CN103555590B - Geotrichum candidum and its application in preparation of (2R, 3S)-ethyl phenyl glycidate - Google Patents
Geotrichum candidum and its application in preparation of (2R, 3S)-ethyl phenyl glycidate Download PDFInfo
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Abstract
The invention provides a new strain Geotrichum candidum ZJUTZQ200 for preparation of optically pure (2R, 3S)-ethyl phenyl glycidate, and its application in preparation of (2R, 3S)-ethyl phenyl glycidate by a microbiological method. The enzyme is preserved in China Center for Type Culture Collection, which is located in Wuhan University, Wuhan, China with a postcode of 430072 on April 1, 2013. The preservation number is CCTCC NO:M2013114. The invention provides the new strain that has stereoselectivity and can be used for preparation of high optical purity (2R, 3S)-ethyl phenyl glycidate, also provides a new method for preparation of (2R, 3S)-ethyl phenyl glycidate from the strain. The method has the advantages of mild reaction conditions, high stereoselectivity, and environmental friendliness.
Description
(1) technical field
The present invention relates to geotrichum candidum (Geotrichum candidum) ZJUTZQ200, and the application in (2R, the 3S)-phenylglicidic acid ethyl ester shown in microbial method preparation formula (I) and derivative thereof.
(2) background technology
The chemical structure of phenylglicidic acid ethyl ester is:
Racemic phenylglicidic acid ethyl ester is made up of (2R, 3S)-phenylglicidic acid ethyl ester and (2S, 3R)-phenylglicidic acid ethyl ester two enantiomorphs.
Phenylglicidic acid ethyl ester (Ethyl phenyl glycidate, EPG) be raw material and the key intermediate of many pharmaceutical synthesis, as (2R, 3S)-EPG is the intermediate of synthesis anti-cancer medicine paclitaxel side chain and Docetaxel side chain, and (2S, 3R)-EPG is synthesis cancer therapy drug aminopeptidase N inhibitor Bestatin, Phebestin, the important intermediate of a series of medicine such as Probestin, MR-387.
Utilize traditional chemical method to prepare the epoxide of chirality, need in reaction, to add a large amount of solvent and expensive metal catalyst, great harm is caused to environment.The epoxide of Biological preparation chirality has that reaction conditions gentleness, stereoselectivity are high, advantages of environment protection.The report preparing phenylglicidic acid ethyl ester with epoxide hydrolase only has two at present, one Ge Shi India scientist utilizes the epoxide hydrolase in mung bean to prepare (2S, 3R)-EPG, another is that Shandong University professor Li Congfa utilizes the epoxide hydrolase in bacterium to prepare (2R, 3S)-EPG.Utilize the epoxide in mould to prepare (2R, 3S)-EPG also not report at present.
(3) summary of the invention
The present invention seeks to provide a strain for the preparation of optical purity (2R, the new strains of 3S)-phenylglicidic acid ethyl ester---geotrichum candidum (Geotrichum candidum) ZJUTZQ200, and the application in microbial method preparation (2R, 3S)-phenylglicidic acid ethyl ester.
The technical solution used in the present invention is:
Geotrichum candidum (Geotrichum candidum) ZJUTZQ200, is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, preservation date on April 1st, 2013, deposit number CCTCC NO:M2013114.
The strain morphology following (see Fig. 1) of this bacterial strain: cultivate 72h for 30 DEG C, bacterium colony is irregular circle on PDA plate culture medium, white, central elevation, hair shape mycelia, surface drying, difficult picking, there are radioactive rays, when bacterium colony surface is by disturbance, become yeastlike thick.Basis of microscopic observation, does not have obvious mycelia and sporocyst, the rounded or tubbiness of the arthrospore of formation, the blunt circle in two ends.
