CN106811432A - A kind of oerskovia turbata and the application in (R) 3 chlorophenethylol is prepared - Google Patents
A kind of oerskovia turbata and the application in (R) 3 chlorophenethylol is prepared Download PDFInfo
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Abstract
The invention discloses a kind of oerskovia turbata and the application in (R) 3 chlorophenethylol is prepared, preparing the chlorophenethylol of optical voidness (R) 3 using the novel bacterial catalysis has concentration of substrate high, stereoselectivity is good, the advantages of product optical purity is high.The present invention is the chiral living things catalysis of catalyst by using oerskovia turbata (Oerskovia turbata) ZJPH1604 cells, when concentration of substrate is 80mmol/L, the e.e of the chlorophenethylol of purpose product (R) 3 reaches 99.9%, and yield reaches 98.1%.
Description
(1) technical field
Can be used for living things catalysis asymmetric reduction 3- chloro-acetophenones the present invention relates to one plant and prepare high-optical-purity (R) -3-
The novel bacterial of chlorophenethylol --- oerskovia turbata (Oerskovia turbata) ZJPH1604 and its application.
(2) background technology
(R) chemical structural formula of -3- chlorophenethylols is：
(R) -3- chlorophenethylols are a kind of important chiral intermediates, are the structure modules of multi-medicament molecule, for example, it
Can be used for synthesis treatment myeloid form muscular dystrophy reactive compound (R) -5- [1- (3- chlorphenyls) ethyoxyl] quinazoline -
2,4- diamines, can be also used for the β of synthesis treatment depression, diabetes and obesity3- adrenoceptor agonists, including
SR58611, Solabegron, CL316243, AJ-9677 etc..
(R) -3- chlorophenethylols are prepared using chemical method, it is necessary to use expensive such as rhodium, ruthenium metallic catalyst, meeting
Environment is polluted.The biological asymmetric reduction method being catalyzed using microbe whole-cell prepares (R) -3- chlorophenethylols to be had
Reaction condition is gentle, stereoselectivity is high, advantages of environment protection.
(J.Med.Chem.2008,51,449-469) such as existing document report, John Thurmond utilizes chiral catalysis
Agent ((S)-MeCBS and (R)-MeCBS) catalysis reduction 3- chloro-acetophenones prepare the (J.of such as (R) -3- chlorophenethylols, Perna
Mol.Catal.B:Enzym.,2016,124:29-37) using Lactobacillus reuteri resting cells catalysis reduction 3-
Chloro-acetophenone prepares (R) -3- chlorophenethylols, conversion ratio 100%, enantiomeric excess value (ee)>99%, work as concentration of substrate>5g/L
When, conversion ratio is less than 70%.
(3) content of the invention
Can be used for living things catalysis asymmetric reduction 3- chloro-acetophenones it is an object of the present invention to provide one plant and prepare high-optical-purity
(R) the microorganism novel bacterial of -3- chlorophenethylols --- oerskovia turbata (Oerskovia turbata) ZJPH1604, with
And application of the strain in catalysis asymmetric syntheses prepares (R) -3- chlorophenethylols.
The technical solution adopted by the present invention is：
The present invention provides one plant of new strains -- oerskovia turbata (Oerskovia turbata) ZJPH1604, preservation
In China typical culture collection center, deposit number：CCTCC NO：M 2016541, preservation date：On September 30th, 2016,
Address：China, Wuhan, Wuhan University, postcode：430072.
The present invention also provides a kind of oerskovia turbata ZJPH1604 in microorganism catalysis asymmetric reduction 3- chlorine
Acetophenone prepares the application in (R) -3- chlorophenethylols, and the specific application is：With 3- chloro-acetophenones as substrate, be in a tumult strategic point this
It is enzyme source to examine the wet thallus cell that the fermented cultures of dimension bacterium ZJPH1604 obtain, and adds cosubstrate, in pH be 6.0~9.0
In buffer solution, conversion reaction is carried out at 25~35 DEG C, after reaction terminates, the separated purifying of reaction solution obtains (R) -3- chlorobenzene second
Alcohol；The cosubstrate is the one kind in glucose, sucrose, maltose, isopropanol, methyl alcohol or ethanol.
