CN101314784A - Method for biological catalysis preparation of (R)-2-hydroxyl-4-phenyl ethyl butyrate - Google Patents
Method for biological catalysis preparation of (R)-2-hydroxyl-4-phenyl ethyl butyrate Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000006555 catalytic reaction Methods 0.000 title abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 55
- 239000000758 substrate Substances 0.000 claims abstract description 45
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- 239000011347 resin Substances 0.000 claims abstract description 32
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- 241000222124 [Candida] boidinii Species 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
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- 239000007788 liquid Substances 0.000 claims description 85
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 60
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- 239000008103 glucose Substances 0.000 claims description 41
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
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- OBNCKNCVKJNDBV-UHFFFAOYSA-N butanoic acid ethyl ester Natural products CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 abstract description 8
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- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 16
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
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- OBKXEAXTFZPCHS-UHFFFAOYSA-N 4-phenylbutyric acid Chemical compound OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing (R)-2-hydroxyl-4- phenylbutyric ethyl butyrate by adopting biological catalysis, which belongs to the technology field of the biocatalytic anisomerous reducing preparation medical chiral intermediate. The method comprises the following steps: utilizing produced bacteria Candida boidinii of high antimer selective carbonyl reductase screened etc.; fermenting and producing enzyme under the optimizing condition; taking 2-hydroxyl-4-phenylbutyric ethyl butyrate as a substrate in a single water phase system, a water/organic two-phase system or a water/resin two-phase system; adding grape sugar to wet thalli; and preparing (R)-2- hydroxyl-4- phenylbutyric ethyl butyrate. When the concentration of the substrate of 2-hydroxyl-4-phenylbutyric ethyl butyrate is 1 to 50g/L, (R)-2- hydroxyl-4- phenylbutyric ethyl butyrate is subjected to transformation for 1h to 48h, so as to ensure that the enantiomeric excess of the (R)-2- hydroxyl-4- phenylbutyric ethyl butyrate reaches 84.9 to 98.88 percent, the transformation rate of Moore reaches 72.0 to 84.6 percent, and coenzyme is not needed during the whole reaction process.
Description
Technical field
The present invention relates to a kind of method of biocatalysis preparation (R)-2-hydroxy-4-phenyl ethyl butyrate, belong to the biocatalysis asymmetric reduction and prepare medical chiral intermediate technical field.
Background technology
Chirality (R)-2-hydroxy-4-phenyl ethyl butyrate (Benzenebutanoic acid, a-hydroxy-, ethylester, (aR)-), molecular formula is C
12H
16O
3, molecular weight is 208.25, CAS number: 90315-82-5.(R)-2-hydroxy-4-phenyl ethyl butyrate is the crucial chiral intermediate of synthetic numerous angiotensin-convertion enzyme inhibitors (ACEI) Puli class medicine.Since (R)-and the importance of 2-hydroxy-4-phenyl ethyl butyrate in such medicine is synthetic, attracted many people that extensive studies has been carried out in its preparation, reported a large amount of preparation methods.
At present, (R)-production of 2-hydroxy-4-phenyl ethyl butyrate can be by chemistry or biological resolution of racemates, chiral source is synthetic and method such as asymmetric reduction is carried out.
1. chemosynthesis and chemical method for splitting.This method needs the cost of chiral auxiliary(reagent) than higher, and by product is many, pollutes more serious.
2. chiral source synthetic method.This method needs expensive chiral source such as D-oxysuccinic acid (EP0759424A) or severe reaction conditions, react (JP62212329A) as the L-Serine under anhydrous cold condition, thereby practical value is little.
3. enzyme process or microorganism are put in order cell asymmetric reduction method.Though the enzyme process reaction has reaction conditions gentleness, stereoselectivity height, advantages of environment protection.But, the expensive redox coenzyme NAD of oxydo-reductase reaction needed
+Perhaps NADP
+Participation, compare with pure enzyme, utilize the whole cell of microorganism to carry out catalyzed reaction and have clear superiority.
Based on these top reasons, utilizing microbial process that alpha-carbonyl is carried out the enantioselectivity reduction is a kind of present relatively effective means.In the process of research and practice, find that multiple biomaterial can be used for the conversion of 2-carbonyl-4-phenylbutyrate.
