CN106811432B

CN106811432B - Erysivelum pratense and application thereof in preparation of (R) -3-chlorobenzene ethanol

Info

Publication number: CN106811432B
Application number: CN201611266505.8A
Authority: CN
Inventors: 何军邀; 王普; 白东亚
Original assignee: Zhejiang University of Technology ZJUT; Zhejiang Medical College
Current assignee: Zhejiang University of Technology ZJUT; Zhejiang University ZJU
Priority date: 2016-12-31
Filing date: 2016-12-31
Publication date: 2020-04-28
Anticipated expiration: 2036-12-31
Also published as: CN106811432A

Abstract

The invention discloses an Ersikovicum harasum and application thereof in preparing (R) -3-chlorobenzene ethanol, and the method for preparing the optically pure (R) -3-chlorobenzene ethanol by adopting the new strain through catalysis has the advantages of high substrate concentration, good stereoselectivity, high optical purity of products and the like. According to the invention, through chiral biological catalysis by adopting the cells ZJPH1604 of the harassing Erssolvia (Oerskovia turbata) as a catalyst, when the concentration of a substrate is 80mmol/L, the e.e of a target product (R) -3-chlorobenzene ethanol reaches 99.9%, and the yield reaches 98.1%.

Description

Erysivelum pratense and application thereof in preparation of (R) -3-chlorobenzene ethanol

(I) technical field

The invention relates to a new strain-perturbed elsholtzia rugosa (Oerskovia turbata) ZJPH1604 which can be used for preparing high-optical-purity (R) -3-chloro-phenylethanol by biologically catalyzing asymmetric reduction of 3-chloro-acetophenone and application thereof.

(II) background of the invention

The chemical structural formula of the (R) -3-chlorophenethanol is as follows:

(R) -3-Chlorobenzeneethanol is an important chiral intermediate, is a building block for a variety of drug molecules, and can be used, for example, for synthesizing an active compound (R) -5- [1- (3-chlorophenyl) ethoxy ] ethoxy for treating spinal muscular atrophy]Quinazoline-2, 4-diamines, and can be used for the synthesis of β for the treatment of depression, diabetes and obesity₃Adrenergic receptor agonists including SR58611, Solabegoron, CL316243, AJ-9677 and the like.

The chemical method for preparing (R) -3-chlorobenzene ethanol needs expensive metal catalysts such as rhodium, ruthenium and the like, and causes pollution to the environment. The (R) -3-chlorobenzene ethanol prepared by the biological asymmetric reduction method of microbial whole-cell catalysis has the advantages of mild reaction conditions, high stereoselectivity, environmental friendliness and the like.

It has been reported in the literature that John Thurmond et al (J.Med.chem.2008,51, 449-.

Disclosure of the invention

The invention aims to provide a new microbial strain-a disturbance elsholtzia rugosa (Oerskovia turbata) ZJPH1604 which can be used for preparing (R) -3-chlorophenethane with high optical purity by biologically catalyzing asymmetric reduction of 3-chloroacetophenone, and application of the strain in preparing (R) -3-chlorophenethane by catalytic asymmetric synthesis.

The technical scheme adopted by the invention is as follows:

the invention provides a new strain-harassing elsholtzia rugosa (Oerskovia turbata) ZJPH1604, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2016541, date of deposit: year 2016, month 9, and day 30, address: china, wuhan university, zip code: 430072.

the invention also provides an application of the Erosacchus praecox ZJPH1604 in preparation of (R) -3-chlorophenylethanol by microbial catalytic asymmetric reduction of 3-chloroacetophenone, which specifically comprises the following steps: taking 3-chloroacetophenone as a substrate, taking wet somatic cells obtained by fermentation culture of Erysivelum dahliae ZJPH1604 as an enzyme source, adding an auxiliary substrate, performing a conversion reaction in a buffer solution with the pH of 6.0-9.0 at 25-35 ℃, and after the reaction is finished, separating and purifying the reaction solution to obtain (R) -3-chlorophenethylalcohol; the auxiliary substrate is one of glucose, sucrose, maltose, isopropanol, methanol or ethanol.

