CN101985609B - Pseudomonas and method for performing microbial transformation on pregabalin intermediate by using same - Google Patents
Pseudomonas and method for performing microbial transformation on pregabalin intermediate by using same Download PDFInfo
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- CN101985609B CN101985609B CN2010105201795A CN201010520179A CN101985609B CN 101985609 B CN101985609 B CN 101985609B CN 2010105201795 A CN2010105201795 A CN 2010105201795A CN 201010520179 A CN201010520179 A CN 201010520179A CN 101985609 B CN101985609 B CN 101985609B
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Abstract
The invention discloses pseudomonas and a method for performing microbial transformation on a pregabalin intermediate by using the same. The collection number of the pseudomonas is CGMCC No. 4184; and the method for performing the microbial transformation on the pregabalin intermediate by using the pseudomonas comprises the following steps of: (1) adding the pseudomonas into a culture medium for fermentation culturing; (2) performing transformation reaction on the pregabalin intermediate by using the cultured fermentation solution to obtain transformation solution, wherein the pregabalin intermediate is racemic 2-carboxyethyl-3-cyano-5-methyl ethyl caproate; and (3) extracting the transformation solution with a solvent, dehydrating the organic phase obtained by the extraction and then evaporating the solvent to obtain (S)-2-carboxyethyl-3-cyano-5-methyl ethyl caproate. In the method, the pseudomonas transforms the racemic 2-carboxyethyl-3-cyano-5-methyl ethyl caproate to prepare the (S)-2-carboxyethyl-3-cyano-5-methyl ethyl caproate; the yield can reach 43.0 percent; and the enantiomeric excess (ees) of the substrate reaches 98.3 percent.
Description
Technical field
The method that the present invention relates to a kind of pseudomonas (Pseudomonas sp.) and the lyrica midbody is carried out microbial transformation is particularly to method that racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester carries out microbial transformation.
Background technology
(S)-(be called for short (S)-CNDE) is that (Pregabalin, crucial chiral intermediate PGB), its racemic mixture are 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester (being called for short CNDE) to synthetic lyrica to 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester.PGB is three isobutyl-substituents of γ-An Jidingsuan (GABA), the assisting therapy of neuropathic pain, insane pain disease is demonstrated powerful advantages, and the neuropathic pain patient with somnopathy is had result of treatment.
The preparation method of lyrica and chiral intermediate thereof mainly can be divided into racemic modification Split Method and asymmetric synthesis method.The method of asymmetric synthesis of report often needs expensive transition metal or chiral ligand at present, and operational condition is relatively harsher, and the organic solvent usage quantity is too big.In Split Method, chemical fractionation will be found out very suffering of a suitable chiral separation agent, and difficulty is big, and expense is high, and essential earlier synthetic racemize title product, and the highest yield of fractionation generally can not surpass 50%.Relatively, it is strong to utilize mikrobe or enzyme Split Method to possess stereoselectivity, mild condition, and cost is lower, and is environmentally friendly, reduces advantages such as wastage of material.2008; People such as the Martinez of Pfizer utilize lypase as optionally hydrolysis of resolving agent (S)-CNDE; (be called for short the sylvite of (S)-CNME), chemosynthesis prepares lyrica as raw material with it again, and this chemo-enzymatic process operational path can make the lyrica yield up to 45% to obtain (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid; And can significantly reduce the usage quantity of organic solvent, and realize effective recycle of (R)-CNDE.Yet these enzyme catalyst prices are higher, and also there is certain limitation in being suitable for of substrate.Relatively,, then can reduce raw materials cost greatly, not see as yet at present relevant for utilizing pseudomonas (Pseudomonas sp.) to method that 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester carries out microbial transformation if utilize mikrobe as resolving agent.
Summary of the invention
The purpose of this invention is to provide a kind of pseudomonas and the lyrica midbody is carried out the method for microbial transformation with it.
For realizing above-mentioned purpose, contriver's separation and purification first is to the microorganism strains of strain ability highly selective hydrolysis CNDE.Identify that through 16S rDNA and Physiology and biochemistry this bacterial strain is that pseudomonas (Pseudomonassp.) belongs to called after pseudomonas (Pseudomonas sp.) YZ05.This bacterial strain is preserved in Chinese common micro-organisms culture presevation administrative center, and preservation date is on September 17th, 2010, and preserving number is CGMCCNo.4184.
