mentioned recombination bacillus coli.
The technical problem that the present invention also will solve provides above-mentioned recombination bacillus coli and prepares application in (S)-4-chloro-ethyl 3-hydroxybutanoate in asymmetric conversion.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of recombination bacillus coli of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate; it is to import carbonyl acyl reductase PsCR (carbonyl reductase from Pichia stipitis; PsCR) gene and Hexose phosphate dehydrogenase GDH (Glucosedehydrogenase from Bacillus megaterium, the GDH) intestinal bacteria of gene.
Above-mentioned carbonyl acyl reductase PsCR gene contains the 849bp base; is its number of including in Genbank XM_001387250 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? val=XM_001387250.1), its gene order is shown in SEQ ID NO:1.By the carbonyl acyl reductase of this genes encoding, comprise 282 amino acid, is its number of including in Genbank XP_001387287 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=protein﹠amp; Id=126273732), its aminoacid sequence is shown in SEQ ID NO:2.
Above-mentioned Hexose phosphate dehydrogenase GDH gene contains the 783bp base, is its number of including in Genbank 142974 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=nuccore﹠amp; Id=142974), its gene order is shown in SEQ ID NO:3.By the Hexose phosphate dehydrogenase of this genes encoding, comprise 261 amino acid, is its number of including in Genbank 729328 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=protein﹠amp; Id=729328), its aminoacid sequence is shown in SEQ ID NO:4.
The construction process method of above-mentioned recombination bacillus coli is: clone's carbonyl acyl reductase PsCR gene and Hexose phosphate dehydrogenase GDH gene; make up two enzyme coupling expression vectors; and in intestinal bacteria, carry out coexpression, obtain the recombination bacillus coli of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate.
Utilize the method for above-mentioned recombination bacillus coli asymmetric reduction preparation (S)-4-chloro-ethyl 3-hydroxybutanoate as follows:
With the 4-chloroacetyl acetacetic ester is substrate; with glucose is cosubstrate; with reduced form two nucleoside of nicotinamide adenine monophosphate monophosphate (oxidized form of nicotinamide-adenine dinucleotide I; NADP) be cofactor, carry out conversion reaction by the recombination bacillus coli that has imported carbonyl acyl reductase PsCR gene and Hexose phosphate dehydrogenase GDH gene and prepare (S)-4-chloro-ethyl 3-hydroxybutanoate.
Wherein, add NADP and promptly generate NADPH, and reaction cycle is carried out, avoid adding continuously NADPH to reduce production costs by the Hexose phosphate dehydrogenase reduction.
Wherein, the initial reaction concentration of substrate 4-chloroacetyl acetacetic ester is 1.5～300g/L, the initial reaction concentration of glucose is 0.2～2mol/L, oxidized form two nucleoside of nicotinamide adenine monophosphate monophosphate (oxidized form of nicotinamide-adenine dinucleotide I, NADP) initial reaction concentration is 0.05～0.5mmol/L, and the consumption of recombination bacillus coli is counted 20～200g/L with wet bacterium.
Wherein, described temperature of reaction is 20～30 ℃, and the reaction times is 16～32h.
Wherein, aqueous phase system conversion method or organic solvent/water diphasic system conversion method are adopted in described conversion reaction.Described aqueous phase system conversion method is: weight in wet base group intestinal bacteria carry out bio-transformation in the phosphate buffer solution of pH 6～7.5; Described organic solvent/water diphasic system conversion method is: weight in wet base group intestinal bacteria carry out bio-transformation in the diphasic system of the phosphoric acid buffer/n-butyl acetate that contains pH6～7.5.
Beneficial effect: the present invention clones gene fragment SD-PsCR and the SD-BmGDH that has the SD sequence from pET-22b-PsCR and pET-22b-BmGDH carrier, make up two enzyme coupling expression vectors, be applied among COBE asymmetric reduction preparation (S)-CHBE, obtain good effect, the enzyme of PsCR is lived and is 18U/mg in the reorganization bacterium (pET-22b-BmGDH-PsCR), the BmGDH enzyme is lived and is 9U/mg, expensive pure enzyme and coenzyme NADP 11 have been avoided adding, only needing to add coenzyme NAD P just can be so that the coenzyme NADP 11 highly efficient regeneration greatly reduces production cost.
Description of drawings
Fig. 1 makes up synoptic diagram for recombinant plasmid pET-22b-BmGDH-PsCR.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the structure of recombination bacillus coli.
1, obtaining of carbonyl acyl reductase gene:
Pichia spp Pichia Stipitis CBS 6054 (purchasing Centre) in Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry, substratum YPD (gL
-1): yeast extract 10g, peptone 20g, glucose 20g mends distilled water to 1L.
Pichia spp Pichia Stipitis CBS 6054 is inoculated in the 5mLYPD liquid nutrient medium 30 ℃ is cultured to logarithmic phase, use genome DNA extracting reagent kit (sky, Beijing is that biological Engineering Co., Ltd yeast genes group is extracted test kit) to extract genome.