Further, the initial concentration of the substrate 3-chloroacetophenone is 20-200 mmol/L buffer solution (preferably 25-150mmol/L), and the addition amount of wet cells of the Erwinia carotovora ZJPH1604 is 50-300 g/L buffer solution (preferably 100-300g/L) in terms of wet weight; when the auxiliary substrate is glucose, sucrose or maltose, the addition amount is 20-300 g/L buffer solution (preferably 100-300g/L), and when the auxiliary substrate is methanol, ethanol or isopropanol, the addition volume is 10-30% (preferably 20%) of the buffer solution volume.

Further, the enzyme source is prepared as follows: 1) slant culture: inoculating Erysiveromyces bravayi ZJPH1604 into a slant culture medium, and culturing at 30 ℃ for 1-2 days to obtain slant thalli; the slant culture medium comprises the following components: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)₄)₂SO₄2g/L，KH₂PO₄1g/L，NaCl 0.5g/L，MgSO₄·7H₂0.5g/L of O, 20g/L of agar, water as a solvent and pH of 6.5;

2) seed culture: selecting a ring of thalli from the inclined plane, inoculating the thalli into a 250mL shake flask filled with 100mL seed culture medium, and culturing at 30 ℃ and 200r/min for 24 hours to obtain seed liquid; the seed culture medium comprises the following components: 10-20g/L glucose, 5-10g/L peptone, 4-8g/L yeast extract, (NH)₄)₂SO₄2-4g/L，KH₂PO₄2-4g/L，NaCl 0.5-2g/L，MgSO₄·7H₂O0.5-2g/L, water as solvent, pH 6.5-8.0; preferably, the seed culture medium consists of: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)₄)₂SO₄2g/L，KH₂PO₄2g/L，NaCl 0.5g/L，MgSO₄·7H₂O is 0.5g/L, the solvent is water, and the pH value is 6.5;

3) fermentation culture: transferring the seed solution into a 250mL shake flask filled with 100mL fermentation medium by an inoculation amount with the volume concentration of 4-10% (preferably 8%), culturing for 36-48h (preferably 48h) at 30 ℃ and 180-220r/min (preferably 200r/min), centrifuging the fermentation liquid, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 20-30 g/L glucose, 10-40 g/L yeast extract, (NH)₄)₂SO₄1.0-6.0g/L，KH₂PO₄2.0～6.0g/L，MgSO₄·7H₂0.3-0.6 g/L of O, 0.2-0.8 g/L of NaCl, water as a solvent, and 6.0-7.5 of pH, preferably the final concentration of the fermentation medium is as follows: 15g/L glucose, 30g/L yeast extract, (NH)₄)₂SO₄2.5g/L，KH₂PO₄4g/L，MgSO₄·7H₂O0.6 g/L, NaCl 0.4g/L, water as solvent, pH 6.5.

Adopting Box-Behnken rotation center combined experimental design method to test glucose concentration, yeast extract concentration and KH₂PO₄The concentration of three factors which have obvious influence on enzyme production is optimized to obtain Erschvicella irritans (Oerskovia) ZJPH 1604. The optimized culture medium of the strain comprises the following components: 20-30 g/L glucose, 10-40 g/L yeast extract, (NH)₄)₂SO₄1.0-6.0g/L，KH₂PO₄2.0～6.0g/L，MgSO₄·7H₂0.3-0.6 g/L of O, 0.2-0.8 g/L of NaCl, water as solvent, and 6.0-7.5 of pH.

The culture conditions of Erschoervitae acinthinus (Oerskovia) ZJPH1604 are as follows: the initial pH is 6.0-7.5, the liquid loading amount of a shake flask is 80-120 mL/250mL, the culture temperature is 30 ℃, the rotation speed of a shaking table is 180-220 rpm, the inoculation amount is 4-10%, and the culture time is 36-48 h.

The reaction liquid separation and purification method comprises the following steps: after the reaction is finished, extracting the conversion solution by using ethyl acetate with the same volume, centrifuging to obtain a supernatant, and analyzing the contents of a target product and a residual substrate and the optical purity of the product by adopting a chiral gas chromatography.