The present invention utilizes said pseudomonas to the method that the lyrica midbody carries out microbial transformation, comprises the steps:
(1) be that the pseudomonas of CGMCC No.4184 joins and carries out fermentation culture in the substratum with preserving number;
(2) by following option A or option b the lyrica midbody is carried out conversion reaction, obtain conversion fluid, said lyrica midbody is racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester:
Option A: racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is directly carried out conversion reaction in the fermented liquid of adding after step (1) is cultivated, obtain conversion fluid;
Option b: will earlier said wet thallus be joined in the phosphate buffer solution through the centrifugal wet thallus that obtains of fermented liquid after step (1) is cultivated, after add racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester again and carry out conversion reaction, obtain conversion fluid;
(3) said conversion fluid is used SX, the organic phase dehydration back evaporating solvent that extraction is obtained obtains (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester.
Further, in the option A of step of the present invention (2), said racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is 1～50g/L with respect to the add-on of said fermented liquid; In the option b of step (2), said racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is 1～50g/L with respect to the add-on of said phosphate buffer solution.
Further, in the option b of step of the present invention (2), said wet thallus is 20～100g/L with respect to the add-on of said phosphate buffer solution.
Further, in step of the present invention (2), the conversion reaction temperature is 25～45 ℃, and the conversion reaction time is 10～60h.
Further, in the option b of step of the present invention (2), the concentration of said phosphate buffer solution is that 0.1M, pH are 5～9.
Further, in step of the present invention (1), 25～40 ℃ of the culture temperature of said fermentation culture were cultivated 1～4 day.
Further; In step of the present invention (1); The composition of said substratum is: peptone 5～15g/L, glycerine 0～6g/L, yeast extract paste 1～5g/L, ammonium sulfate 0～4g/L, potassium primary phosphate 0～4g/L, sodium-chlor 0.5～10g/L and sal epsom 0～0.5g/L, the pH value of said substratum is 6～8.
The inventive method has the following advantages with respect to traditional chemical method for splitting and asymmetric synthesis method:
(1) because chemical resolution method and asymmetric synthesis method have the shortcoming that severe reaction conditions, difficulty are big, need a large amount of organic solvents of use, environment is polluted; And by contrast; The present invention adopts mikrobe pseudomonas (Pseudomonas sp.) YZ05 conversion of substrate (racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester); Not only have the gentle advantage of efficient selective and reaction conditions, and can be from a large amount of uses that reduce organic solvents on the technology, environmentally friendly.
(2) microbe transformation method of the present invention is to utilize microbial cells as catalyzer, fermentative prepn voluntarily, and steady quality, and can reduce raw materials cost greatly, have vast application prospect;
(3) adopt microbe transformation method of the present invention, final its enantiomeric excess value of (S)-CNDE that generates is up to more than 99%; And the portion of product (2-propyloic-3-cyanic acid-5-methylhexanoic acid) that reaction generates can be racemized to substrate (racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester) after the esterification again, thereby improves substrate utilization ratio.
The preservation information of biological material specimens:
The biological material specimens of preservation: pseudomonas (Pseudomonas sp.) YZ05;
Depositary institution: (be called for short: CGMCC) at China Committee for Culture Collection of Microorganisms common micro-organisms center;
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica (postcode: 100101);
Preservation date: on September 17th, 2010;
Preservation registration number: CGMCC No.4184.
Description of drawings
Fig. 1 is the gas chromatographic analysis spectrogram of substrate (2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester) and product (2-propyloic-3-cyanic acid-5-methylhexanoic acid).From left to right the pairing compound in each peak is respectively: product (R)-2-propyloic-3-cyanic acid-5-methylhexanoic acid go out the peak position 13.007min with (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid goes out the cutting edge of a knife or a sword position at 13.907min, substrate (R)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester go out the peak position 50.957min with (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester goes out the peak position at 52.490min.
Fig. 2 is the change curve of transformation efficiency and substrate enantiomeric excess value under the differential responses time.