Comparing of the sequence of the 18S rDNA in the sequence of the 18S rDNA of ZJUTZQ200 and NCBI, find the homology having the sequence of 99% with geotrichum candidum, the 18SrDNA partial nucleotide sequence of geotrichum candidum ZJUTZQ200 is as follows:
TGGTTCTAGTATAAGCATTATACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATATTACATTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTAAAACGGCCGGGTTCCCGGCTGGTATTTATTAGATAAAAAACCAATGCCTTCGGGCTCTATGGTGAATCATAATAACTTGTCGAATCGCATGGCCTTGTGCTGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTTTTAACGGGTAACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACAATAAATAACGATACGGGGCCTATTAGGTCTCGTAATTGGAATGAGAACAATTTAAATACCTTAACGAGGAACAATTAGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCTGATAGTATATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAAACTTGGGTGTGTAGGGGCGGTCTCTTTTAGAGTACTACCCTGAAACATCTTTCTTTGGTGTAAACTCTTTATTCACTTAAGGAGTGTAAACCAAACATTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATGGAATAATAGAATAGGACGTATGGTTCTATTTTGTTGGTTTCTAGGACCGTCGTAATGATTAATAGGGACGGTCGGGGGCATCAGTATTCAGTTGTCAGAGGTGAAATTCTTGGATTTACTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGACGTTTTCATTAATCAAGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGGAGGGCGTTATAATAACCTCTCCGGCACCTTACGAGAAATCAAAGTTTTTGGGTTCTGGGGGGAGTATGGTTGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCATGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCTGTAACAATAGATTATTGTTTTGACAGCTTCTTAGAGGGACCTATCCGATTTCCAA。
Comprehensive the above results, this bacterial strain is accredited as geotrichum candidum (Geotrichum candidum).
Microorganism involved in the present invention is obtained by the screening of following program:
(1) be inoculated in primary dcreening operation substratum by the soil sample that all parts of the country are adopted back, at 30 DEG C, the shaking table of 200rpm cultivated 3 days, draw the line of a small amount of nutrient solution on PDA plate culture medium, 30 DEG C of thermostat containers are cultivated.Then picking list bacterium colony is transferred into test tube slant, takes out, be stored in 4 DEG C of refrigerators after 2 ~ 3d cultivated by 30 DEG C of thermostat containers.The formula of primary dcreening operation substratum: NaNO
32.0g/L, K
2hPO
43H
2o1.0g/L, KCl0.5g/L, MgSO
47H
2o0.5g/L, analysis for soybean powder 1g/L, 0.1%(v/v) phenylglicidic acid ethyl ester, solvent is water, pH4.0.
(2) appropriate thalline is transferred from test tube slant to fermention medium, 30 DEG C, 200rpm shaking culture 72 hours; Fermention medium is composed as follows: NaNO
32.0g/L, K
2hPO
43H
2o1.0g/L, KCl0.5g/L, MgSO
47H
2o0.5g/L, glucose 30g/L, analysis for soybean powder 10g/L, solvent is water pH4.0.
(3) collected by centrifugation thalline, take 1g wet thallus and be suspended in 10mL potassium phosphate buffer (0.1M, pH70) in, join in above-mentioned suspension after 10 μ L phenylglicidic acid ethyl esters are dissolved in 40 μ L dimethyl sulfoxide (DMSO) (DMSO), 30 DEG C, 200rpm transforms after 6h, get conversion fluid 200 μ L, add the substrate that ethyl acetate (2 × 1000mL) extraction is remaining.With adding 1mL AG normal hexane/Virahol (80/20, v/v), filtering with microporous membrane after evaporation concentration.Filtrate is by the transformation efficiency of Normal-phase HPLC detection of active bacterial strain and stereoselectivity.
Normal-phase HPLC is analyzed: chiral column AS-H(46cm × 25cm, 4 μm, DaicelCHIRALPAK), moving phase is normal hexane/Virahol (80/20, V/V, 0.8mL/min), and determined wavelength is 220nm.The appearance time of phenylglicidic acid ethyl ester (R, R), (S, S) configuration is respectively 5.994min and 7.744min.
The calculation formula of substrate e.e.s: e.e.s (%)=(nR, S-nS, R)/(nR, S+nS, R) × 100%, nR, S represents (2R, 3S) configuration peak area in substrate peak, nS, R represent the peak area of (2S, 3R) configuration in substrate peak.
The invention still further relates to the application of described geotrichum candidum ZJUTZQ200 in (2R, the 3S)-phenylglicidic acid ethyl ester shown in microbial method preparation formula (I) and derivative thereof;
In formula (I), R is-H ,-CH
3,-OCH
3,-NH
2,-CN ,-Cl or-Br.
Concrete, described is applied as: with corresponding racemize phenylglicidic acid ethyl ester for substrate, with the wet thallus cell of geotrichum candidum ZJUTZQ200 for catalyzer, under 25 ~ 40 DEG C (being preferably 30 DEG C), in the preferred pH7.2 of pH6.0 ~ 8.4() damping fluid in (preferably phosphoric acid salt buffer) react 1 ~ 12 hour (preferred reaction 4 hours), obtained described (2R, 3S)-phenylglicidic acid ethyl ester.