The used primer of construction of expression vector adds restriction enzyme site, and primer sequence is as follows:
Upstream primer (PsCR-sense contains Nde I) is: GGATCCTAAGGAGGATATACATATGACCAACAAC,
Downstream primer (PsCR-anti contains BamH I) is: CCGCTCGAGTTACTATGGCGCACAGT,
All primers can betting office synthesize by the Shen, Shanghai.
The PCR condition of gene:
94 ℃ of sex change 7min, circulate 30 times by following parameter: 94 ℃ of sex change 1min, 60 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min.
Gel electrophoresis separation and purification PCR product, glue reclaims the PsCR goal gene.The CR goal gene is connected on the PMD18T-vector, becomes cloned plasmids PMD 18 T-PsCR, connect product transformed into escherichia coli DH5 α competent cell, coat ammonia benzyl resistant panel.
Single bacterium colony of PMD18 T-PsCR reorganization bacterium is inoculated in the 50mL centrifuge tube that the 5mLLB liquid nutrient medium is housed on the picking LB flat board, and 37 ℃, 220rpm cultivated 8-12 hour down.Utilize the test kit of vast Tektronix Ltd. to carry out plasmid extraction according to the operational manual that its company provides.
The PMD18 T-PsCR that is structured among the simple T is carried out BamH I/Xho I double digestion respectively, and glue reclaims the purpose fragment of double digestion
With BamH I and Xho I expression vector pET-22b is carried out double digestion, glue reclaims the purpose fragment of double digestion.
The expression vector of double digestion is connected with the T4 ligase enzyme with goal gene GDH spends the night, connect product pET-22b-PsCR transformed into escherichia coli DH5 α competent cell, cut the Screening and Identification positive colony with plasmid electrophoresis and enzyme.
2, obtaining of glucose dehydrogenase gene:
Bacillus megaterium DSMZ2894 (purchasing CellCultures) in German Collection of Microorganisms and, substratum LB (gL
-1): yeast extract 5g, peptone 10g, NaCl 5g/L mends distilled water to 1L.
Bacillus megaterium is inoculated in the 5mLLB liquid nutrient medium 37 ℃ is cultured to logarithmic phase, use genome DNA extracting reagent kit (sky, Beijing is that biological Engineering Co., Ltd bacterial genomes is extracted test kit) to extract genome.
The used primer of construction of expression vector adds restriction enzyme site, and primer sequence is as follows:
Upstream primer (GDH-sense contains Nde I) is: AATTCCATATGTATACAGATTTAAAAGAT,
Downstream primer (GDH-anti contains BamH I) is: TATGGATCCCTATTAGCCTCTTC.
The PCR condition of gene:
94 ℃ of sex change 7min, circulate 30 times by following parameter: 94 ℃ of sex change 1min, 60 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min.
Gel electrophoresis separation and purification PCR product, glue reclaims the BmGDH goal gene.The GDH goal gene is connected on the PMD18 T-vector, becomes cloned plasmids PMD18 T-BmGDH, connect product transformed into escherichia coli DH5 α competent cell respectively, coat ammonia benzyl resistant panel.
Single bacterium colony of PMD18 T-GDH reorganization bacterium is inoculated in the 50mL centrifuge tube that the 5mLLB liquid nutrient medium is housed on the picking LB flat board, and 37 ℃, 220rpm cultivated 8-12 hour down.Utilize the test kit of vast Tektronix Ltd. to carry out plasmid extraction according to the operational manual that its company provides.
The PMD18 T-BmGDH that is structured among the simple T is carried out BamH I/Xho I double digestion respectively, and glue reclaims the purpose fragment of double digestion
With BamH I and Xho I expression vector pET-22b is carried out double digestion, glue reclaims the purpose fragment of double digestion.
The expression vector of double digestion is connected with the T4 ligase enzyme with goal gene BmGDH spends the night, connect product pET-22b-BmGDH and be transformed into the bacillus coli DH 5 alpha competent cell, cut the Screening and Identification sun with plasmid electrophoresis and enzyme and clone.
3, the coexpression of carbonyl acyl reductase gene and glucose dehydrogenase gene:
Pcr amplification has the PsCR gene SD-PsCR of restriction enzyme site and SD sequence, and primer sequence is as follows:
Upstream primer (GDH-sense contains Bam I) is: GGATCCTAAGGAGGATATACATATGACCAACAAC,
Downstream primer (GDH-anti contains Xho I) is: CCGCTCGAGTTACTATGGCGCACAGT.
The PCR condition of gene:
94 ℃ of sex change 7min, circulate 30 times by following parameter: 94 ℃ of sex change 1min, 60 ℃ of annealing 50s, 72 ℃ are extended 1.5min.Last 72 ℃ are extended 10min.
With BamH and Xho I respectively enzyme cut SD-PsCR and pET-22b-BmGDH (the carbonyl acyl reductase carrier of pET-22b-BmGDH) for having built; glue reclaims the expression vector and the purpose fragment of double digestion respectively; the expression vector pET-22b-BmGDH of double digestion is connected with the T4 ligase enzyme with SD-PsCR spends the night; the connection product pET-22b-BmGDH-PsCR of 10uL is added in Rosetta (DE3) competent cell of 100uL; place 30min on ice; 42 ℃ of heat shock 90sec place 2min on ice.The 900uLLB substratum that adds preheating.220rpm?37℃1h。200uL bacterium liquid is coated on the LB flat board of the penbritin that contains 100 μ g/mL and paraxin 37 ℃ of incubated overnight 12-16h.Make up collection of illustrative plates and see accompanying drawing.