Embodiment
In following examples, the lyrica midbody is being carried out in the conversion reaction process, reaction solution is being carried out substrate conversion efficiency, product enantiomeric excess value (ee
p) and substrate enantiomeric excess value (ee
s) measuring method (wherein, said substrate is racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester, is abbreviated as CNDE) specific as follows:
In the conversion reaction process, the 5mL reaction solution is regulated pH to 2-3 with Hydrogen chloride, uses ethyl acetate extraction again, and centrifugal (4 ℃, 10000 * g 5min) obtains organic phase, after anhydrous sodium sulfate drying spends the night, substrate and converted product thereof is carried out the chirality gas chromatographic analysis.The system of this chirality gas chromatographic analysis is following:
Detecting instrument: GC-950 gas chromatograph;
Chirality gas phase post: Astec CHIRALDEX
TMG-TA (30m * 0.25mm * 0.12 μ m);
Testing conditions: 125 ℃ of column temperatures, detector and sampler temperature are 250 ℃, carrier gas (N
2) 30mL/min, air 300mL/min, hydrogen 30mL/min.
Calculation formula:
Substrate conversion efficiency (%)=(initial substrate concentration-residue concentration of substrate)/initial substrate concentration * 100%;
ee
p(％)＝[(A
R-A
S)/(A
S+A
R)]×100％
ee
s＝c×ee
p/(1-c)×100％
E＝ln[1-c(1+ee
p％)]/ln[1-c(1-ee
p％)]
Wherein, c is a transformation efficiency, ee
pBe product enantiomeric excess value, wherein A
S, A
RBe respectively (S)-configuration product in the gc institute test sample article with (R)-peak area of configuration product, E is the enantiomorph selection rate.
Embodiment 1: bacterial screening and evaluation
The present invention is from Hangzhou and collection soil such as oil chemistry factory in the neighbourhood, pharmaceutical factories, through enrichment, primary dcreening operation, sieve three steps again, carries out the screening of pseudomonas (Pseudomonas sp.) YZ05.Method is following:
Enrichment culture: medium component is (g/L): yeast extract paste 1.0, and ammonium sulfate 2.0, potassium hydrogenphosphate 1.0, sodium-chlor 0.5, sal epsom 0.5, ferrous sulfate 0.01, pH are 7.0.120 ℃ of sterilization 20min.After the sterilization of substrate CNDE uv irradiating, be added in the good substratum of sterilization, concentration is 10g/L.Take by weighing fresh soil sample 1g and join the 250mL that the 80ml enrichment medium is housed and shake in the bottle, place 30 ℃, 200r/min shaking table to cultivate.Inoculum size by 5% is every other day once transferred triplicate.Bacterium liquid after the enrichment for the third time is stored in the glycerine pipe 4 ℃ of refrigerations.
Dull and stereotyped primary dcreening operation: medium component is (g/L): yeast extract paste 1.0, and glycerine 6.0, ammonium sulfate 4.0, potassium hydrogenphosphate 4.0, sodium-chlor 2.0, sal epsom 0.4, agar 20.0, pH are 7.0.120 ℃ of sterilization 20min.Substrate CNDE and tween are processed emulsion with 2: 1 (w/w), after the uv irradiating sterilization, are added into the good temperature of sterilization in 50 ℃ substratum, fall dull and stereotyped, cooled and solidified.Adopt dilution coating partition method,, respectively get 200 μ L and be coated on the plate culture medium, cultivated 1～3 day for 37 ℃ the enrichment bacterium liquid appropriateness dilution of having preserved.Single colony inoculation is on fresh plate culture medium on the picking flat board, and 37 ℃ are cultured to the plentiful somatic cells of appearance and are placed on 4 ℃ of refrigerators preservations.