Switzerland Ciba-Geigy company was once studied with serum lactic dehydrogenase (Lactate dehydrogenase) and hydrogenlyase, carrying out commercial enzyme by the continuous operations of stirred reactor transforms, obtained greater than 99% ee value with greater than 91% transformation efficiency, but the commercial enzyme price is high, and also must add expensive coenzyme NAD in reaction
+As cofactor, so this method can't practical application.
Utilize wild Radix Dauci Sativae (Daucus carota) cell catalysis in addition, can obtain productive rate (90%) and ee value (99%) preferably, but (about 10 days) (Chadha etc. that but need the very long reaction times, Tetrahedron:Asymmetry, 7 (6), 1571-1572,1996).Use the cell of kinds such as peppermint, tobacco, flower of Capa Jasmine to carry out report (Akakabe, the Y. of catalytic reduction in addition; Naoshima, Y.; Phytochem., 1994,35,661).
Utilize the whole cell of microorganism to carry out catalyzed reaction and in water/organic phase, transform 2-carbonyl-4-phenylbutyrate as utilizing little rhodotorula (Rhodotorula minuta IFO 0920) and candiyeast (Candida holmii KPY 124020), the ee value reaches 95% and 94% respectively, shortcoming is that transformation efficiency is not high, the highest by 58% (Shinobu Oda etc., Biosci.Biochem.62:1762-1767,1998); Xia Yongmei etc. utilize bread yeast water and organic solvent mutually in asymmetry catalysis transform 2-carbonyl-4-phenylbutyrate to obtain product enantiomorph value be 82.25%, productive rate is 75.82% (chemical research and application, 2006,18 (4): 426-430; Food and biotechnology journal, 2006,25 (2): 66-69).
Report and research from synthetic (the R)-2-hydroxy-4-phenyl ethyl butyrate of present biocatalysis, the microbial enzyme source that lacks high enantioselectivity and high yield, therefore, also need microorganism is screened, find a kind of stable, higher more economical bacterial strain of catalytic efficiency, and the selection appropriate reaction conditions, improve enantio-selectivity and unit productive rate to greatest extent.
Summary of the invention
The purpose of this invention is to provide the microorganism that produces the highly-solid selectively carbonyl reductase, a kind of method of effectively catalytic asymmetric reduction prepared in reaction chiral hydroxyl group butyric ester newly is provided, and utilize such microorganism strains catalysis 2-carbonyl-4-phenylbutyrate asymmetric reduction, to obtain (R)-2-hydroxy-4-phenyl ethyl butyrate of high antimer value, high reaction molar yield, high production concentration.
The method of preparation of the present invention (R)-2-hydroxy-4-phenyl ethyl butyrate is that to produce bacterium with the selectivity carbonyl reductase be starting strain, is that substrate is prepared with 2-carbonyl-4-phenylbutyrate, these method following steps:
(1) select following selectivity carbonyl reductase to produce bacteria strain as starting strain: Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799, Candida boidinii (Candida boidinii) AS 2.2159, AS 2.2160 or AS 2.2162, or reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2110, AS 2.2111, AS 2.2113, AS 2.2114, AS 2.2115, AS 2.2116, AS 2.2117, AS 2.2118, AS 2.2119, AS 2.2120, AS2.2121 or AS 2.2122;
(2) cultivation of wet thallus: substratum is formed and to be counted with g/L: peptone 10～30, and glucose 10～30, yeast extract paste 5～20, pH5.5～7.5,20～40 ℃ of temperature, with the described strain culturing of step (1) 1～5 day, filtering fermentation liquor obtained wet thallus as the enzyme source;
(3) preparation reaction system: with 2-carbonyl-4-phenylbutyrate is substrate, is mixed with single water react system with the phosphate buffered saline buffer of 0.1M, pH5.5～pH7.5, and concentration of substrate is 1g/L～50g/L; Perhaps the phosphate buffered saline buffer with organic solvent and pH6.5 is mixed with water/organic solvent two-phase system, adds substrate again, concentration of substrate 1g/L～50g/L; Perhaps the phosphate buffered saline buffer with macroporous resin and pH5.5～pH7.5 is mixed with water/resin two-phase system, adds substrate again, concentration of substrate 1g/L～50g/L;
(4) enzymatic conversion reaction: add (2) described wet thallus in reaction system, carry out enzyme reaction in 20～50 ℃, the reaction times is 1～48 hour;
(5) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, the polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, drying and dehydrating evaporates and reclaims solvent again, obtains colourless oil liquid (R)-2-hydroxy-4-phenyl ethyl butyrate.