Embodiment 2: the mensuration that enzyme is lived.
Picking reorganization bacterium E.coli Rosseta (pET-22b-BmGDH-PsCR) and set out intestinal bacteria Rosseta (DE3) to the LB liquid nutrient medium that contains microbiotic penbritin and paraxin, 37 ℃ of shaking culture are spent the night.Be inoculated into respectively in the fresh medium by 2% inoculum size then, 37 ℃ are cultured to OD
600Be about at 0.6 o'clock, add IPTG to final concentration 0.8mmolL
-1, 25 ℃, 220rpm, behind the abduction delivering 10h, centrifugal (4 ℃, 5000rpm, 15min), bacterium mud is resuspended with 100mM potassiumphosphate buffering (pH7.0), ultrasonication cell (power 300W, ultrasonic 5s, intermittently 5s, be total to 5min), centrifugal (4 ℃, 12000rpm, 15min), the enzyme of measuring in the supernatant is lived.
Measure the enzyme of PsCR and live, the enzyme reaction system comprises 100mM potassium phosphate buffer (pH6.0), 5mM NADPH, and 20mM COBE, 30 ℃, the 340nm place measures the decline of light absorption value.It is the enzyme U of unit alive that enzyme work is defined as the interior needed enzyme amount of oxidation 1 μ molNADPH of per minute.
Measure the enzyme of BmGDH and live, the enzyme reaction system comprises 100mM potassium phosphate buffer (pH6.0), 5mM NADP, and 2M glucose, 30 ℃, the 340nm place measures the rising of light absorption value.It is the enzyme U of unit alive that enzyme work is defined as the needed enzyme amount of per minute interior reduction 1 μ mol NADP.Albumen adopts the Brandford method to measure.
The result shows that it is 18U/mg that the enzyme of the middle PsCR of reorganization bacterium (pET-22b-BmGDH-PsCR) is lived, and the BmGDH enzyme is lived and is 9U/mg.
Embodiment 3: the fermentation of recombination bacillus coli E.coli Rosseta (pET22b-BmGDH-PsCR).
Picking reorganization bacterium E.coli Rosseta (pET-22b-BmGDH-PsCR) is to containing antibiotic LB nutrient solution, and 37 ℃ of shaking culture are spent the night.Be inoculated into respectively in the fresh medium by 2% inoculum size then, 37 ℃ are cultured to OD
600Be about at 0.6 o'clock, add IPTG to final concentration 0.8mmolL
-1, 25 ℃, 220rpm, behind the abduction delivering 10h, 8000rpm, 4 ℃ of centrifugal 10min abandon supernatant, precipitate standby.
Embodiment 4:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 6.5) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 1g (dry weight), is suspended in the pH 6.5 potassiumphosphates buffering of 15mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s, 5min altogether) adds glucose 200mmol/L, COBE 1.5g/L, NADP 0.005mmol/L, 20 ℃, 200rpm, 16h.The output of product (S)-CHBE is 1.47g/L, and the yield of product is: 98%, and optical purity e.e% is 100%.The regeneration efficiency TN of coenzyme is 1765.
(S)-and the detection method of CHBE is as follows, and the detection method of product is identical in following examples:
For water react: after reaction finishes, add the equal-volume ethyl acetate, thermal agitation 10min placed two hours then, and the centrifugal 10min of 8000rpm separates organic layer and water layer.The careful upper strata ethyl acetate of drawing is crossed organic membrane, and mark in adding is preserved test sample.
For water/organic two phase reaction: reaction finishes the back centrifugal 10min of 8000rpm and separates organic layer and water layer.The careful upper strata ethyl acetate of drawing is crossed organic membrane, and mark in adding is preserved test sample.
PEG-20M capillary column, internal standard substance are naphthalene.Program is: detector FID, and 210 ℃ of temperature, 210 ℃ of temperature of vaporization chamber, 150 ℃ of column temperatures, column cap is pressed 0.03MPa, hydrogen 0.05MPa, air 0.1MPa, tail blows 0.08MPa.With HPLC the opticity of (S)-4-chloro-ethyl 3-hydroxybutanoate is analyzed (chiral column Chiralcel OB, 4.6 * 250mm; Daicel Chemical Industries, Japan), testing conditions: moving phase is normal hexane: normal hexane (9: 1), and wavelength 214nm, flow are 0.8mL/min,, the appearance time of R type and S type CHBE is respectively: 10.5min and 11.6min.
Embodiment 5:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 7.5) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 2g (dry weight), is suspended in pH 7.5 potassium phosphate buffers of 15mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s, 5min altogether) adds glucose 500mmol/L, COBE 20g/L (0,2,6, each 5g/L of 12h), NADP 0.05mmol/L, 25 ℃, 220rpm, 24h.The output of product (S)-CHBE is 16.8g/L, and the yield of product is: 84%, and e.e is greater than＞99%, and the regeneration efficiency TN of coenzyme is 2017.