Shake the multiple sieve of bottle: medium component is (g/L): peptone 5.0, and glycerine 3.0, yeast extract paste 2.0, ammonium sulfate 2.0, potassium primary phosphate 2.0, sodium-chlor 1.0, sal epsom 0.2, pH are 7.0.120 sterilization 20min.The sterilization of substrate CNDE uv irradiating.Single bacterium colony access 10mL that primary dcreening operation is obtained sieves in the substratum again, and 30 ℃, after 200rpm grew 1 day down; Adding the substrate CNDE sterilized in the 10mL fermented liquid, is 10g/L with respect to the add-on of fermented liquid, 30 ℃; Transform 18～36h under the 200rpm, add Hydrogen chloride and regulate pH value to 2～3, add the extraction of ETHYLE ACETATE mixing; The centrifugal organic phase that obtains is used for the chirality gc and analyzes, and the bacterial strain preservation that substrate conversion efficiency and product enantiomeric excess value is all higher is subsequent use.
Strain identification: oily sem observation, 16S rDNA identify and Physiology and biochemistry is identified.
Through screening, be numbered the bacterial strain of YZ05, its enantiomorph is the highest to value, and has relative better conversion rate, and the gas phase spectrogram is seen Fig. 1.Can know from Fig. 1; Product (R)-2-propyloic-3-cyanic acid-5-methylhexanoic acid goes out the peak position at 13.007min; Product (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid goes out the cutting edge of a knife or a sword position at 13.907min; Substrate (R)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester goes out the peak position at 50.957min, and substrate (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester goes out the peak position at 52.490min.Bacterial strain YZ05 is done 16S rDNA PCR sequencing analysis and Physiology and biochemistry experiment (seeing table 1); Sequencing result is shown in SEQ No.1; Can confirm that by sequencing analysis and Physiology and biochemistry result of experiment the bacterial strain that is filtered out is a Rhodopseudomonas, this bacterial strain called after pseudomonas (pseudomonas sp.) YZ05.
The Physiology and biochemistry experimental result of table 1 pseudomonas (pseudomonas sp.) YZ05
Embodiment 2:
Select the fermentation culture based component to be: peptone 10g/L, yeast extract paste 5g/L, sodium-chlor 10g/L regulates pH to 6.0.The seed liquor of pseudomonas (pseudomonas sp.) YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size; Place 25 ℃, 200r/min constant temperature shaking table fermentation after 4 days; Taking out the 5mL fermented liquid in the good small-sized Erlenmeyer flask of sterilization, add substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester, is 1g/L with respect to the add-on of fermented liquid; Place 25 ℃, 200r/min constant temperature shaking table oscillatory reaction 10h; Go out substrate and product with ethyl acetate extraction, after the organic phase that dry extraction obtains, carry out gas chromatographic analysis again; The transformation efficiency that obtains final substrate is 58.5%, product enantiomeric excess value and substrate enantiomeric excess value difference 70.7% and 99.7%.
Embodiment 3:
Select the fermentation culture based component to be: peptone 5, glycerine 3, yeast extract paste 1, ammonium sulfate 4, potassium primary phosphate 4, sodium-chlor 0.5, sal epsom 0.5 is regulated pH to 8.0.The seed liquor of bacterial strain YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size; Place 40 ℃, 200r/min constant temperature shaking table fermentation after 1 day; Taking out the 5mL fermented liquid in the good small-sized Erlenmeyer flask of sterilization, add substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester, is 50g/L with respect to the add-on of fermented liquid; Place 45 ℃, 200r/min constant temperature shaking table oscillatory reaction 60h; Go out substrate and product with ethyl acetate extraction, after the organic phase that dry extraction obtains, carry out gas chromatographic analysis again; The transformation efficiency that obtains final substrate is 13.7%, product enantiomeric excess value and substrate enantiomeric excess value difference 93.9% and 17.4%.
Embodiment 4:
Select the fermentation culture based component to be: peptone 15, glycerine 6, yeast extract paste 2, ammonium sulfate 2, potassium primary phosphate 2, sodium-chlor 1, sal epsom 0.2 is regulated pH to 7.0.The seed liquor of pseudomonas (pseudomonas sp.) YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size; Place 30 ℃, 200r/min constant temperature shaking table fermentation after 3 days; Taking out the 5mL fermented liquid in the good small-sized Erlenmeyer flask of sterilization, add substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester, is 5g/L with respect to the add-on of fermented liquid; Place 30 ℃, 200r/min constant temperature shaking table oscillatory reaction 48h; Go out substrate and product with ethyl acetate extraction, after the organic phase that dry extraction obtains, carry out gas chromatographic analysis again; The transformation efficiency that obtains final substrate is 57.2%, product enantiomeric excess value and substrate enantiomeric excess value difference 74.2% and 99.1%.