In aforesaid method, wherein the changing effect of Candida boidinii (Candida boidinii) AS 2.2159 is more excellent, as an example in further research starting strain.
In step (4) catalyzed reaction of aforesaid method: the wet thallus amount that adds in reaction system is 10～60g/g substrate.
Can replenish the interpolation substrate in the enzymatic conversion reaction process of aforesaid method, making its concentration is 1～50g/L.
In above-mentioned enzymatic conversion reaction process, can add glucose as cosubstrate, its concentration is 10～100g/L.And in the conversion reaction process, the conversion fluid of gained is carried out chirality gas chromatographic analysis (GC) measure, the detection reaction process is determined the reaction deadline.
The macroporous resin that adds in above-mentioned steps (3) is to be used for adsorbing substrate, product, to suppress substrate, the product toxicity to thalline.The mass ratio of resin and substrate is 0.6～6: 1, macroporous resin is macroporous adsorbent resins such as DA201, Hz816, Hz803, AB-8, D4006, HPD300, HPD100A, NAK-II, X-5, D201GF, D392, D315, D290, D280, D293 large pore anion resin, HD-2, D61, D001, HD-1, D001-CC, D113 macropore cation resin, the aperture of described macroporous resin are 100～180
Above-mentioned resin is commercially available, as products such as Shanghai East China University of Science China shake scientific and technological development company, Chemical Plant of Nankai Univ..
In above-mentioned steps (3) during preparation water/organic solvent two-phase system, phosphate buffered saline buffer with organic solvent and pH5.5～pH7.5, be mixed with water/organic solvent two-phase system in 0.1: 1 by volume～1: 0.1, add concentration of substrate 1g/L～50g/L again, used organic solvent is a dioctyl phthalate (DOP), or dibutyl phthalate, n-butyl acetate, n-amyl acetate, hexanaphthene, normal hexane, normal heptane, octane, octane-iso, decane, nonane, octanol, propyl carbinol or isopropylcarbinol etc., be commercially available chemical pure product.
When carrying out above-mentioned steps (5), the concrete operations that product (R)-2-hydroxy-4-phenyl ethyl butyrate is extracted are: after conversion reaction finishes, with reaction solution centrifugal (8,000g * 20min, 4 ℃), supernatant liquor extracts with isopyknic ethyl acetate, for example extracts the polymeric adsorbent eluent ethyl acetate three times, combined ethyl acetate extraction liquid and elutriant, to the anhydrous sodium sulphate that wherein adds 1%～5% (w/v), stir evenly the back standing over night again, drying is removed remaining moisture content.The processing of decolouring in case of necessity.With the organic phase filter paper filtering, collect organic phase.40 ℃ of water-bath rotary evaporations reclaim solvent, obtain colourless oil liquid (R)-2-hydroxy-4-phenyl ethyl butyrate.
Get (R)-2-hydroxy-4-phenyl ethyl butyrate 0.05g, be dissolved in the ethyl acetate, be settled to 5mL, carry out qualitative analysis with GC-MASS, the contrast of sample and standard substance (Sigma Co.) mass spectrum is defined as same substance.