Embodiment 6:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 7.0) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 4g (dry weight), is suspended in pH 7.0 potassium phosphate buffers of 50mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s, 5min altogether) adds glucose 600mmol/L, COBE 35g/L (0,1,2,8, each 7g/L of 14h), NADP 0.08mmol/L, 30 ℃, 240rpm, 28h.The output of product (S)-CHBE is 33.8g/L, and the yield of product is: 96.6%, and optical purity e.e% is 100%.The regeneration efficiency TN of coenzyme is 2536.
Embodiment 7:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 6.0) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 10g (dry weight), is suspended in pH 6.0 potassium phosphate buffers of 50mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s is total to 5min), add glucose 1.5mol/L, the 50mL n-butyl acetate (can promote the dissolving of COBE and remove substrate and product to enzyme and cell inhibiting effect), add COBE 100g/L (0,2,4,6,10 each 20g/L), NADP 0.12mmol/L, 20 ℃, 240rpm, 28h.The output of product (S)-CHBE is 97.5g/L, and the yield of product is: 97.5%, and optical purity e.e% is 100%.The regeneration efficiency TN of coenzyme is 4877.
Embodiment 8:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 6.5) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 5g (dry weight), is suspended in pH 6.5 potassium phosphate buffers of 15mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s, 5min altogether), add the 15mL n-butyl acetate (can promote the dissolving of COBE and remove substrate and product to enzyme and cell inhibiting effect), add glucose 1mol/L, COBE 50g/L (0,2,4,6,10 each 10g/L), NADP 0.09mmol/L, 20 ℃, 240rpm, 28h.The output of product (S)-CHBE is 47.5g/L, and the yield of product is: 95.0%, and optical purity e.e% is 100%.The regeneration efficiency TN of coenzyme is 3168.
Embodiment 9:
The precipitation of getting embodiment 3 cushions (100mmolL-1, pH 6.5) washed twice with potassiumphosphate, takes by weighing the intestinal bacteria bacterium mud of 20g (dry weight), is suspended in pH 6.5 potassium phosphate buffers of 200mL.Supersound process cell (power 300W, ultrasonic 5s, intermittently 5s, 5min altogether), add the 200mL n-butyl acetate (can promote the dissolving of COBE and remove substrate and product to enzyme and cell inhibiting effect), add glucose 2mol/L, COBE 300g/L (0,2,4,6,10 each 60g/L), NADP 0.18mmol/L, 25 ℃, 280rpm, 32h.The output of product (S)-CHBE is 292.7g/L, and the yield of product is: 97.5%, and optical purity e.e% is 100%.The regeneration efficiency TN of coenzyme is 9760.
Sequence table
＜110〉Nanjing University of Technology
Nanjing TongKai ZhaoYe biotechnology Co.,Ltd
＜120〉a kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate
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＜213〉pichia spp (Pichia stipitis CBS 6054)
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atg?acc?aac?aac?ccg?agc?atc?acc?tct?cat?atc?aac?gct?gcc?gtg?ggt 48