Further, the initial concentration of the substrate 3- chloro-acetophenones is 20~200mmol/L buffer solutions (preferably 25-
150mmol/L), the addition of oerskovia turbata ZJPH1604 wet thallus cell is calculated as 50~300g/L buffer solutions with weight in wet base
(preferably 100-300g/L)；When the cosubstrate is glucose, sucrose or maltose, addition is 20~300g/L buffer solutions
(preferably 100-300g/L), the cosubstrate be methyl alcohol, ethanol or isopropanol when, add volume for buffer solution volume 10~
30% (preferably 20%).
Further, the enzyme source is prepared as follows：1) inclined-plane culture：By oerskovia turbata ZJPH1604 inoculations
Into slant medium, 30 DEG C are cultivated 1-2 days, obtain inclined-plane thalline；The slant medium is constituted：Glucose 10g/L, egg
White peptone 5g/L, yeast extract 4g/L, (NH4)2SO42g/L, KH2PO41g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L,
Agar 20g/L, solvent is water, pH 6.5；
2) seed culture：Chosen in ring thalline 250mL shaking flasks of the access equipped with 100mL seed culture mediums from inclined-plane, 30 DEG C,
200r/min is cultivated 24 hours, obtains seed liquor；The seed culture medium is constituted：Glucose 10-20g/L, peptone 5-
10g/L, yeast extract 4-8g/L, (NH4)2SO42-4g/L, KH2PO42-4g/L, NaCl 0.5-2g/L, MgSO4·7H2O
0.5-2g/L, solvent is water, pH 6.5-8.0；It is preferred that the seed culture medium composition is：Glucose 10g/L, peptone 5g/L,
Yeast extract 4g/L, (NH4)2SO42g/L, KH2PO42g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, solvent is water,
pH 6.5；
3) fermented and cultured：Seed liquor is transferred to the inoculum concentration of volumetric concentration 4-10% (preferably 8%) is sent out equipped with 100mL
In the 250mL shaking flasks of ferment culture medium, 30 DEG C, 180-220r/min (preferably 200r/min) cultivates 36-48h (preferably 48h), will send out
Zymotic fluid is centrifuged, and collects wet thallus；The fermentation medium final concentration composition is as follows：20~30g/L of glucose, yeast extract 10~
40g/L, (NH4)2SO41.0-6.0g/L, KH2PO42.0~6.0g/L, MgSO4·7H2O 0.3~0.6g/L, NaCl 0.2
~0.8g/L, solvent is water, and pH 6.0~7.5, preferably described fermentation medium final concentration composition is as follows：Glucose 15g/L, ferment
Female cream 30g/L, (NH4)2SO42.5g/L, KH2PO44g/L, MgSO4·7H2O 0.6g/L, NaCl 0.4g/L, solvent is water,
pH 6.5。
Using Box-Behnken pivot combination experiment design methods to concentration of glucose, yeast extract concentration and KH2PO4It is dense
Degree three influences significant factor to optimize producing enzyme, obtains oerskovia turbata (Oerskovia) ZJPH1604.Bacterial strain
Optimal Medium composition be：20~30g/L of glucose, yeast extract 10~40g/L, (NH4)2SO41.0-6.0g/L, KH2PO4
2.0~6.0g/L, MgSO4·7H2O 0.2~0.8g/L of 0.3~0.6g/L, NaCl, solvent is water, pH 6.0~7.5.
The condition of culture of oerskovia turbata (Oerskovia) ZJPH1604 is：Initial pH 6.0~7.5, shaking flask dress
Liquid measure 80~120mL/250mL conical flasks, 30 DEG C of cultivation temperature, 180~220rpm of shaking speed, inoculum concentration 4~10%, culture
36~48h of time.
Reaction solution isolation and purification method of the present invention is：After reaction terminates, conversion fluid is extracted with isometric ethyl acetate
Take, supernatant is obtained after centrifugation, using chiral gas chromatography analysis purpose product and the content of remaining substrate, and product
Optical purity.
The preferably cosubstrate of the invention is isopropanol, now the optical purity of products therefrom and yield highest.
Present invention screening from soil sample obtains the microorganism novel bacterials different from similar research report in the past, and is catalyzed
The concentration of substrate and yield of conversion are higher, so as to prepare chiral intermediate (R) -3- chlorophenethylols in microorganisms catalysis method
Aspect provides beneficial reference.