The interpolation concentration of described geotrichum candidum ZJUTZQ200 wet thallus cell is 100 ~ 500g/L, and the starting point concentration of substrate racemize phenylglicidic acid ethyl ester is 1 ~ 10g/L.
For improving transformation efficiency, the solubility promoter that volume is damping fluid volume 1 ~ 10% also can be added in described damping fluid, described solubility promoter is one of following: dimethyl sulfoxide (DMSO) (DMSO), DMF (DMF), tetrahydrofuran (THF) (THF), acetone, Virahol (IPN), ethanol (EtOH).
More preferred, described solubility promoter is dimethyl sulfoxide (DMSO).
Preferably, described geotrichum candidum ZJUTZQ200 wet thallus cell obtains by the following method: geotrichum candidum ZJUTZQ200 is seeded to culture medium, in 25 ~ 40 DEG C, shaking culture 48 ~ 96 hours under 100 ~ 200rpm, medium centrifugal gets precipitation through brine, collects and obtains described geotrichum candidum ZJUTZQ200 wet thallus cell; Described culture medium final concentration is composed as follows: glycerine 5 ~ 20g/L, analysis for soybean powder 10 ~ 30g/L, SODIUMNITRATE 1 ~ 10g/L, anhydrous magnesium sulfate 0.1 ~ 1.0g/L, potassium primary phosphate 0.5 ~ 2.0g/L, solvent is water, pH4 ~ 10.
The slant medium of bacterial strain is PDA substratum.
Concrete, described application can be as follows:
(1) the final concentration composition of culture medium: glycerine 10g/L, analysis for soybean powder 15g/L, SODIUMNITRATE 3g/L, anhydrous magnesium sulfate 0.5g/L, potassium primary phosphate 1g/L, solvent is water, pH5.0; Shaking flask liquid amount 30%, inoculation geotrichum candidum ZJUTZQ200, in 30 DEG C, 200rpm shaking culture 48h, will be centrifugal through brine in thalline, collect wet thallus cell;
(2) the phosphate buffer 1 0mL of 100mM pH7.0 is got, add the wet thallus cell 1 ~ 5g of step (1) gained, add racemize phenylglicidic acid ethyl ester 0.01 ~ 0.1g, 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 6 ~ 8 hours, be extracted with ethyl acetate after reaction terminates, obtain (2R, 3S)-phenylglicidic acid ethyl ester.
Beneficial effect of the present invention is mainly reflected in: the invention provides one and have stereoselectivity, can prepare high optical purity (2R, the new strains of 3S)-phenylglicidic acid ethyl ester---geotrichum candidum (Geotrichum candidum) ZJUTZQ200, additionally providing one utilizes this bacterial strain to prepare (2R, the novel method of 3S)-phenylglicidic acid ethyl ester, reaction conditions is gentle, stereoselectivity is high, environmental friendliness.
(4) accompanying drawing explanation
Fig. 1 is bacterial strain thalli morphology figure of the present invention; A: dull and stereotyped front; B: arthrospore.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the separation of bacterial strain
Produce the separation of epoxide hydrolase bacterium: take 1g soil in 100mL sterilized water, then add some granulated glass spherees, fully make Soil Slurry after vibration, get 1mL supernatant liquor in 50ml primary dcreening operation substratum, 30 DEG C, shaking culture 3 days on 200rpm shaking table.Draw a small amount of fermented liquid to rule respectively on bacterium and mould plate culture medium, obtain single bacterium colony.
The formula of primary dcreening operation substratum: NaNO
32.0g/L, K
2hPO
43H
2o1.0g/L, KCl0.5g/L, MgSO
47H
2o0.5g/L, analysis for soybean powder 1g/L, 0.1%(v/v) phenylglicidic acid ethyl ester, the bottled 50mL of pH4.0,250mL triangle, solvent is water, 121 DEG C of sterilizing 20min.
After screening, detect the enantiomeric excess value (e.e. value) of chirality phenylglicidic acid ethyl ester with chiral hplc, the e.e. value of described geotrichum candidum ZJUTZQ160 is 85.5%.