Met?Thr?Asn?Asn?Pro?Ser?Ile?Thr?Ser?His?Ile?Asn?Ala?Ala?Val?Gly
1 5 10 15
cct?ctc?cct?act?aag?gct?ccc?aaa?ctt?gcg?tct?aac?gtg?ttg?gat?tta 96
Pro?Leu?Pro?Thr?Lys?Ala?Pro?Lys?Leu?Ala?Ser?Asn?Val?Leu?Asp?Leu
20 25 30
ttt?tct?ctc?aag?ggt?aag?gta?gct?tcc?att?act?ggc?tct?tca?gcc?ggg 144
Phe?Ser?Leu?Lys?Gly?Lys?Val?Ala?Ser?Ile?Thr?Gly?Ser?Ser?Ala?Gly
35 40 45
ata?ggt?ctt?gct?gta?gcc?gag?gca?tac?gct?cag?gct?ggt?gct?gat?gtt 192
Ile?Gly?Leu?Ala?Val?Ala?Glu?Ala?Tyr?Ala?Gln?Ala?Gly?Ala?Asp?Val
50 55 60
gca?atc?tgg?tac?aac?tcc?cag?cct?gct?aag?gaa?aag?gcc?gac?aag?ata 240
Ala?Ile?Trp?Tyr?Asn?Ser?Gln?Pro?Ala?Lys?Glu?Lys?Ala?Asp?Lys?Ile
65 70 75 80
gcc?aag?acg?tat?ggt?gta?cgt?tgc?aga?gct?tat?aag?tgt?aat?gtt?tca 288
Ala?Lys?Thr?Tyr?Gly?Val?Arg?Cys?Arg?Ala?Tyr?Lys?Cys?Asn?Val?Ser
85 90 95
gat?cag?cag?gat?gtt?gaa?acc?act?gtg?gct?cag?atc?gag?gct?gac?ttt 336
Asp?Gln?Gln?Asp?Val?Glu?Thr?Thr?Val?Ala?Gln?Ile?Glu?Ala?Asp?Phe
100 105 110
ggc?acc?ata?gat?atc?ttt?gtt?gcc?aat?gcc?ggt?gtt?ccc?tgg?act?gaa 384
Gly?Thr?Ile?Asp?Ile?Phe?Val?Ala?Asn?Ala?Gly?Val?Pro?Trp?Thr?Glu
115 120 125
ggc?gaa?agt?gta?gaa?att?gac?aac?ttt?gac?tcc?tgg?aaa?aag?gtc?ata 432
Gly?Glu?Ser?Val?Glu?Ile?Asp?Asn?Phe?Asp?Ser?Trp?Lys?Lys?Val?Ile
130 135 140
gac?tta?gac?ttg?tct?ggg?gct?tac?tac?tgt?gca?cat?gcg?gct?ggt?aag 480
Asp?Leu?Asp?Leu?Ser?Gly?Ala?Tyr?Tyr?Cys?Ala?His?Ala?Ala?Gly?Lys
145 150 155 160
atc?ttt?aag?aaa?aac?ggc?aag?ggc?tcc?atg?att?ttc?acc?gct?tct?atg 528
Ile?Phe?Lys?Lys?Asn?Gly?Lys?Gly?Ser?Met?Ile?Phe?Thr?Ala?Ser?Met
165 170 175
tct?ggt?cac?att?gtg?aat?att?cct?caa?ttc?cag?gct?cct?tac?aac?gct 576
Ser?Gly?His?Ile?Val?Asn?Ile?Pro?Gln?Phe?Gln?Ala?Pro?Tyr?Asn?Ala
180 185 190
gcc?aag?gct?gcg?gtg?ttg?cac?ttg?agc?aaa?tcg?ttg?gct?ata?gaa?tgg 624
Ala?Lys?Ala?Ala?Val?Leu?His?Leu?Ser?Lys?Ser?Leu?Ala?Ile?Glu?Trp
195 200 205
gct?cct?ttt?gcc?aga?gtc?aat?acg?att?tcg?cca?gga?tac?att?gtc?acc 672
Ala?Pro?Phe?Ala?Arg?Val?Asn?Thr?Ile?Ser?Pro?Gly?Tyr?Ile?Val?Thr
210 215 220
gag?atc?tcg?gac?ttt?gtc?tca?gac?gac?atc?aag?tcc?aag?tgg?tgg?cag 720
Glu?Ile?Ser?Asp?Phe?Val?Ser?Asp?Asp?Ile?Lys?Ser?Lys?Trp?Trp?Gln
225 230 235 240
ttt?att?cct?ctt?ggt?aga?gag?gga?gtc?aca?caa?gag?ttg?gtt?ggt?gcc 768
Phe?Ile?Pro?Leu?Gly?Arg?Glu?Gly?Val?Thr?Gln?Glu?Leu?Val?Gly?Ala
245 250 255
tac?ttg?tac?ttt?gct?tct?gat?gcc?tct?aca?tat?act?acg?gga?tca?gat 816
Tyr?Leu?Tyr?Phe?Ala?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ser?Asp
260 265 270
ctt?atc?gtc?gat?gga?ggc?tac?tgt?gcg?cca?tag 849
Leu?Ile?Val?Asp?Gly?Gly?Tyr?Cys?Ala?Pro
275 280
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＜213〉pichia spp (Pichia stipitis CBS 6054)
<400>2
Met?Thr?Asn?Asn?Pro?Ser?Ile?Thr?Ser?His?Ile?Asn?Ala?Ala?Val?Gly
1 5 10 15
Pro?Leu?Pro?Thr?Lys?Ala?Pro?Lys?Leu?Ala?Ser?Asn?Val?Leu?Asp?Leu
20 25 30
Phe?Ser?Leu?Lys?Gly?Lys?Val?Ala?Ser?Ile?Thr?Gly?Ser?Ser?Ala?Gly
35 40 45
Ile?Gly?Leu?Ala?Val?Ala?Glu?Ala?Tyr?Ala?Gln?Ala?Gly?Ala?Asp?Val
50 55 60
Ala?Ile?Trp?Tyr?Asn?Ser?Gln?Pro?Ala?Lys?Glu?Lys?Ala?Asp?Lys?Ile
65 70 75 80