The beneficial effects are mainly as follows：Can be used for microorganism catalysis asymmetric reduction the invention provides one plant
3- chloro-acetophenones prepare the microorganism novel bacterial of (R) -3- chlorophenethylols, and optical voidness (R) -3- chlorine is prepared using the novel bacterial catalysis
Benzyl carbinol has concentration of substrate high, and stereoselectivity is good, the advantages of product optical purity is high.The present invention by using disturbance strategic point this
It is the chiral living things catalysis of catalyst to examine dimension bacterium (Oerskovia turbata) ZJPH1604 cells, when concentration of substrate is
During 80mmol/L, the e.e of purpose product (R) -3- chlorophenethylols reaches 99.9%, and yield reaches 98.1%.
(4) illustrate
Fig. 1 is substrate standard items gas chromatogram (containing the internal standard dodecane)；
Fig. 2 is raceme product standard items gas chromatogram (containing the internal standard dodecane)；
Fig. 3 is oerskovia turbata (Oerskovia turbata) ZJPH1604 bacterial strain bioreduction extracts
Gas chromatogram (containing the internal standard dodecane).
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
The consumption of substrate described in the embodiment of the present invention, cosubstrate and wet thallus is with buffer solution stereometer.
Embodiment 1：The screening and identification of bacterial strain
Strain source：Oerskovia turbata (Oerskovia turbata) ZJPH1604 bacterial strains system from Zhejiang Province east money
Separation screening is obtained in soil sample near lake, and specific screening technique is as follows：
The soil sample 1g of collection is added in the 250mL shaking flasks equipped with 50mL enriched mediums, 30 DEG C, 200rpm, culture 5
~6d, after nutrient solution becomes muddiness, takes 1mL nutrient solutions and is forwarded in fresh enriched medium, continues to cultivate 5~6d, so
Repeat enrichment culture 3~4 times.With 3- chloro-acetophenones as sole carbon source in enriched medium.Enrichment culture based formulas are as follows：3- chlorine
Acetophenone 25mmol/L, (NH4)2SO42g/L, KH2PO42g/L, NaCl0.5g/L, MgSO4·7H2O 0.5g/L, solvent is
Water, pH 6.5.
Subsequent enrichment culture liquid is applied on separation flat board that (plating medium composition is enriched medium through gradient dilution
The agar of middle addition 15-20g/L), single bacterium colony bacterial strain is obtained after separating for several times culture.Picking single bacterium colony inoculation is to seed
Culture medium, 30 DEG C are cultivated 24 hours, then are forwarded in culture medium 30 DEG C and are cultivated 42 hours.With the 3- chlorobenzenes of final concentration 5mM
Ethyl ketone is substrate, and the microbial cell that screening is obtained is catalyst (final concentration 100g/L), in the phosphate buffer 10ml of pH 6.5
In, after 30 DEG C of bioconversion 24h.After conversion terminates, conversion fluid is extracted with isometric ethyl acetate, and supernatant is obtained after centrifugation
Using chiral gas chromatography detection conversion fluid in purpose product (R) -3- chlorophenethylols enantiomeric excess value (e.e. values) and
Yield.With inner mark method ration, internal standard compound is dodecane.Taking 1mL extracts adds 1 μ L dodecanes to be analyzed.Gas-chromatography bar
Part：Japanese Shimadzu GC-2014 gas chromatographs, Zhejiang University's N2000 chromatographic work stations；U.S.'s Varian CP-Chirasil-Dex hands
Property capillary gas chromatographic column (25m × 0.25mm × 0.25 μm).Carrier gas is high pure nitrogen, and flow is 2mL/min；The μ of sample size 1
L, split ratio is 15:1；Detector and injector temperature are 250 DEG C；120~160 DEG C of chromatogram column temperature；Programming rate：8℃/
min；Detector is FID.Sample is detected with conditions above, the retention time of substrate is 4.1min, such as Fig. 1；Raceme
The retention time of product standard items, R types product is 6.5min, and S types product is 6.7min, such as Fig. 2；The conversion of ZJPH1604 bacterial strains is produced
The retention time of thing is 6.5min, such as Fig. 3.The retention time of internal standard compound dodecane is 3.0min.
Calculate the concentration of the substrate and product in reaction solution respectively with relative correction factor.And then obtain the yield of reaction
(Yield).Calculating formula is:
Yield=Ci/C0× 100%
C in formula0、CiThe molar concentration of product respectively at the end of the molar concentration of reaction starting material and reaction.