In addition, in aforesaid method, also substrate directly can be joined contain in the wet thallus cultivation and fermentation liquid and carry out enzymatic conversion reaction.Therefore, it is the further method of substrate biocatalysis preparation (R)-2-hydroxyl-4-butyl ethyl butyrate that the present invention also provides with 2-carbonyl-4-phenylbutyrate, and this method may further comprise the steps:
(1) select following selectivity carbonyl reductase to produce bacteria strain as starting strain: Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799, Candida boidinii (Candida boidinii) AS 2.2159, AS 2.2160 or AS 2.2162, or reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2110, AS 2.2111, AS 2.2113, AS 2.2114, AS 2.2115, AS 2.2116, AS 2.2117, AS 2.2118, AS 2.2119, AS 2.2120, AS2.2121 or AS 2.2122;
(2) cultivation of wet thallus: substratum is formed and to be counted with g/L: peptone 10～30, and glucose 10～30, yeast extract paste 5～20, pH5.5～7.5,20～40 ℃ of temperature with the described strain culturing of step (1) 1～5 day, obtain containing the fermented liquid of wet thallus;
(3) in above-mentioned wet thallus cultivation and fermentation liquid, add 2-carbonyl-4-phenylbutyrate of 1～50g/L as substrate; Carry out enzyme reaction in 20～50 ℃, the reaction times is 1～48 hour;
(4) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, the polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, drying and dehydrating evaporates and reclaims solvent again, obtains colourless oil liquid (R)-2-hydroxy-4-phenyl ethyl butyrate.
Analytical procedure
Product (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomer excessive value (e.e.) is measured and is adopted chiral gas chromatography.Chiral chromatographic column: CP-Chirasil Dex CB 25m * 0.25mm * 0.25 μ m; Chromatographic condition is: 10 ℃ keep 2min, are warming up to 140 ℃ with 2 ℃/min and keep 2min; Sample size 0.5 μ L; Column flow rate 2.0mL/min; Carrier gas H
2Flow velocity 30mL/min, burning gas H
2Flow velocity 30mL/min, air velocity 300mL/min, tail blows N
2Flow velocity: 25mL/min; Injection port: 270 ℃, detector: 270 ℃, splitting ratio: 50: 1.
Beneficial effect of the present invention
The present invention screens and has obtained optionally Candida boidinii (Candida boidinii) etc. of high antimer, and product (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value that the asymmetric also 2-carbonyl-4-phenylbutyrate of catalysis generates in single water or two-phase system reaches more than the 98%e.e; Simultaneously, the energize material adds high concentration substrate, can obtain high antimer value, high reaction molar yield, high production concentration, and main is not need to add coenzyme in entire reaction course.
Microbe transformation method of the present invention is with respect to traditional chemical asymmetric synthesis method, bread yeast oxide-reduction method commonly used or with adding coenzyme NAD H/NAD
+Enzyme catalysis asymmetric reduction method have the following advantages: 1. (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value height of Sheng Chenging reaches more than the 98%e.e; 2. biological catalyst is a microbial cells, fermentative production voluntarily, and steady quality, with low cost; 3. add resin or organic solvent in reaction system, energize material glucose etc. add high concentration substrate, can obtain high-optical-purity, high reaction conversion ratio, high production concentration; 4. in entire reaction course, do not need to add coenzyme; 5. reaction conditions gentleness, environmental friendliness.
The biological material specimens explanation
Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799 that the present invention is used, Candida boidinii (Candida boidinii) AS 2.2159, AS 2.2160, AS 2.2162, reddish brown shadow yeast (Sporobolomycessalmonicolor) AS 2.2110, AS 2.2111, AS 2.2113, AS 2.2114, AS 2.2115, AS 2.2116, AS 2.2117, AS 2.2118, AS 2.2119, AS 2.2120, AS 2.2121, AS 2.2122 etc. are all available from the common culture presevation of Chinese microorganism administrative center (CGMCC).
Embodiment
Below being that to produce bacterium with the selectivity carbonyl reductase be starting strain, in single aqueous phase system or two-phase system, is substrate with 2-carbonyl-4-phenylbutyrate, the embodiment of microorganism catalysis preparation (R)-2-hydroxy-4-phenyl ethyl butyrate.But the present invention is not limited to listed several examples.
Embodiment 1
Choose Candida boidinii (Candida boidinii) AS 2.2159 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 1.5g is changed in the identical potassium phosphate buffer that 5mL contains glucose (5%), add 2-carbonyl-4-phenylbutyrate of 5g/L simultaneously, in 30 ℃, 180rpm is reaction down.React end in 22 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 98.7% with molar yield 80.1%.