According to the invention, the auxiliary substrate is preferably isopropanol, and the optical purity and yield of the obtained product are highest.

The invention screens and obtains new microbial strains different from the previous similar research reports from the soil sample, and the concentration and the yield of the substrate for catalytic conversion are higher, thereby providing beneficial reference in the aspect of preparing the chiral intermediate (R) -3-chlorobenzene ethanol by researching the microbial catalytic method.

The invention has the following beneficial effects: the invention provides a new microbial strain for preparing (R) -3-chlorophenylethanol by catalyzing asymmetric reduction of 3-chloroacetophenone by microorganisms, and the preparation of the optically pure (R) -3-chlorophenylethanol by the catalysis of the new strain has the advantages of high substrate concentration, good stereoselectivity, high optical purity of the product and the like. According to the invention, through chiral biological catalysis by adopting the cells ZJPH1604 of the harassing Erssolvia (Oerskovia turbata) as a catalyst, when the concentration of a substrate is 80mmol/L, the e.e of a target product (R) -3-chlorobenzene ethanol reaches 99.9%, and the yield reaches 98.1%.

(IV) description of the drawings

FIG. 1 is a gas chromatogram of a substrate standard (containing internal standard dodecane);

FIG. 2 is a gas chromatogram of a racemate standard (containing internal standard dodecane);

FIG. 3 is a gas chromatogram of the bioreduction extract (containing internal standard dodecane) from the strain ZJPH1604 of Ascomyces terrestris (Oerskovia turbata).

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the dosage of the substrate, the auxiliary substrate and the wet bacteria in the embodiment of the invention is calculated by the volume of the buffer solution.

Example 1: screening and identification of strains

The strain source is as follows: the Elsholtzia furiosu (Oerskovia turbata) ZJPH1604 strain is obtained by separating and screening soil samples near the east lake of Zhejiang province, and the specific screening method is as follows:

adding 1g of the collected soil sample into a 250mL shake flask filled with 50mL of enrichment medium, culturing for 5-6 days at 30 ℃ and 200rpm, transferring 1mL of culture solution into a fresh enrichment medium after the culture solution becomes turbid, continuously culturing for 5-6 days, and repeating the enrichment culture for 3-4 times. 3-chloroacetophenone is used as a unique carbon source in the enrichment medium. The enrichment medium formula is as follows: 25mmol/L (NH) of 3-chloroacetophenone₄)₂SO₄2g/L，KH₂PO₄2g/L，NaCl0.5g/L，MgSO₄·7H₂O0.5 g/L, solvent is water, pH 6.5.

And then the enrichment culture solution is diluted in a gradient way, coated on a separation plate (the plate culture medium is formed by adding agar of 15-20g/L into the enrichment culture medium), and a single colony strain is obtained after multiple times of separation culture. The single colony strain is selected and inoculated to a seed culture medium, cultured for 24 hours at 30 ℃, and then transferred to an enzyme production culture medium for 42 hours at 30 ℃. The microbial cells obtained by screening were used as a catalyst (final concentration 100g/L) in 10ml of a phosphate buffer solution of pH 6.5 at 30 ℃ for 24 hours using 3-chloroacetophenone of final concentration 5mM as a substrate. After the conversion is finished, extracting the conversion solution by using ethyl acetate with the same volume, centrifuging to obtain supernatant, and detecting the enantiomeric excess value (e.e. value) and yield of the target product (R) -3-chlorobenzene ethanol in the conversion solution by using chiral gas chromatography. The quantification was done by internal standard method, where the internal standard was dodecane. 1mL of the extract was analyzed by adding 1. mu.L of dodecane. Gas chromatography conditions: shimadzu, japan GC-2014 gas chromatograph, N2000 chromatography workstation, zheda; walian CP-Chirasil-Dex chiral capillary gas chromatography column (25 m. times.0.25 mm. times.0.25 μm) in USA. The carrier gas is high-purity nitrogen, and the flow rate is 2 mL/min; the sample injection amount is 1 mu L, and the split ratio is 15: 1; the temperature of the detector and the injection port is 250 ℃; the temperature of the chromatographic column is 120-160 ℃; temperature rise rate: 8 ℃/min; the detector is FID. Detecting the sample under the above conditions, with substrate retention time of 4.1min, as shown in FIG. 1; retention time of racemate standard, R-type product 6.5min, and S-type product 6.7min, as shown in FIG. 2; the retention time of the ZJPH1604 strain transformation product was 6.5min, as shown in FIG. 3. The internal standard dodecane has a retention time of 3.0 min.