Ala?Lys?Thr?Tyr?Gly?Val?Arg?Cys?Arg?Ala?Tyr?Lys?Cys?Asn?Val?Ser
85 90 95
Asp?Gln?Gln?Asp?Val?Glu?Thr?Thr?Val?Ala?Gln?Ile?Glu?Ala?Asp?Phe
100 105 110
Gly?Thr?Ile?Asp?Ile?Phe?Val?Ala?Asn?Ala?Gly?Val?Pro?Trp?Thr?Glu
115 120 125
Gly?Glu?Ser?Val?Glu?Ile?Asp?Asn?Phe?Asp?Ser?Trp?Lys?Lys?Val?Ile
130 135 140
Asp?Leu?Asp?Leu?Ser?Gly?Ala?Tyr?Tyr?Cys?Ala?His?Ala?Ala?Gly?Lys
145 150 155 160
Ile?Phe?Lys?Lys?Asn?Gly?Lys?Gly?Ser?Met?Ile?Phe?Thr?Ala?Ser?Met
165 170 175
Ser?Gly?His?Ile?Val?Asn?Ile?Pro?Gln?Phe?Gln?Ala?Pro?Tyr?Asn?Ala
180 185 190
Ala?Lys?Ala?Ala?Val?Leu?His?Leu?Ser?Lys?Ser?Leu?Ala?Ile?Glu?Trp
195 200 205
Ala?Pro?Phe?Ala?Arg?Val?Asn?Thr?Ile?Ser?Pro?Gly?Tyr?Ile?Val?Thr
210 215 220
Glu?Ile?Ser?Asp?Phe?Val?Ser?Asp?Asp?Ile?Lys?Ser?Lys?Trp?Trp?Gln
225 230 235 240
Phe?Ile?Pro?Leu?Gly?Arg?Glu?Gly?Val?Thr?Gln?Glu?Leu?Val?Gly?Ala
245 250 255
Tyr?Leu?Tyr?Phe?Ala?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ser?Asp
260 265 270
Leu?Ile?Val?Asp?Gly?Gly?Tyr?Cys?Ala?Pro
275 280
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<211>783
<212>DNA
＜213〉bacillus megaterium (Bacillus subtilis)
<220>
<221>CDS
<222>(1)..(783)
<400>3
atg?tat?aca?gat?tta?aaa?gat?aaa?gta?gtt?gta?att?aca?ggt?gga?tca 48
Met?Tyr?Thr?Asp?Leu?Lys?Asp?Lys?Val?Val?Val?Ile?Thr?Gly?Gly?Ser
1 5 10 15
aca?ggt?tta?gga?cgc?gca?atg?gct?gtt?cgt?ttc?ggt?caa?gaa?gaa?gca 96
Thr?Gly?Leu?Gly?Arg?Ala?Met?Ala?Val?Arg?Phe?Gly?Gln?Glu?Glu?Ala
20 25 30
aaa?gtt?gtt?att?aac?tat?tac?aac?aat?gaa?gaa?gaa?gct?tta?gat?gcg 144
Lys?Val?Val?Ile?Asn?Tyr?Tyr?Asn?Asn?Glu?Glu?Glu?Ala?Leu?Asp?Ala
35 40 45
aaa?aaa?gaa?gta?gaa?gaa?gca?ggc?gga?caa?gca?atc?atc?gtt?caa?ggc 192
Lys?Lys?Glu?Val?Glu?Glu?Ala?Gly?Gly?Gln?Ala?Ile?Ile?Val?Gln?Gly
50 55 60
gac?gta?aca?aaa?gaa?gaa?gac?gtt?gta?aat?ctt?gtt?cag?aca?gct?act 240
Asp?Val?Thr?Lys?Glu?Glu?Asp?Val?Val?Asn?Leu?Val?Gln?Thr?Ala?Thr
65 70 75 80
aaa?gaa?ttc?ggt?aca?tta?gac?gtt?atg?att?aac?aac?gct?ggt?gtt?gaa 288
Lys?Glu?Phe?Gly?Thr?Leu?Asp?Val?Met?Ile?Asn?Asn?Ala?Gly?Val?Glu
85 90 95
aat?cca?gtt?cct?tct?cat?gag?tta?tct?cta?gat?aac?tgg?aac?aaa?gtt 336
Asn?Pro?Val?Pro?Ser?His?Glu?Leu?Ser?Leu?Asp?Asn?Trp?Asn?Lys?Val
100 105 110
att?gat?aca?aac?tta?aca?ggt?gca?ttc?tta?gga?agc?cgt?gaa?gca?att 384
Ile?Asp?Thr?Asn?Leu?Thr?Gly?Ala?Phe?Leu?Gly?Ser?Arg?Glu?Ala?Ile
115 120 125
aaa?tat?ttc?gtt?gaa?aac?gat?att?aaa?gga?aat?gtt?atc?aac?atg?tct 432
Lys?Tyr?Phe?Val?Glu?Asn?Asp?Ile?Lys?Gly?Asn?Val?Ile?Asn?Met?Ser
130 135 140
agc?gtt?cac?gaa?atg?att?cct?tgg?cca?tta?ttt?gtt?cac?tat?gca?gca 480
Ser?Val?His?Glu?Met?Ile?Pro?Trp?Pro?Leu?Phe?Val?His?Tyr?Ala?Ala
145 150 155 160
agt?aaa?ggc?ggc?atg?aaa?cta?atg?acg?gaa?aca?ttg?gct?ctt?gaa?tat 528
Ser?Lys?Gly?Gly?Met?Lys?Leu?Met?Thr?Glu?Thr?Leu?Ala?Leu?Glu?Tyr
165 170 175
gcg?cca?aaa?ggt?atc?cgc?gta?aat?aac?att?gga?cca?ggt?gcg?atg?aac 576
Ala?Pro?Lys?Gly?Ile?Arg?Val?Asn?Asn?Ile?Gly?Pro?Gly?Ala?Met?Asn
180 185 190
aca?cca?att?aac?gca?gag?aaa?ttt?gca?gat?cca?gaa?caa?cgt?gca?gac 624
Thr?Pro?Ile?Asn?Ala?Glu?Lys?Phe?Ala?Asp?Pro?Glu?Gln?Arg?Ala?Asp