Embodiment 2
Choose Candida boidinii (Candida boidinii) AS 2.2160 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.0, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.7) clean twice, the above-mentioned thalline of 1.2 grams is changed in the identical potassium phosphate buffer that 5mL contains glucose (6%), add 2-carbonyl-4-phenylbutyrate of 8g/L simultaneously, in 28 ℃, 180rpm is reaction down.React end in 40 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 1% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 95.9% with molar yield 80%.
Embodiment 3
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2121 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.0) clean twice, the above-mentioned thalline of 1.5g is changed in the identical potassium phosphate buffer that 5mL contains glucose (7%), add 2-carbonyl-4-phenylbutyrate of 7g/L simultaneously, in 19 ℃, 180rpm is reaction down.React end in 48 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 85.4% with molar yield 72.1%.
Embodiment 4
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2120 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 1.0g is changed in the identical phosphoric acid buffer that 5mL contains glucose (5%), add 2-carbonyl-4-phenylbutyrate of 8g/L simultaneously, in 20 ℃, 180rpm is reaction down.React end in 5 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 85.5% with molar yield 73.5%.
Embodiment 5
Choose Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 1.8g is changed in the identical phosphoric acid buffer that 5mL contains glucose (5%), add 2-carbonyl-4-phenylbutyrate of 11g/L simultaneously, in 40 ℃, 180rpm is reaction down.React end in 36 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 3% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 84.9% with molar yield 76.2%.
Embodiment 6
Choose Candida boidinii (Candida boidinii) AS 2.2159 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) collect thalline, (0.1M pH6.5) cleans twice with potassium phosphate buffer, take by weighing above-mentioned 2.0g wet thallus and be added on the bottled 0.1M that contains glucose (7%) of 100mL triangle, pH6.5 potassium phosphate buffer 10mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 20g/L, adds X-5 resin 0.6g in the reaction system, in 30 ℃, carry out conversion reaction under the 180rpm.Finish reaction after 12 hours, filter paper filtering is collected resin.With the resin collected ethyl acetate extraction, in 30 ℃, more than the 180rpm shaking table vibration 4h with 10ml.Behind the centrifugal removal resin, collect ethyl acetate, add 2% (W/V) anhydrous Na SO
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 98.5% with molar yield be 82.7%.
Embodiment 7
Choose Candida boidinii (Candida boidinii) AS 2.2159 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, (0.1M pH6.5) cleans twice, takes by weighing above-mentioned 1.7g wet thallus and is added on the bottled 0.1M that contains 5% glucose of 100mL triangle with potassium phosphate buffer, pH6.5 potassiumphosphate-dibutyl phthalate (volume ratio 0.1: 1) damping fluid 5mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 8g/L, in 30 ℃, carries out conversion reaction under the 180rpm.Finish reaction after 32 hours.Centrifugal thalline and the water of going out obtains dibutyl phthalate, intact a small amount of substrate, R configuration and the S configuration mixture of products of unreacted, adds 2% (W/V) anhydrous Na SO again
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 98.88% with molar yield be 83.7%.
Embodiment 8
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2114 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, (0.1M pH6.5) cleans twice, takes by weighing above-mentioned 2.2g wet thallus and is added on the bottled 0.1M that contains 5% glucose of 100mL triangle with potassium phosphate buffer, pH6.5 potassiumphosphate-n-butyl acetate (volume ratio 1: 0.1) damping fluid 5mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 25g/L, in 35 ℃, carries out conversion reaction under the 180rpm.Finish reaction after 10 hours.Centrifugal thalline and the water of going out obtains n-butyl acetate, intact a small amount of substrate, R configuration and the S configuration mixture of products of unreacted, adds 2% (W/V) anhydrous Na SO again
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 85.2% with molar yield be 78.3%.
Embodiment 9
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2119 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, (0.1M pH6.5) cleans twice, takes by weighing above-mentioned 2.5g wet thallus and is added on the bottled 0.1M that contains 5% glucose of 100mL triangle with potassium phosphate buffer, pH6.5 potassiumphosphate-n-butyl acetate (volume ratio 0.9: 0.1) damping fluid 5mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 40g/L, in 27 ℃, carries out conversion reaction under the 180rpm.Finish reaction after 6 hours.Centrifugal thalline and the water of going out obtains n-butyl acetate, intact a small amount of substrate, R configuration and the S configuration mixture of products of unreacted, adds 2% (W/V) anhydrous Na SO again
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 77.2% with molar yield be 75%.