The concentrations of the substrate and the product in the reaction solution were calculated using the relative correction factors, respectively. The Yield (Yield) of the reaction was then determined. The calculation formula is as follows:

yield ═ C_i/C₀×100％

In the formula C₀、C_iRespectively, the molar concentration of the starting substrate of the reaction and the molar concentration of the product at the end of the reaction.

The optical purity of the product is indicated by enantiomeric excess (e.e.).

e.e.＝(C_R-C_S)/(C_R+C_S)×100％

In the formula C_RAnd C_SThe molar concentrations of R-type 3-chlorophenethanol and S-type 3-chlorophenethanol are shown respectively.

The characteristics of the new strain are as follows:

the biochemically physiological characteristics are gram-positive, non-sporulating, non-motile, non-capsulated, acid-producing by carbohydrate fermentation, non-gas-producing, oxidase-positive, catalase-positive, obligate aerobic, 59 carbon sources that can be utilized include dextrin, D-maltose, trehalose, cellobiose, gentiobiose, sucrose, stachyose, raffinose, lactose, D-melibiose, D- (-) -salicin, N-acetyl-D-glucosamine, N-acetyl- α -D-mannosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, D-galactose, methyldipretone, D-fucose, L-fucose, glycyl-L-proline, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, L-serine, L-galactonolactone, D-gluconic acid, glucuronic acid, glucuronamide, mucic acid, quinic acid, glucaric acid, p-cresol-phenylacetic acid, methyl lactate, L-glutamic acid, L-serine, L-galactono-galactaric acid, L-galactono-D-L-D-L-D-L-D-L-D-L-D-L-D-L.

16S rDNA sequence characteristics of the strain: the extracted total DNA of the cells is used as a template, the 16S rDNA gene of the strain is amplified by using universal primers P1 and P2, and then the PCR product is subjected to 1 percent agarose gel electrophoresis. The 16S rDNA gene sequence (SEQ ID NO.1) of the strain ZJPH1604 is confirmed to be submitted to GenBank (GenBank accession No. KY302675) through sequencing, and the 16S rRNA sequence of the strain ZJPH1604 is subjected to homology alignment (BLAST) on NCBI website (http:// www.ncbi.nlm.nih.gov), and the results show that: the ZJPH1604 strain has high sequence homology with a part of strains of Erschoervita haracterium (Oerskovia sp.). The sequence homology of ZJPH1604 strain and Oerskovia strain (GenBank accession No. HM56054.1) reaches 99%.

Based on physiological and biochemical characteristics combined with molecular biological identification, the strain was identified as elsholtzia aekei (oerkovia sp.), named as elsholtzia furazai (oerkovia sp.) ZJPH1604, deposited at the chinese type culture collection, address: china, wuhan university, zip code: 430072; the preservation number is: CCTCC NO: m2016541, date of deposit: 2016, 9 months and 30 days.

Example 2: obtaining wet somatic cells

The seed culture medium formula comprises: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)₄)₂SO₄2g/L，KH₂PO₄2g/L，NaCl 0.5g/L，MgSO₄·7H₂O0.5 g/L, solvent is water, pH 6.5.

The fermentation medium formula comprises: 15g/L glucose, 30g/L yeast extract, (NH)₄)₂SO₄4g/L，KH₂PO₄4g/L，NaCl0.6g/L，MgSO₄·7H₂O0.8 g/L, phosphate buffer as solvent, pH 6.5.