Embodiment 2: the cultivation of bacterial strain
Dull and stereotyped and the slant medium of PDA: peeling cleaned by potato, then take 200g potato and be cut into small pieces, add 600mL poach rotten, by eight layers of filtered through gauze, get filtrate heating, add agar 20g, continue heated and stirred mixing, after agar has dissolved, add glucose 20g, stir, slightly supply moisture to 1000 milliliter again, packing after cooling, 121 DEG C of sterilizings 20 minutes, bevel or flat board, for subsequent use.
On flat board, picking geotrichum candidum ZJUTZQ200, is seeded to inclined-plane, and 3d cultivated by 30 DEG C of thermostat containers, and the line of picking list bacterium colony, to corresponding bacterium and mould inclined-plane, is taken out to put in 4 DEG C of refrigerators after 3d cultivated by 30 DEG C of thermostat containers and preserved.
Embodiment 3: the acquisition of wet thallus cell
Substratum is prepared: glycerine 10g/L, analysis for soybean powder 15g/L, SODIUMNITRATE 3g/L, anhydrous magnesium sulfate 0.5g/L, potassium primary phosphate 1g/L, and solvent is water, pH5.0; The bottled 50mL of 250mL triangle, 121 DEG C of sterilizing 20min;
Inoculate a ring geotrichum candidum ZJUTZQ200 in 30 DEG C, 200rpm shaking table shaking culture 48h; Cultivate terminate secondary fermentation liquid with 4% volume ratio be seeded to fermention medium, 30 DEG C, 200rpm shaking table shaking culture 48h, centrifugal and with brine twice, collection wet thallus cell, for subsequent use.
Embodiment 4:
In the phosphate buffered saline buffer of 10mL, 100mM (pH7.0), add 1g embodiment 3 gained wet thallus cell, add the racemic phenylglicidic acid ethyl ester of 0.01g (being dissolved in 0.1mL dimethyl sulfoxide (DMSO)), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 6 hours, get conversion fluid 200 μ L, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), (2R, the e.e. value of 3S)-phenylglicidic acid ethyl ester is 85.5%, enantio-selectivity E is 3(wherein E=ln [(1-c) (1-e.e.s)]/ln [(1-c) (1+e.e.s)], e.e.s is the enantiomeric excess value of substrate, c is transformation efficiency).
Embodiment 5:
In the phosphate buffered saline buffer of 10mL, 100mM (pH7.0), add 1g embodiment 4 gained wet thallus cell; Add the racemic phenylglicidic acid ethyl ester of 0.1g (being dissolved in 1mL dimethyl sulfoxide (DMSO)), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 1 ~ 6 hour, get conversion fluid 200 μ L, add ethyl acetate (2 × 1000mL) extraction, after organic layer concentrated by rotary evaporation, add 1mL moving phase (normal hexane/Virahol: 80/20, V/V), finally determine that optimum transformation time is 4h, the e.e. value of (2R, 3S)-phenylglicidic acid ethyl ester is 93.4%, E is 8.0.
Embodiment 6:
In the phosphate buffered saline buffer of 10mL, 100mM (pH7.0), add 1g embodiment 4 gained wet thallus cell; Add 0.01g racemic m-methoxyphenyl glycidic acid ethyl ester (being dissolved in the dimethyl sulfoxide (DMSO) of 0.1mL), respectively at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 4 hours, get conversion fluid 200 μ L respectively, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), e.e. value is respectively 81%, 91%, 95%, 89%, 75%.Finally determine that optimal conversion temperature is 30 DEG C, the e.e. value of (2R, 3S)-m-methoxyphenyl glycidic acid ethyl ester is 95%, E is 8.
Embodiment 7:
Respectively in the sodium citrate buffer solution of 10mL, 100mM (pH4.4,4.8,5.2,6.0,6.4), 1g embodiment 4 gained wet thallus cell is added; Add 0.01g racemic m-methoxyphenyl glycidic acid ethyl ester (being dissolved in the dimethyl sulfoxide (DMSO) of 0.1mL), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 4 hours, get conversion fluid 200 μ L respectively, add ethyl acetate (2 × 1000mL) extraction, add 1mL moving phase (normal hexane/Virahol: 80/20, V/V) after organic layer concentrated by rotary evaporation, result shows, the e.e. value of (2R, 3S)-phenylglicidic acid ethyl ester is respectively 71%, 76%, 80%, 85%, 82%.And pH is when being 6.0, E is 26.