Embodiment 10
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2118 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 1.4g is changed in the identical potassium phosphate buffer that 5mL contains glucose (7%), add 2-carbonyl-4-phenylbutyrate of 15g/L simultaneously, in 25 ℃, 180rpm is reaction down.React end in 24 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 90% with molar yield 72%.
Embodiment 11
Choose Candida boidinii (Candida boidinii) AS 2.2162 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 2.3g is changed in the identical potassium phosphate buffer that 5mL contains glucose (5%), add 2-carbonyl-4-phenylbutyrate of 45g/L simultaneously, in 20 ℃, 180rpm is reaction down.React end in 28 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 88.5% with molar yield 81%.
Embodiment 12
Choose Candida boidinii (Candida boidinii) AS 2.2160 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, centrifugal 10 minutes (8,000 rev/min) collect thalline, (0.1M pH6.5) cleans twice with potassium phosphate buffer, take by weighing above-mentioned 1.3g wet thallus and be added on the bottled 0.1M that contains glucose (5%) of 100mL triangle, pH6.5 potassium phosphate buffer 10mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 13g/L, adds HD-1 resin 0.8g in the reaction system, in 30 ℃, carry out conversion reaction under the 180rpm.Finish reaction after 36 hours, filter paper filtering is collected resin.With the resin collected ethyl acetate extraction, in 30 ℃, more than the 180rpm shaking table vibration 6h with 10ml.Behind the centrifugal removal resin, collect ethyl acetate, add 2% (W/V) anhydrous Na SO
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 89.3% with molar yield be 84.6%.
Embodiment 13
Choose Candida boidinii (Candida boidinii) AS 2.2159 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 3 days for 26 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 28 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.6g, centrifugal 10 minutes (8,000 rev/min) collect thalline, (0.1M pH6.5) cleans twice with potassium phosphate buffer, take by weighing above-mentioned 2.4g wet thallus and be added on the bottled 0.1M that contains glucose (5%) of 100mL triangle, pH6.5 potassium phosphate buffer 10mL, initial substrate 2-carbonyl-4-phenylbutyrate amount is 38g/L, adds D392 resin 0.7g in the reaction system, in 25 ℃, carry out conversion reaction under the 180rpm.Finish reaction after 20 hours, filter paper filtering is collected resin.With the resin collected ethyl acetate extraction, in 30 ℃, more than the 180rpm shaking table vibration 5h with 10ml.Behind the centrifugal removal resin, collect ethyl acetate, add 2% (W/V) anhydrous Na SO
4Dry, filter laggard promoting the circulation of qi analysis of hplc (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 95.5% with molar yield be 82.9%.
Embodiment 14
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2111 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 5 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 25 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 2.0g, centrifugal 10 minutes (8,000 rev/min) the collection thalline, with potassium phosphate buffer (0.1M, pH6.5) clean twice, the above-mentioned thalline of 0.8g is changed in the identical phosphoric acid buffer that 5mL contains glucose (5%), add 2-carbonyl-4-phenylbutyrate of 8g/L simultaneously, in 30 ℃, 180rpm is reaction down.React end in 48 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 94.1% with molar yield 78.5%.
Embodiment 15
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2121 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 5 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 25 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, the substrate of 1g/L is added in the thalline fermented liquid of 50ml, and adding glucose to glucose concn is 5%, and in 30 ℃, 180rpm is reaction down.React end in 42 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 81.7% with molar yield 77.5%.
Embodiment 16
Choose reddish brown shadow yeast (Sporobolomyces salmonicolor) AS 2.2116 as the catalysis bacterial strain
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and pH6.0 sterilized 15 minutes for 121 ℃, and the inoculation of sterilization postcooling was cultivated 5 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, pH6.5, liquid amount is the bottled liquid 50mL of 250mL triangle, sterilizes 20 minutes sterilization postcooling inoculation inclined-plane seed for 120 ℃, the shaking table of 180rpm was cultivated 48 hours for 25 ℃, as seed or fermenting enzyme liquid.