Slant culture: inoculating Erysiveromyces bravayi ZJPH1604 into a slant culture medium, and culturing at 30 ℃ for 1-2 days to obtain slant thalli; the slant culture medium comprises the following components: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)₄)₂SO₄2g/L，KH₂PO₄1g/L，NaCl 0.5g/L，MgSO₄·7H₂0.5g/L of O, 20g/L of agar, water as a solvent and pH of 6.5;

selecting a ring of thalli from a slant, inoculating the thalli into a 250mL shake flask filled with 100mL of seed culture medium, culturing for 24 hours at 30 ℃ and 200rpm to obtain seed liquid, then inoculating the seed liquid into a 250mL shake flask filled with 100mL of fermentation culture medium according to the inoculation amount with the volume concentration of 8%, and culturing for 48 hours at 30 ℃ and 200 rpm. After the culture is finished, the fermentation liquor is centrifuged, the precipitate is washed once by phosphate buffer solution with the pH value of 6.5, and wet thalli cells are collected for later use.

Example 3:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 100g/L in terms of wet weight; adding 3-chloroacetophenone of 25mmol/L buffer solution as a substrate, adding isopropanol accounting for 10% of the volume of the buffer solution as an auxiliary substrate, and placing the mixture in a shaking table at 30 ℃ and 200r/min for reaction for 24 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 17.8mmol/L, the optical purity ee was 99.9%, and the yield was 70.9%.

Example 4:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 100g/L in terms of wet weight; adding 3-chloroacetophenone with 25mmol/L of buffer solution as a substrate, adding isopropanol accounting for 10% of the volume of the buffer solution as an auxiliary substrate, and placing the mixture in a shaking table at 37 ℃ and 200r/min for reaction for 24 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 16.8mmol/L, the optical purity ee was 99.9%, and the yield was 67.3%.

Example 5

The wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L in terms of wet weight; adding 3-chloroacetophenone of 25mmol/L buffer solution as a substrate, adding isopropanol accounting for 10% of the volume of the buffer solution as an auxiliary substrate, and placing the mixture in a shaking table at 30 ℃ and 200r/min for reaction for 24 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 19.2mmol/L, the optical purity ee was 99.9%, and the yield was 76.7%.

Example 6:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L in terms of wet weight; adding 3-chloroacetophenone with 25mmol/L of buffer solution as a substrate, adding isopropanol accounting for 10% of the volume of the buffer solution as an auxiliary substrate, and placing the mixture in a shaking table at 37 ℃ and 200r/min for reaction for 24 hours. By the detection method of example 1, the concentration of the product (R) -3-chlorophenethanol was 20.4mmol/L, the optical purity ee was 99.9%, and the yield was 88.8%.

Example 7:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 7.0) at a concentration of 150g/L in terms of wet weight; adding 25mmol/L buffer solution of 3-chloroacetophenone as a substrate, adding 300g/L glucose as an auxiliary substrate, and reacting in a shaking table at 37 ℃ and 200r/min for 48 h. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 3.4mmol/L, the optical purity ee was 99.9%, and the yield was 13.7%.

Example 8:

the wet cells obtained in example 2 were suspended in 15mL of a phosphate buffer (pH 6.5) at a concentration of 200g/L in terms of wet weight; adding 3-chloroacetophenone of 80mmol/L buffer solution as a substrate, adding isopropanol accounting for 20% of the volume of the buffer solution as an auxiliary substrate, and placing the mixture in a shaking table at 37 ℃ and 200r/min for reaction for 48 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 78.5mmol/L, the optical purity ee was 99.9%, and the yield was 98.1%.

Example 9:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 7.0) at a concentration of 100g/L in terms of wet weight; adding 35mmol/L buffer solution of 3-chloroacetophenone as a substrate, adding 100g/L buffer solution of glucose as an auxiliary substrate, and reacting in a shaker at 30 ℃ and 200r/min for 24 h. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 7.7mmol/L, the optical purity ee was 99.9%, and the yield was 22.2%.

Example 10:

the wet cells obtained in example 2 were suspended in 20mL of a phosphate buffer (pH 7.0) at a concentration of 200g/L in terms of wet weight; 3-chloroacetophenone in 35mmol/L buffer solution was added as substrate, 2mL (10% by volume of buffer solution, v/v) of isopropanol was added as co-substrate, and the mixture was placed in a shaker at 37 ℃ and 200r/min for reaction for 48 h. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 34.6mmol/L, the optical purity ee was 99.9%, and the yield was 99%.