The optical purity of product is represented by enantiomeric excess value (enantiomeric excess, e.e.).
E.e.=(CR-CS)/(CR+CS) × 100%
C in formulaRAnd CSThe respectively molar concentration of R types and S type 3- chlorophenethylols.
The feature of the novel bacterial is as follows：
Physiological and biochemical property：Gram-positive, spore of not sprouting, is not moved, and without pod membrane, carbohydrate fermentation is produced
Acid, aerogenesis, oxidase positive, do not contact enzyme positive, obligate aerobic.Utilizable carbon source has 59 kinds：Dextrin, D-Maltose,
Extra large bath sugar, cellobiose, gentiobiose, sucrose, stachyose, gossypose, lactose, D- melibioses, D- (-)-salicin, N- second
Acyl-D- Glucosamines, N- acetyl-α-D-MANNOSE amine, N- acetyl-D-galactosamine, N- acetyl-nerve ammonia (sugar) acid, D-
Galactolipin, methyl diacetone, D- fucoses, L-fucose, glycyl-L-PROLINE, ASPARTIC ACID, Pidolidone,
L-Glutimic acid, Serine, L-GaA lactone, D- gluconic acids, glucuronic acid, glucuronamide, glactaric acid, Kui Ni
Acid, glucaric acid, p- cresols-phenylacetic acid, methyl pyruvate, lactic acid methyl esters, D-lactic acid, citric acid, α -one-penta two
Acid, D-malic acid, rhamnose, inosine, mannitol, arabite, inositol, glycerine, G-6-P, D-Fructose-
6- phosphoric acid, asparatate, D-Ser, left-handed malic acid, bromosuccinic acid, butyrine, AHIB, α-hydroxyl
Base-D, L- butyric acid, α -one butyric acid, acetoacetate, propionic acid, acetic acid, formic acid.
The 16S rDNA sequence characteristics of strain：It is template with the cell STb gene for extracting, is expanded using universal primer P1 and P2
Increase the 16S rDNA genes of bacterial strain, then the agarose gel electrophoresis that PCR primer is carried out 1%.Confirm the bacterial strain through sequencing
The 16S rDNA gene orders (SEQ ID NO.1) of ZJPH1604 have been filed on GenBank, and (GenBank accession number is
No.KY302675), by the 16S rRNA sequences of ZJPH1604 bacterial strains in NCBI websites (http://
Www.ncbi.nlm.nih.gov sequence analysis (BLAST) are carried out on), is as a result shown：ZJPH1604 bacterial strains with disturbance strategic point this
The part strain sequence homology for examining dimension bacterium (Oerskovia sp.) is higher.ZJPH1604 bacterial strains and Oerskovia bacterial strains
The sequence homology of (GenBank accession number is No.HM563054.1) reaches 99%.
According to physio-biochemical characteristics and binding molecule Biology identification, the bacterial strain is accredited as E Sikaowei bacterium
(Oerskovia sp.), is named as oerskovia turbata (Oerskovia sp.) ZJPH1604, is preserved in Chinese Typical Representative training
Support thing collection, address：China, Wuhan, Wuhan University, postcode：430072；Deposit number：CCTCC NO：M 2016541,
Preservation date：On September 30th, 2016.
Embodiment 2：The acquisition of wet thallus cell
Seed culture based formulas：Glucose 10g/L, peptone 5g/L, yeast extract 4g/L, (NH4)2SO42g/L, KH2PO4
2g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, solvent is water, pH 6.5.
Fermentative medium formula：Glucose 15g/L, yeast extract 30g/L, (NH4)2SO44g/L, KH2PO44g/L, NaCl
0.6g/L, MgSO4·7H2O 0.8g/L, solvent is phosphate buffer, pH 6.5.
Inclined-plane culture：Oerskovia turbata ZJPH1604 is seeded in slant medium, 30 DEG C are cultivated 1-2 days, are obtained
Obtain inclined-plane thalline；The slant medium is constituted：Glucose 10g/L, peptone 5g/L, yeast extract 4g/L, (NH4)2SO4
2g/L, KH2PO41g/L, NaCl 0.5g/L, MgSO4·7H2O 0.5g/L, agar 20g/L, solvent are water, pH 6.5；
Chosen in ring thalline 250mL shaking flasks of the access equipped with 100mL seed culture mediums from inclined-plane, 30 DEG C, 200rpm cultures
Seed liquor is obtained, then seed liquor is transferred to equipped with 100mL fermentation mediums with the inoculum concentration of volumetric concentration 8% within 24 hours
In 250mL shaking flasks, 30 DEG C, 200rpm is cultivated 48 hours.Culture terminates the centrifugation of after fermentation liquid, the precipitation phosphate of pH value 6.5
Buffer solution washed once, and collect wet thallus cell, standby.