Obtaining containing the wet thallus amount in every 50mL fermenting enzyme liquid is 1.5g, the substrate of 1.2g/L is added in the thalline fermented liquid of 50ml, and adding glucose to glucose concn is 5%, and in 25 ℃, 180rpm is reaction down.React end in 24 hours, the centrifugal thalline of removing gets supernatant liquor.Supernatant liquor equal volume of ethyl acetate, water phase separated and organic phase.Add 2% (W/V) anhydrous Na SO to organic phase
4Dried overnight, gas chromatography chiral post assay products (R)-2-hydroxy-4-phenyl ethyl butyrate enantiomeric excess value be 83.4% with molar yield 78.6%.
Claims (8)
1. one kind is the method for substrate biocatalysis preparation (R)-2-hydroxy-4-phenyl ethyl butyrate with 2-carbonyl-4-phenylbutyrate, and the method comprising the steps of:
(1) select following selectivity carbonyl reductase to produce bacteria strain as starting strain: Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799, Candida boidinii (Candida boidinii) AS2.2159, AS 2.2160 or AS 2.2162, or reddish brown shadow yeast (Sporobolomycessalmonicolor) AS 2.2110, AS 2.2111, AS 2.2113, AS 2.2114, AS 2.2115, AS 2.2116, AS 2.2117, AS 2.2118, AS 2.2119, AS 2.2120, AS2.2121 or AS2.2122;
(2) cultivation of wet thallus: substratum is formed and to be counted with g/L: peptone 10～30, and glucose 10～30, yeast extract paste 5～20, pH5.5～7.5,20～40 ℃ of temperature, with the described strain culturing of step (1) 1～5 day, filtering fermentation liquor obtained wet thallus as the enzyme source;
(3) preparation reaction system: with 2-carbonyl-4-phenylbutyrate is substrate, is mixed with single water react system with the phosphate buffered saline buffer of 0.1M, pH5.5～pH7.5, and concentration of substrate is 1g/L～50g/L; Perhaps the phosphate buffered saline buffer with organic solvent and pH6.5 is mixed with water/organic solvent two-phase system, adds substrate again, concentration of substrate 1g/L～50g/L; Perhaps the phosphate buffered saline buffer with macroporous resin and pH5.5～pH7.5 is mixed with water/resin two-phase system, adds substrate again, concentration of substrate 1g/L～50g/L;
(4) enzymatic conversion reaction: add the described wet thallus of step (2) in reaction system, carry out enzyme reaction in 20～50 ℃, the reaction times is 1～48 hour;
(5) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, the polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, drying and dehydrating evaporates and reclaims solvent again, obtains colourless oil liquid (R)-2-hydroxy-4-phenyl ethyl butyrate.
2. method according to claim 1 is characterized in that described bacterial strain is Candida boidinii bacterium (Candida boidinii) AS 2.2159.
3. method according to claim 1 is characterized in that the wet thallus amount that adds in the step (4) is 10～60g/g substrate.
4. method according to claim 1 is characterized in that described enzymatic conversion reaction, adds glucose as cosubstrate in reaction process, and its concentration is 10～100g/L.
5. method according to claim 1 is characterized in that described enzymatic conversion reaction, replenishes in reaction process and adds substrate to 1～50g/L.
6. method according to claim 1, it is characterized in that used macroporous resin is DA201, Hz816, Hz803, AB-8, D4006, HPD300, HPD100A, NAK-II, X-5, Hz004, Hz202, Hz016 or Hz802 macroporous adsorbent resin, or D201GF, D392, D315, D290, D280 or D293 large pore anion resin, or HD-2, D61, D001, HD-1, D001-CC or D113 macropore cation resin, the aperture of described macroporous resin is 100～180
The mass ratio of resin and substrate is 0.6～6: 1.
7. method according to claim 1, the compound method that it is characterized in that water/organic solvent two-phase system is the phosphate buffered saline buffer with organic solvent and pH5.5～pH7.5, be mixed with water/organic solvent two-phase system in 0.1: 1 by volume～1: 0.1, add concentration of substrate 1g/L～50g/L again, used organic solvent is a dioctyl phthalate (DOP), or dibutyl phthalate, n-butyl acetate, n-amyl acetate, hexanaphthene, normal hexane, normal heptane, octane, octane-iso, decane, nonane, octanol, propyl carbinol or isopropylcarbinol.