Example 11:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 8.0) at a concentration of 300g/L in terms of wet weight; adding 3-chloroacetophenone of 25mmol/L buffer solution as a substrate, adding 1mL (accounting for 10% of the buffer solution by volume) of isopropanol as an auxiliary substrate, and placing the mixture in a shaking table at 30 ℃ and 200r/min for reaction for 24 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 19.9mmol/L, the optical purity ee was 99.9%, and the yield was 79.7%.

Example 12

The wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 8.0) at a concentration of 200g/L in terms of wet weight; adding 150mmol/L buffer solution of 3-chloroacetophenone as a substrate, adding 2mL (20% of the buffer solution in volume) of isopropanol as an auxiliary substrate, and reacting in a shaking table at 30 ℃ and 200r/min for 24 h. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 98.1mmol/L, the optical purity ee was 99.9%, and the yield was 65.4%.

Example 13

The wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 8.0) at a concentration of 250g/L in terms of wet weight; adding 150mmol/L buffer solution of 3-chloroacetophenone as a substrate, adding 2mL (20% of the buffer solution by volume) of isopropanol as an auxiliary substrate, and reacting for 36h in a shaking table at 30 ℃ and 200 r/min. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 121.8mmol/L, the optical purity ee was 99.9%, and the yield was 71.2%.

Example 14:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 9.0) at a concentration of 100g/L in terms of wet weight; adding 3-chloroacetophenone in 25mmol/L buffer solution as a substrate, adding 2.5mL (accounting for 20% of the buffer solution by volume), namely isopropanol as an auxiliary substrate, and placing the mixture in a shaking table at 30 ℃ and 200r/min for reaction for 48 hours. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 11.2mmol/L, the optical purity ee was 99.9%, and the yield was 44.1%.

Example 15:

the wet cells obtained in example 2 were suspended in 10mL of a phosphate buffer (pH 9.0) at a concentration of 100g/L in terms of wet weight; 3-chloroacetophenone in 35mmol/L buffer solution was added as substrate, 2mL (20% by volume of buffer solution, v/v) of isopropanol was added as co-substrate, and the mixture was placed in a shaker at 30 ℃ and 200r/min for reaction for 48 h. By the examination method of example 1, the concentration of the product (R) -3-chlorophenethanol was 34.6mmol/L, the optical purity ee was 99.9%, and the yield was 99.1%.

SEQUENCE LISTING

<110> Zhejiang medical high specialty school; zhejiang industrial university

<120> Erschoervilia incarnata and application thereof in preparation of (R) -3-chlorobenzene ethanol

<130>

<160>1

<170>PatentIn version 3.5

<210>1

<211>1395

<212>DNA

<213>Oerskovia turbata

<400>1

gatcactggg gaacgggtga gtaacacgtg agtaacctgc cccagactcc gggataagcc 60

ttggaaacga ggtctaatac tggatatgag atgcccctgc atggggagtg tctggaaaga 120

tttatcggtc tgggatggac tcgcggccta tcagcttgtt ggtggggtaa tggcctacca 180

aggcgacgac gggtagccgg cctgagaggg cgaccggcca cactgggact gagacacggc 240

ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgaaa gcctgatgca 300

gcgacgccgc gtgagggatg aaggccttcg ggttgtaaac ctctttcagc agggaagaag 360

cgcaagtgac ggtacctgca gaagaagcgc cggctaacta cgtgccagca gccgcggtaa 420

tacgtagggc gcaagcgttg tccggaatta ttgggcgtaa agagctcgta ggcggtttgt 480

cgcgtctggt gtgaaaactc aaggctcaac cttgagcttg catcgggtac gggcagacta 540

gagtgcggta ggggtgactg gaattcctgg tgtagcggtg gaatgcgcag atatcaggag 600

gaacaccgat ggcgaaggca ggtctctggg ccgcaactga cgctgaggag cgaaagcatg 660

gggagcgaac aggattagat accctggtag tccatgccgt aaacgttggg cactaggtgt 720

ggggctcatt ccacgagttc cgtgccgcag caaacgcatt aagtgccccg cctggggagt 780

acggccgcaa ggctaaaact caaaggaatt gacgggggcc cgcacaagcg gcggagcatg 840

cggattaatt cgatgcaacg cgaagaacct taccaaggct tgacatacac cggaaacttc 900

cagagatggt tgccccgcaa ggtcggtgta caggtggtgc atggttgtcg tcagctcgtg 960

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc tcgtcttatg ttgccagcac 1020