Embodiment 5:
Select the fermentation culture based component to be: peptone 15, glycerine 6, yeast extract paste 2, ammonium sulfate 2, potassium primary phosphate 2, sodium-chlor 1, sal epsom 0.2 is regulated pH to 7.0.The seed liquor of pseudomonas (pseudomonas sp.) YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size; Place 30 ℃, 200r/min constant temperature shaking table fermentation after 2 days, centrifugal fermented liquid is collected thalline, is suspended in pH and is 7.0 phosphate buffer solution (0.1M; 5mL); Wet thallus is 75g/L with respect to the add-on of phosphate buffer solution, adds substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester again, is 10g/L with respect to the add-on of phosphoric acid buffer; Place 30 ℃, the oscillatory reaction of 200r/min constant temperature shaking table, in the oscillatory reaction process, monitor substrate conversion efficiency, product enantiomeric excess value (ee
p) and substrate enantiomeric excess value (ee
s).Result such as table 2 and Fig. 2.Can know all that from table 2 data and Fig. 2 curve conversion reaction proceeds to 48h, substrate conversion efficiency reaches 56.3%, and the substrate enantiomeric excess value explains that more than 98% the optical purity of product has reached desired value.
The hydrolysis of table 2 pseudomonas YZ05 resting cell splits substrate CNDE and generates (S)-CNDE
Embodiment 6:
Select the fermentation culture based component to be: peptone 5, glycerine 5, yeast extract paste 2, ammonium sulfate 2, potassium primary phosphate 2, sodium-chlor 3, sal epsom 0.2 is regulated pH to 6.0.The seed liquor of pseudomonas (pseudomonas sp.) YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size, places 40 ℃, 200r/min constant temperature shaking table fermentation after 1 day, centrifugal fermented liquid is collected thalline; Be suspended in pH and be 5.0 phosphate buffer solution (0.1M; 5mL), wet thallus is 20g/L with respect to the add-on of phosphate buffer solution, adds substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester again; Add-on with respect to phosphoric acid buffer is 1g/L; Place 45 ℃, the oscillatory reaction of 200r/min constant temperature shaking table, process monitoring substrate conversion efficiency, product enantiomeric excess value (ee
p) and substrate enantiomeric excess value (ee
s).Result such as table 3 can know that from table 3 data conversion reaction proceeds to 10h, and substrate conversion efficiency reaches 59.4%, and the substrate enantiomeric excess value is 99.8%.
The hydrolysis of table 3 pseudomonas YZ05 resting cell splits substrate CNDE and generates (S)-CNDE
Embodiment 7:
Select the fermentation culture based component to be: peptone 5, glycerine 5, yeast extract paste 3, ammonium sulfate 1, potassium primary phosphate 3, sodium-chlor 2, sal epsom 0.2 is regulated pH to 8.0.The seed liquor of pseudomonas (pseudomonas sp.) YZ05 is inoculated in the above-mentioned fermention medium of 120mL with 1% (v/v) inoculum size, places 25 ℃, 200r/min constant temperature shaking table fermentation after 4 days, centrifugal fermented liquid is collected thalline; Be suspended in pH and be 9.0 phosphate buffer solution (0.1M; 5mL), wet thallus is 100g/L with respect to the add-on of phosphate buffer solution, adds substrate racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester again; Add-on with respect to phosphoric acid buffer is 50g/L; Place 25 ℃, the oscillatory reaction of 200r/min constant temperature shaking table, process monitoring substrate conversion efficiency, product enantiomeric excess value (ee
p) and substrate enantiomeric excess value (ee
s).Result such as table 4 can know that from table 4 data conversion reaction proceeds to 60h, and substrate conversion efficiency reaches 45.6%, and the substrate enantiomeric excess value is 73.6%.
The hydrolysis of table 4 pseudomonas YZ05 resting cell splits substrate CNDE and generates (S)-CNDE
Claims (8)
- A pseudomonas ( Pseudomonas sp.) YZ05, it is characterized in that: this bacterial strain is preserved in Chinese common micro-organisms culture presevation administrative center, and preservation date is on September 17th, 2010, and its preserving number is CGMCC No.4184.