8. one kind is the method for substrate biocatalysis preparation (R)-2-hydroxy-4-phenyl ethyl butyrate with 2-carbonyl-4-phenylbutyrate, this method steps:
(1) select following selectivity carbonyl reductase to produce bacteria strain as starting strain: Bacillus foecalis alkaligenes (Alcaligenes faecalis) AS 1.1799, Candida boidinii (Candida boidinii) AS2.2159, AS 2.2160 or AS 2.2162, or reddish brown shadow yeast (Sporobolomycessalmonicolor) AS 2.2110, AS 2.2111, AS 2.2113, AS 2.2114, AS 2.2115, AS 2.2116, AS 2.2117, AS 2.2118, AS 2.2119, AS 2.2120, AS2.2121 or AS2.2122;
(2) cultivation of wet thallus: substratum is formed and to be counted with g/L: peptone 10～30, and glucose 10～30, yeast extract paste 5～20, pH5.5～7.5,20～40 ℃ of temperature with the described strain culturing of step (1) 1～5 day, obtain containing the fermented liquid of wet thallus;
(3) in above-mentioned wet thallus cultivation and fermentation liquid, add 2-carbonyl-4-phenylbutyrate of 1～50g/L as substrate; Carry out enzyme reaction in 20～50 ℃, the reaction times is 1～48 hour;
(4) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, the polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, drying and dehydrating evaporates and reclaims solvent again, obtains colourless oil liquid (R)-2-hydroxy-4-phenyl ethyl butyrate.
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| CN102382780A (en) * | 2010-09-03 | 2012-03-21 | 中国科学院成都生物研究所 | Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same |
| CN102925500A (en) * | 2012-11-05 | 2013-02-13 | 遵义医学院 | Preparation method of chiral 4-hydroxy-4 phenyl butyric acid ester |
| CN102994403A (en) * | 2012-08-20 | 2013-03-27 | 常州大学 | Rhodotorula glutinis and application of Rhodotorula glutinis in preparation of (R)-2-hydroxy-4- phenyl ethyl butyrate through asymmetric catalysis |
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| CN102382780A (en) * | 2010-09-03 | 2012-03-21 | 中国科学院成都生物研究所 | Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same |
| CN102382780B (en) * | 2010-09-03 | 2013-02-13 | 中国科学院成都生物研究所 | Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same |
| CN102994403A (en) * | 2012-08-20 | 2013-03-27 | 常州大学 | Rhodotorula glutinis and application of Rhodotorula glutinis in preparation of (R)-2-hydroxy-4- phenyl ethyl butyrate through asymmetric catalysis |
| CN102994403B (en) * | 2012-08-20 | 2014-08-13 | 常州大学 | Rhodotorula glutinis and application of Rhodotorula glutinis in preparation of (R)-2-hydroxy-4- phenyl ethyl butyrate through asymmetric catalysis |
| CN102925500A (en) * | 2012-11-05 | 2013-02-13 | 遵义医学院 | Preparation method of chiral 4-hydroxy-4 phenyl butyric acid ester |
| CN102925500B (en) * | 2012-11-05 | 2014-07-30 | 遵义医学院 | Preparation method of chiral 4-hydroxy-4 phenyl butyric acid ester |
| CN104313064A (en) * | 2014-10-01 | 2015-01-28 | 青岛科技大学 | Method for producing chiral bromophenyl methyl propionate by virtue of cell method |
| CN106244044A (en) * | 2016-08-29 | 2016-12-21 | 无锡万能胶粘剂有限公司 | A kind of seccotine |
| CN115074404A (en) * | 2022-06-28 | 2022-09-20 | 吉尔多肽生物制药(大连市)有限公司 | A kind of synthetic method of (2R, 4R)-2-amino-4-methylnonanoic acid |
| CN115074404B (en) * | 2022-06-28 | 2025-02-18 | 吉尔多肽生物制药(大连市)有限公司 | A method for synthesizing (2S, 4R)-2-amino-4-methylnonanoic acid |
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