195 200 205
gta?gaa?agc?atg?att?cca?atg?ggc?tac?atc?ggt?aaa?cca?gaa?gaa?gta 672
Val?Glu?Ser?Met?Ile?Pro?Met?Gly?Tyr?Ile?Gly?Lys?Pro?Glu?Glu?Val
210 215 220
gca?gca?gtt?gca?gca?ttc?tta?gca?tca?tca?caa?gca?agc?tat?gta?aca 720
Ala?Ala?Val?Ala?Ala?Phe?Leu?Ala?Ser?Ser?Gln?Ala?Ser?Tyr?Val?Thr
225 230 235 240
ggt?att?aca?tta?ttt?gct?gat?ggt?ggt?atg?acg?aaa?tac?cct?tct?ttc 768
Gly?Ile?Thr?Leu?Phe?Ala?Asp?Gly?Gly?Met?Thr?Lys?Tyr?Pro?Ser?Phe
245 250 255
caa?gca?gga?aga?ggc 783
Gln?Ala?Gly?Arg?Gly
260
<210>4
<211>261
<212>PRT
＜213〉bacillus megaterium (Bacillus subtilis)
<400>4
Met?Tyr?Thr?Asp?Leu?Lys?Asp?Lys?Val?Val?Val?Ile?Thr?Gly?Gly?Ser
1 5 10 15
Thr?Gly?Leu?Gly?Arg?Ala?Met?Ala?Val?Arg?Phe?Gly?Gln?Glu?Glu?Ala
20 25 30
Lys?Val?Val?Ile?Asn?Tyr?Tyr?Asn?Asn?Glu?Glu?Glu?Ala?Leu?Asp?Ala
35 40 45
Lys?Lys?Glu?Val?Glu?Glu?Ala?Gly?Gly?Gln?Ala?Ile?Ile?Val?Gln?Gly
50 55 60
Asp?Val?Thr?Lys?Glu?Glu?Asp?Val?Val?Asn?Leu?Val?Gln?Thr?Ala?Thr
65 70 75 80
Lys?Glu?Phe?Gly?Thr?Leu?Asp?Val?Met?Ile?Asn?Asn?Ala?Gly?Val?Glu
85 90 95
Asn?Pro?Val?Pro?Ser?His?Glu?Leu?Ser?Leu?Asp?Asn?Trp?Asn?Lys?Val
100 105 110
Ile?Asp?Thr?Asn?Leu?Thr?Gly?Ala?Phe?Leu?Gly?Ser?Arg?Glu?Ala?Ile
115 120 125
Lys?Tyr?Phe?Val?Glu?Asn?Asp?Ile?Lys?Gly?Asn?Val?Ile?Asn?Met?Ser
130 135 140
Ser?Val?His?Glu?Met?Ile?Pro?Trp?Pro?Leu?Phe?Val?His?Tyr?Ala?Ala
145 150 155 160
Ser?Lys?Gly?Gly?Met?Lys?Leu?Met?Thr?Glu?Thr?Leu?Ala?Leu?Glu?Tyr
165 170 175
Ala?Pro?Lys?Gly?Ile?Arg?Val?Asn?Asn?Ile?Gly?Pro?Gly?Ala?Met?Asn
180 185 190
Thr?Pro?Ile?Asn?Ala?Glu?Lys?Phe?Ala?Asp?Pro?Glu?Gln?Arg?Ala?Asp
195 200 205
Val?Glu?Ser?Met?Ile?Pro?Met?Gly?Tyr?Ile?Gly?Lys?Pro?Glu?Glu?Val
210 215 220
Ala?Ala?Val?Ala?Ala?Phe?Leu?Ala?Ser?Ser?Gln?Ala?Ser?Tyr?Val?Thr
225 230 235 240
Gly?Ile?Thr?Leu?Phe?Ala?Asp?Gly?Gly?Met?Thr?Lys?Tyr?Pro?Ser?Phe
245 250 255
Gln?Ala?Gly?Arg?Gly
260
Claims (9)
1, a kind of recombination bacillus coli of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate is characterized in that it is the intestinal bacteria that import carbonyl acyl reductase PsCR gene and Hexose phosphate dehydrogenase GDH gene.
2, the recombination bacillus coli of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate according to claim 1 is characterized in that described carbonyl acyl reductase PsCR gene order is shown in SEQ ID NO:1.
3, make up the method for the described recombination bacillus coli of claim 1; it is characterized in that this method is: clone's carbonyl acyl reductase PsCR gene and Hexose phosphate dehydrogenase GDH gene; make up two enzyme coupling expression vectors; and in intestinal bacteria, carry out coexpression, the recombination bacillus coli that acquisition can asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate.
4, claim 1 described can be asymmetric the application of recombination bacillus coli in 4-chloroacetyl acetacetic ester asymmetric reduction preparation (S)-4-chloro-ethyl 3-hydroxybutanoate of conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate.