gtcatggtgg ggactcataa gagactgccg gggtcaactc ggaggaaggt ggggatgacg 1080

tcaaatcatc atgcccctta tgtcttgggc ttcacgcatg ctacaatggc cggtacaaag 1140

ggctgcgata ccgtaaggtg gagcgaatcc caaaaagccg gtctcagttc ggattggggt 1200

ctgcaactcg accccatgaa gtcggagtcg ctagtaatcg cagatcagca acgctgcggt 1260

gaatacgttc ccgggccttg tacacaccgc ccgtcaagtc acgaaagtcg gtaacacccg 1320

aagccggtgg cccaacccct tgtgggaggg agccgtcgaa agtgggactg gcgattggga 1380

ctaagtcgta acaag 1395

Claims

1. The Erschoervitae acinthini (Oerskovia turbata) ZJPH1604, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2016541, date of deposit: year 2016, month 9, and day 30, address: china, wuhan university, zip code: 430072.

2. use of the Ersikov perturbed Ersikov strain ZJPH1604 of claim 1 in the preparation of (R) -3-chlorophenethanol by the microbial catalytic asymmetric reduction of 3-chloroacetophenone.

3. The use according to claim 2, characterized in that the use is: taking 3-chloroacetophenone as a substrate, taking wet somatic cells obtained by fermentation culture of Erysivelum dahliae ZJPH1604 as an enzyme source, adding an auxiliary substrate, performing a conversion reaction in a buffer solution with the pH of 6.0-9.0 at 25-35 ℃, and after the reaction is finished, separating and purifying the reaction solution to obtain (R) -3-chlorophenethylalcohol; the co-substrate is isopropanol.

4. The application as claimed in claim 3, characterized in that the initial concentration of the substrate 3-chloroacetophenone is 20-200 mmol/L buffer, and the addition amount of wet cells of Erysiveromyces rimosus ZJPH1604 is 50-300 g/L buffer by wet weight; the volume of the auxiliary substrate is 10-30% of the volume of the buffer solution.

5. Use according to claim 3, characterized in that the enzyme source is prepared as follows: 1) slant culture: inoculating Erysiveromyces bravayi ZJPH1604 into a slant culture medium, and culturing at 30 ℃ for 1-2 days to obtain slant thalli; the slant culture medium comprises the following components: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)₄)₂SO₄2g/L，KH₂PO₄1g/L，NaCl0.5g/L，MgSO₄·7H₂0.5g/L of O, 20g/L of agar, water as a solvent and pH of 6.5;

2) seed culture: selecting a ring of thalli from the inclined plane, inoculating the thalli into a 250mL shake flask filled with 100mL seed culture medium, and culturing at 30 ℃ and 200r/min for 24 hours to obtain seed liquid; the seed culture medium comprises the following components: 10-20g/L glucose, 5-10g/L peptone, 4-8g/L yeast extract, (NH)₄)₂SO₄2-4g/L，KH₂PO₄2-4g/L，NaCl 0.5-2g/L，MgSO₄·7H₂O0.5-2g/L, water as solvent, pH 6.5-8.0;

3) fermentation culture: transferring the seed solution into a 250mL shake flask filled with 100mL fermentation medium by an inoculation amount with the volume concentration of 4-10%, culturing for 36-48 hours at 30 ℃ and 220r/min at 180-; the final concentration of the fermentation medium is as follows: 20-30 g/L glucose, 10-40 g/L yeast extract, (NH)₄)₂SO₄1.0-6.0g/L，KH₂PO₄2.0～6.0g/L，MgSO₄·7H₂O 0.3～0.6g/LNaCl 0.2-0.8 g/L, water as solvent, and pH 6.0-7.5.