Embodiment 3：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 6.5), wet thallus are in terms of weight in wet base
Concentration is 100g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add and account for buffer solution volume
10% isopropanol is placed in 30 DEG C as cosubstrate, and 24h is reacted in the shaking table of 200r/min.Using the detection side of embodiment 1
Method, the concentration of product (R) -3- chlorophenethylols is 17.8mmol/L, optical purity ee values 99.9%, yield 70.9%.
Embodiment 4：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 6.5), wet thallus are in terms of weight in wet base
Concentration is 100g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add and account for buffer solution volume
10% isopropanol is placed in 37 DEG C as cosubstrate, and 24h is reacted in the shaking table of 200r/min.Using the detection side of embodiment 1
Method, the concentration of product (R) -3- chlorophenethylols is 16.8mmol/L, optical purity ee values 99.9%, yield 67.3%.
Embodiment 5
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 6.5), wet thallus are in terms of weight in wet base
Concentration is 200g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add and account for buffer solution volume
10% isopropanol is placed in 30 DEG C as cosubstrate, and 24h is reacted in the shaking table of 200r/min.Using the detection side of embodiment 1
Method, the concentration of product (R) -3- chlorophenethylols is 19.2mmol/L, optical purity ee values 99.9%, yield 76.7%.
Embodiment 6：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 6.5), wet thallus are in terms of weight in wet base
Concentration is 200g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add and account for buffer solution volume
10% isopropanol is placed in 37 DEG C as cosubstrate, and 24h is reacted in the shaking table of 200r/min.Using the detection side of embodiment 1
Method, the concentration of product (R) -3- chlorophenethylols is 20.4mmol/L, optical purity ee values 99.9%, yield 88.8%.
Embodiment 7：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 7.0), wet thallus are in terms of weight in wet base
Concentration is 150g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add the grape of 300g/L
Sugar is placed in 37 DEG C as cosubstrate, and 48h is reacted in the shaking table of 200r/min.Using the detection method of embodiment 1, product
(R) concentration of -3- chlorophenethylols is 3.4mmol/L, optical purity ee values 99.9%, yield 13.7%.
Embodiment 8：
The wet thallus of the gained of embodiment 2 are suspended in 15mL phosphate buffers (pH 6.5), wet thallus are in terms of weight in wet base
Concentration is 200g/L buffer solutions；Add the 3- chloro-acetophenones of 80mmol/L buffer solutions as substrate, add and account for buffer solution volume
20% isopropanol is placed in 37 DEG C as cosubstrate, and 48h is reacted in the shaking table of 200r/min.Using the detection side of embodiment 1
Method, the concentration of product (R) -3- chlorophenethylols is 78.5mmol/L, optical purity ee values 99.9%, yield 98.1%.
Embodiment 9：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 7.0), wet thallus are in terms of weight in wet base
Concentration is 100g/L buffer solutions；Add the 3- chloro-acetophenones of 35mmol/L buffer solutions as substrate, add 100g/L buffer solutions
Glucose as cosubstrate, be placed in 30 DEG C, react 24h in the shaking table of 200r/min.Using the detection method of embodiment 1,
The concentration of product (R) -3- chlorophenethylols is 7.7mmol/L, optical purity ee values 99.9%, yield 22.2%.
Embodiment 10：
The wet thallus of the gained of embodiment 2 are suspended in 20mL phosphate buffers (pH 7.0), wet thallus are in terms of weight in wet base
Concentration is 200g/L buffer solutions；Add the 3- chloro-acetophenones of 35mmol/L buffer solutions as substrate, add 2mL and (account for buffer solution
Volume 10%, v/v) isopropanol as cosubstrate, be placed in 37 DEG C, react 48h in the shaking table of 200r/min.Using embodiment
1 detection method, the concentration of product (R) -3- chlorophenethylols is 34.6mmol/L, optical purity ee values 99.9%, yield 99%.