- 2. a pseudomonas that utilizes claim 1 comprises the steps: the method that the lyrica midbody carries out microbial transformation(1) be that the pseudomonas of CGMCC No.4184 joins and carries out fermentation culture in the substratum with preserving number;(2) by following option A or option b the lyrica midbody is carried out conversion reaction, obtain conversion fluid, said lyrica midbody is racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester:Option A: racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is directly carried out conversion reaction in the fermented liquid of adding after step (1) is cultivated, obtain conversion fluid;Option b: will earlier said wet thallus be joined in the phosphate buffer solution through the centrifugal wet thallus that obtains of fermented liquid after step (1) is cultivated, after add racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester again and carry out conversion reaction, obtain conversion fluid;(3) said conversion fluid is used SX, the organic phase dehydration back evaporating solvent that extraction is obtained obtains (S)-2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester.
- 3. the method that the lyrica midbody is carried out microbial transformation as claimed in claim 2 is characterized in that:In the option A of step (2), said racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is 1 ~ 50g/L with respect to the add-on of said fermented liquid;In the option b of step (2), said racemization 2-propyloic-3-cyanic acid-5-methylhexanoic acid ethyl ester is 1 ~ 50g/L with respect to the add-on of said phosphate buffer solution.
- 4. like claim 2 or the 3 described methods that the lyrica midbody is carried out microbial transformation, it is characterized in that: in the option b of step (2), said wet thallus is 20 ~ 100g/L with respect to the add-on of said phosphate buffer solution.
- 5. like claim 2 or the 3 described methods that the lyrica midbody is carried out microbial transformation, it is characterized in that: in the step (2), the conversion reaction temperature is 25 ~ 45 ℃, and the conversion reaction time is 10 ~ 60h.
- 6. like claim 2 or the 3 described methods that the lyrica midbody is carried out microbial transformation, it is characterized in that: in the option b of step (2), the concentration of said phosphate buffer solution is that 0.1M, pH are 5 ~ 9.
- 7. the method that the lyrica midbody is carried out microbial transformation as claimed in claim 2 is characterized in that: in the step (1), 25 ~ 40 ℃ of the culture temperature of said fermentation culture were cultivated 1 ~ 4 day.
- 8. like claim 2 or 7 described methods of the lyrica midbody being carried out microbial transformation; It is characterized in that: in the step (1); The composition of said substratum is: peptone 5 ~ 15g/L, glycerine 0 ~ 6g/L, yeast extract paste 1 ~ 5g/L, ammonium sulfate 0 ~ 4g/L, potassium primary phosphate 0 ~ 4g/L, sodium-chlor 0.5 ~ 10g/L and sal epsom 0 ~ 0.5g/L, the pH value of said substratum is 6 ~ 8.
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| CN1995327A (en) * | 2006-10-21 | 2007-07-11 | 浙江工业大学 | Pseudomonas zjwp-14 and its uses in asymmetrical hydrolyzed preparation of L-cysteine |
| CN101268037A (en) * | 2005-09-19 | 2008-09-17 | 特瓦制药工业有限公司 | Novel asymmetric synthesis of (S)-(+)-3-(aminomethyl)-5-methylhexanoic acid |
| CN101346461A (en) * | 2005-11-24 | 2009-01-14 | 株式会社晓星 | Pseudomonas sp. HN-72 and purification method of 2,6-naphthalene dicarboxylic acid using the same |
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| CN101268037A (en) * | 2005-09-19 | 2008-09-17 | 特瓦制药工业有限公司 | Novel asymmetric synthesis of (S)-(+)-3-(aminomethyl)-5-methylhexanoic acid |
| CN101346461A (en) * | 2005-11-24 | 2009-01-14 | 株式会社晓星 | Pseudomonas sp. HN-72 and purification method of 2,6-naphthalene dicarboxylic acid using the same |
| CN1995327A (en) * | 2006-10-21 | 2007-07-11 | 浙江工业大学 | Pseudomonas zjwp-14 and its uses in asymmetrical hydrolyzed preparation of L-cysteine |
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