The method of 5, a kind of production (S)-4-chloro-ethyl 3-hydroxybutanoate; it is characterized in that this method is a substrate with the 4-chloroacetyl acetacetic ester; with glucose is cosubstrate; with reduced form two nucleoside of nicotinamide adenine monophosphate monophosphate is cofactor, carries out conversion reaction by the recombination bacillus coli that has imported carbonyl acyl reductase PsCR gene and Hexose phosphate dehydrogenase GDH gene and prepares (S)-4-chloro-ethyl 3-hydroxybutanoate.
The method of 6, production according to claim 5 (S)-4-chloro-ethyl 3-hydroxybutanoate, the initial reaction concentration that it is characterized in that substrate 4-chloroacetyl acetacetic ester is 1.5～300g/L, the initial reaction concentration of glucose is 0.2～2mol/L, oxidized form two nucleoside of nicotinamide adenine monophosphate monophosphate initial reaction concentration is 0.05～0.5mmol/L, and the consumption of recombination bacillus coli is counted 20～200g/L with wet bacterium.
The method of 7, production according to claim 5 (S)-4-chloro-ethyl 3-hydroxybutanoate is characterized in that described temperature of reaction is 20～30 ℃, and the reaction times is 16～32h.
The method of 8, production according to claim 5 (S)-4-chloro-ethyl 3-hydroxybutanoate is characterized in that described conversion reaction employing aqueous phase system conversion method or organic solvent/water diphasic system conversion method.
The method of 9, production according to claim 8 (S)-4-chloro-ethyl 3-hydroxybutanoate, it is characterized in that described aqueous phase system conversion method is: weight in wet base group intestinal bacteria carry out bio-transformation in the phosphate buffer solution of pH 6～7.5; Described organic solvent/water diphasic system conversion method is: weight in wet base group intestinal bacteria carry out bio-transformation in the diphasic system of the phosphoric acid buffer/n-butyl acetate that contains pH 6～7.5.
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| CN101962661A (en) * | 2010-06-29 | 2011-02-02 | 南京工业大学 | Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate |
| CN102604904A (en) * | 2012-03-14 | 2012-07-25 | 苏州汉酶生物技术有限公司 | Production method of glucose dehydrogenase |
| CN102605011A (en) * | 2012-03-16 | 2012-07-25 | 苏州汉酶生物技术有限公司 | (S)-4-chloride-3-ethyl 3-hydroxybutyrate biological preparation method |
| CN102676590A (en) * | 2011-03-16 | 2012-09-19 | 苏州国镝医药科技有限公司 | Chiral synthesis of Lipitor intermediate ATS-4 by using bio-enzyme |
| CN102827850A (en) * | 2012-08-31 | 2012-12-19 | 杭州师范大学 | Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application |
| CN103014088A (en) * | 2012-12-19 | 2013-04-03 | 苏州汉酶生物技术有限公司 | Biological preparation method of (S)-3-methylamino-(2-thienyl)-1-propyl alcohol |
| CN103911406A (en) * | 2014-01-14 | 2014-07-09 | 苏州国镝医药科技有限公司 | Method for synthesis of (S)-3-hydroxypyrrolidine and its derivatives by enzyme method reduction |
| CN104450801A (en) * | 2014-12-10 | 2015-03-25 | 江南大学 | Application of double-enzyme method to asymmetric preparation of (R)-2-chloro-1-(3-chlorphenyl) ethyl alcohol |
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| CN101962661A (en) * | 2010-06-29 | 2011-02-02 | 南京工业大学 | Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate |
| CN101962661B (en) * | 2010-06-29 | 2012-05-23 | 南京工业大学 | Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate |
| CN102676590A (en) * | 2011-03-16 | 2012-09-19 | 苏州国镝医药科技有限公司 | Chiral synthesis of Lipitor intermediate ATS-4 by using bio-enzyme |
| CN102604904A (en) * | 2012-03-14 | 2012-07-25 | 苏州汉酶生物技术有限公司 | Production method of glucose dehydrogenase |
| CN102605011A (en) * | 2012-03-16 | 2012-07-25 | 苏州汉酶生物技术有限公司 | (S)-4-chloride-3-ethyl 3-hydroxybutyrate biological preparation method |
| CN102827850A (en) * | 2012-08-31 | 2012-12-19 | 杭州师范大学 | Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application |
| CN103014088A (en) * | 2012-12-19 | 2013-04-03 | 苏州汉酶生物技术有限公司 | Biological preparation method of (S)-3-methylamino-(2-thienyl)-1-propyl alcohol |
| WO2014094462A1 (en) * | 2012-12-19 | 2014-06-26 | 苏州汉酶生物技术有限公司 | Biological preparation method of (s)-3-methylamino-1-(2-thienyl)-1-propyl alcohol |
| CN103911406A (en) * | 2014-01-14 | 2014-07-09 | 苏州国镝医药科技有限公司 | Method for synthesis of (S)-3-hydroxypyrrolidine and its derivatives by enzyme method reduction |
| CN104450801A (en) * | 2014-12-10 | 2015-03-25 | 江南大学 | Application of double-enzyme method to asymmetric preparation of (R)-2-chloro-1-(3-chlorphenyl) ethyl alcohol |
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