Embodiment 11：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 8.0), wet thallus are in terms of weight in wet base
Concentration is 300g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add 1mL and (account for buffer solution
Volume 10%, v/v) isopropanol as cosubstrate, be placed in 30 DEG C, react 24h in the shaking table of 200r/min.Using embodiment
1 detection method, the concentration of product (R) -3- chlorophenethylols is 19.9mmol/L, optical purity ee values 99.9%, yield
79.7%.
Embodiment 12
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 8.0), wet thallus are in terms of weight in wet base
Concentration is 200g/L buffer solutions；Add the 3- chloro-acetophenones of 150mmol/L buffer solutions as substrate, add 2mL and (account for buffer solution
Volume 20%, v/v) isopropanol as cosubstrate, be placed in 30 DEG C, react 24h in the shaking table of 200r/min.Using embodiment
1 detection method, the concentration of product (R) -3- chlorophenethylols is 98.1mmol/L, optical purity ee values 99.9%, yield
65.4%.
Embodiment 13
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 8.0), wet thallus are in terms of weight in wet base
Concentration is 250g/L buffer solutions；Add the 3- chloro-acetophenones of 150mmol/L buffer solutions as substrate, add 2mL and (account for buffer solution
Volume 20%, v/v) isopropanol as cosubstrate, be placed in 30 DEG C, react 36h in the shaking table of 200r/min.Using embodiment
1 detection method, the concentration of product (R) -3- chlorophenethylols is 121.8mmol/L, optical purity ee values 99.9%, yield
71.2%.
Embodiment 14：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 9.0), wet thallus are in terms of weight in wet base
Concentration is 100g/L buffer solutions；Add the 3- chloro-acetophenones of 25mmol/L buffer solutions as substrate, add 2.5mL and (account for buffering
Liquid accumulate 20%, v/v) isopropanol as cosubstrate, be placed in 30 DEG C, react 48h in the shaking table of 200r/min.Using implementation
The detection method of example 1, the concentration of product (R) -3- chlorophenethylols is 11.2mmol/L, optical purity ee values 99.9%, yield
44.1%.
Embodiment 15：
The wet thallus of the gained of embodiment 2 are suspended in 10mL phosphate buffers (pH 9.0), wet thallus are in terms of weight in wet base
Concentration is 100g/L buffer solutions；Add the 3- chloro-acetophenones of 35mmol/L buffer solutions as substrate, add 2mL and (account for buffer solution
Volume 20%, v/v) isopropanol as cosubstrate, be placed in 30 DEG C, react 48h in the shaking table of 200r/min.Using embodiment
1 detection method, the concentration of product (R) -3- chlorophenethylols is 34.6mmol/L, optical purity ee values 99.9%, yield
99.1%.
SEQUENCE LISTING
<110>Zhejiang Pharmaceutical College；Zhejiang Polytechnical University
<120>A kind of oerskovia turbata and the application in (R) -3- chlorophenethylols are prepared
<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 (5)
1. oerskovia turbata (Oerskovia turbata) ZJPH1604, is preserved in China typical culture collection center,
Deposit number：CCTCC NO：M 2016541, preservation date：On September 30th, 2016, address：China, Wuhan, Wuhan University, postal
Compile：430072.
2. oerskovia turbata ZJPH1604 described in a kind of claim 1 is in microorganism catalysis asymmetric reduction 3- chloro-acetophenones
Prepare the application in (R) -3- chlorophenethylols.
3. application as claimed in claim 2, it is characterised in that the application is：With 3- chloro-acetophenones as substrate, with strategic point of being in a tumult
The wet thallus cell that the fermented cultures of scott dimension bacterium ZJPH1604 are obtained is enzyme source, adds cosubstrate, in pH be 6.0~9.0
Buffer solution in, carry out conversion reaction at 25 DEG C~35 DEG C, after reaction terminates, the separated purifying of reaction solution obtains (R) -3- chlorine
Benzyl carbinol；The cosubstrate is the one kind in glucose, sucrose, maltose, isopropanol, methyl alcohol or ethanol.
4. application as claimed in claim 3, it is characterised in that the initial concentration of the substrate 3- chloro-acetophenones is 20~
200mmol/L buffer solutions, the addition of oerskovia turbata ZJPH1604 wet thallus cells is calculated as 50~300g/L with weight in wet base
Buffer solution；When the cosubstrate is glucose, sucrose or maltose, addition is 20~300g/L buffer solutions, the auxiliary
When substrate is methyl alcohol, ethanol or isopropanol, it is the 10~30% of buffer solution volume to add volume.