Embodiment 8:
Respectively in the potassium phosphate buffer of 10mL, 100mM (pH6,6.5,7,7.5,8), 1g embodiment 4 gained wet thallus cell is added; Add 0.01g racemic m-methoxyphenyl glycidic acid ethyl ester (being dissolved in the dimethyl sulfoxide (DMSO) of 0.1mL), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 4 hours; Get conversion fluid 200 μ L respectively, add ethyl acetate (2 × 1000mL) extraction, after organic layer concentrated by rotary evaporation, add 1mL moving phase (normal hexane/Virahol: 80/20, V/V), the e.e. value of (2R, 3S)-p-methylphenyl glycidic acid ethyl ester is respectively 81%, 88%, 90%, 99%, 85%., finally determine that optimal conversion pH be 7.5, E is 17.
Embodiment 9:
In the Tris-HCl damping fluid of 10mL, 100mM (pH8.0), add 1g embodiment 4 gained wet thallus cell; Add 0.01g racemic m-methoxyphenyl glycidic acid ethyl ester (being dissolved in the dimethyl sulfoxide (DMSO) of 0.1mL), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 10 hours, get conversion fluid 200 μ L, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20, V/V) is added, (2R after organic layer concentrated by rotary evaporation, the e.e.>85% of 3S)-adjacent aminophenyl glycidic acid ethyl ester, E is 15.
Embodiment 11:
In the phosphate buffered saline buffer of 10mL, 100mM (pH7.2), add 1g embodiment 4 gained wet thallus cell; Add 0.01g racemic to itrile group epoxy styryl carbinol (being dissolved in respectively in the dimethyl sulfoxide (DMSO) of 0.1mL, tetrahydrofuran (THF), acetone, Virahol, ethanol, DMF six kinds of organic solvents), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 4 hours; Get conversion fluid 200 μ L respectively, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), (2R, 3S)-and 99.3%, 83%, 75%, 28%, 12%, 92% is respectively to the e.e. value of cyanophenyl glycidic acid ethyl ester, finally determine that optimal conversion solubility promoter be DMSO, E is 15.
Embodiment 12:
In the phosphate buffered saline buffer (pH7.2) of 10mL, 100mM, add 1g embodiment 4 gained wet thallus cell; Add 0.1g racemic m-methoxyphenyl glycidic acid ethyl ester (being dissolved in respectively in the methylene dichloride of 1mL, ethyl acetate, normal hexane, hexanaphthene, normal heptane, acetone, octane-iso), in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 4 hours; Get conversion fluid 200 μ L respectively, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), result shows, when adding above-mentioned solubility promoter, and (2R, 3S)-m-methoxyphenyl glycidic acid ethyl ester e.e. value is respectively 49%, 23%, 65%, 48%, 56%, 75,81%, finally causes the selectivity of enzyme to decline.
Embodiment 13:
In the phosphate buffered saline buffer of 10mL, 100mM (pH7.2), add 1g embodiment 4 gained wet thallus cell; Add the racemize p-methoxyphenyl glycidic acid ethyl ester being dissolved in dimethyl sulfoxide (DMSO), make its ultimate density be 1,3,5,7,10g/L, and in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 12 hours, get conversion fluid 200 μ L, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), (2R, 3S)-p-methoxyphenyl glycidic acid ethyl ester e.e. is respectively 89,92,96,99,90%, finally determine that best enzyme/concentration of substrate be 5g/L, E is 45.
Embodiment 14:
In the phosphate buffered saline buffer of 200mL, 100mM (pH7.2), add 20g embodiment 4 gained wet thallus cell; Add 1g racemic rubigan glycidic acid ethyl ester (being dissolved in 10mL dimethyl sulfoxide (DMSO)) in 30 DEG C of water bath with thermostatic control shaking tables oscillatory reaction 8 hours, get conversion fluid 200 μ L, add ethyl acetate (2 × 1000mL) extraction, 1mL moving phase (normal hexane/Virahol: 80/20 is added after organic layer concentrated by rotary evaporation, V/V), the e.e. value of (2R, 3S)-rubigan glycidic acid ethyl ester is 99.9%, E is 71.
Claims (8)
1. geotrichum candidum (Geotrichum candidum) ZJUTZQ 200, is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, preservation date on April 1st, 2013, deposit number CCTCC NO:M 2013114.