5. application as claimed in claim 3, it is characterised in that the enzyme source is prepared as follows：1) inclined-plane culture：To disturb
Dynamic E Sikaowei bacterium ZJPH1604 is seeded in slant medium, and 30 DEG C are cultivated 1-2 days, obtain inclined-plane thalline；The inclined-plane training
Supporting base composition is：Glucose 10g/L, peptone 5g/L, yeast extract 4g/L, (NH4)2SO42g/L, KH2PO41g/L, NaCl
0.5g/L, MgSO4·7H2O 0.5g/L, agar 20g/L, solvent are water, pH 6.5；
2) seed culture：Chosen in ring thalline 250mL shaking flasks of the access equipped with 100mL seed culture mediums from inclined-plane, 30 DEG C,
200r/min is cultivated 24 hours, obtains seed liquor；The seed culture medium is constituted：Glucose 10-20g/L, peptone 5-
10g/L, yeast extract 4-8g/L, (NH4)2SO42-4g/L, KH2PO42-4g/L, NaCl 0.5-2g/L, MgSO4·7H2O
0.5-2g/L, solvent is water, pH 6.5-8.0；
3) fermented and cultured：Seed liquor is transferred to equipped with 100mL fermentation mediums with the inoculum concentration of volumetric concentration 4-10%
In 250mL shaking flasks, 30 DEG C, 180-220r/min is cultivated 36-48 hours, and zymotic fluid is centrifuged, and collects wet thallus；The fermentation training
Support base final concentration composition as follows：20~30g/L of glucose, yeast extract 10~40g/L, (NH4)2SO41.0-6.0g/L, KH2PO4
2.0~6.0g/L, MgSO4·7H2O 0.2~0.8g/L of 0.3~0.6g/L, NaCl, solvent is water, pH6.0~7.5.
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| CN201611266505.8A CN106811432B (en) | 2016-12-31 | 2016-12-31 | Erysivelum pratense and application thereof in preparation of (R) -3-chlorobenzene ethanol |
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| CN201611266505.8A CN106811432B (en) | 2016-12-31 | 2016-12-31 | Erysivelum pratense and application thereof in preparation of (R) -3-chlorobenzene ethanol |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107746861A (en) * | 2017-11-20 | 2018-03-02 | 浙江工业大学 | A kind of biological preparation method of (R) 1 (2 trifluoromethyl) ethanol |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5244289A (en) * | 1975-10-03 | 1977-04-07 | Ajinomoto Co Inc | Preparation of l-amino acids |
| WO2007097336A1 (en) * | 2006-02-21 | 2007-08-30 | Kaneka Corporation | Process for producing (2r,3r)- and (2s,3s)-3-phenylisoserine derivatives |
| CN106148471A (en) * | 2015-04-08 | 2016-11-23 | 中国科学院大连化学物理研究所 | A kind of cellulomonas cartae section is as the application of industrial biocatalytic agent |
-
2016
- 2016-12-31 CN CN201611266505.8A patent/CN106811432B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5244289A (en) * | 1975-10-03 | 1977-04-07 | Ajinomoto Co Inc | Preparation of l-amino acids |
| WO2007097336A1 (en) * | 2006-02-21 | 2007-08-30 | Kaneka Corporation | Process for producing (2r,3r)- and (2s,3s)-3-phenylisoserine derivatives |
| CN106148471A (en) * | 2015-04-08 | 2016-11-23 | 中国科学院大连化学物理研究所 | A kind of cellulomonas cartae section is as the application of industrial biocatalytic agent |
Non-Patent Citations (1)
| Title |
|---|
| F.M. PERNA等: "Cheap and environmentally sustainable stereoselective arylketones reduction by Lactobacillus reuteri whole cells", 《JOURNAL OF MOLECULAR CATALYSIS. B, ENZYMATIC》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107746861A (en) * | 2017-11-20 | 2018-03-02 | 浙江工业大学 | A kind of biological preparation method of (R) 1 (2 trifluoromethyl) ethanol |
| CN107746861B (en) * | 2017-11-20 | 2020-06-23 | 浙江工业大学 | Biological preparation method of (R) -1- (2-trifluoromethylphenyl) ethanol |
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| CN106811432B (en) | 2020-04-28 |
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