2. geotrichum candidum ZJUTZQ 200 as claimed in claim 1, is characterized in that the 18S rDNA partial nucleotide sequence of described geotrichum candidum ZJUTZQ 200 is as follows:
TGGTTCTAGTATAAGCATTATACAGTGAAACTGCGAATGGCTCATT
AAATCAGTTATCGTTTATTTGATATTACATTACTACTTGGATAACCG
TGGTAATTCTAGAGCTAATACATGCTAAAACGGCCGGGTTCCCGG
CTGGTATTTATTAGATAAAAAACCAATGCCTTCGGGCTCTATGGTG
AATCATAATAACTTGTCGAATCGCATGGCCTTGTGCTGGCGATGGT
TCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGC
CTACCATGGTTTTAACGGGTAACGGGGAATCAGGGTTCGATTCCG
GAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAG
CAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACA
ATAAATAACGATACGGGGCCTATTAGGTCTCGTAATTGGAATGAGA
ACAATTTAAATACCTTAACGAGGAACAATTAGAGGGCAAGTCTGG
TGCCAGCAGCCGCGGTAATTCCAGCTCTGATAGTATATATTAAAGT
TGTTGCAGTTAAAAAGCTCGTAGTTGAAACTTGGGTGTGTAGGGG
CGGTCTCTTTTAGAGTACTACCCTGAAACATCTTTCTTTGGTGTAA
ACTCTTTATTCACTTAAGGAGTGTAAACCAAACATTTACTTTGAAA
AAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATG
GAATAATAGAATAGGACGTATGGTTCTATTTTGTTGGTTTCTAGGA
CCGTCGTAATGATTAATAGGGACGGTCGGGGGCATCAGTATTCAG
TTGTCAGAGGTGAAATTCTTGGATTTACTGAAGACTAACTACTGC
GAAAGCATTTGCCAAGGACGTTTTCATTAATCAAGAACGAAAGTT
AGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCGTAAA
CTATGCCGACTAGGGATCGGAGGGCGTTATAATAACCTCTCCGGC
ACCTTACGAGAAATCAAAGTTTTTGGGTTCTGGGGGGAGTATGGT
TGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAG
GAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCA
CCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCA
TGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGT
GATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTA
AATAGCTGTAACAATAGATTATTGTTTTGACAGCTTCTTAGAGGGA
CCTATCCGATTTCCAA。
3. the application of geotrichum candidum ZJUTZQ 200 according to claim 1 in (2R, the 3S)-phenylglicidic acid ethyl ester shown in microbial method preparation formula (I);
In formula (I), R is-H ,-CH
3,-OCH
3,-NH
2,-CN ,-Cl or-Br.
4. apply as claimed in claim 3, it is characterized in that described being applied as: with corresponding racemize phenylglicidic acid ethyl ester for substrate, with the wet thallus cell of geotrichum candidum ZJUTZQ 200 for catalyzer, at 25 ~ 40 DEG C, react 1 ~ 12 hour in the damping fluid of pH 6.0 ~ 8.4, obtained described (2R, 3S)-phenylglicidic acid ethyl ester.
5. apply as claimed in claim 4, it is characterized in that the interpolation concentration of described geotrichum candidum ZJUTZQ 200 wet thallus cell is 100 ~ 500g/L, the starting point concentration of substrate racemize phenylglicidic acid ethyl ester is 1 ~ 10g/L.
6. apply as claimed in claim 4, it is characterized in that in described damping fluid, being also added with the solubility promoter that volume is damping fluid volume 1 ~ 10%, described solubility promoter is one of following: dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetone, Virahol, ethanol, DMF.
7. apply as claimed in claim 6, it is characterized in that described solubility promoter is dimethyl sulfoxide (DMSO).
8. apply as claimed in claim 4, it is characterized in that described geotrichum candidum ZJUTZQ 200 wet thallus cell obtains by the following method: geotrichum candidum ZJUTZQ 200 is seeded to culture medium, in 25 ~ 40 DEG C, shaking culture 48 ~ 96 hours under 100 ~ 200rpm, medium centrifugal gets precipitation through brine, collects and obtains described geotrichum candidum ZJUTZQ 200 wet thallus cell; Described culture medium final concentration is composed as follows: glycerine 5 ~ 20g/L, analysis for soybean powder 10 ~ 30g/L, SODIUMNITRATE 1 ~ 10g/L, anhydrous magnesium sulfate 0.1 ~ 1.0g/L, potassium primary phosphate 0.5 ~ 2.0g/L, and solvent is water, pH 4 ~ 10.
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