CN107881202B - Biological preparation method of key chiral intermediate of levonorgestrel - Google Patents
Biological preparation method of key chiral intermediate of levonorgestrel Download PDFInfo
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- CN107881202B CN107881202B CN201711155745.5A CN201711155745A CN107881202B CN 107881202 B CN107881202 B CN 107881202B CN 201711155745 A CN201711155745 A CN 201711155745A CN 107881202 B CN107881202 B CN 107881202B
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- WWYNJERNGUHSAO-XUDSTZEESA-N (+)-Norgestrel Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](CC)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 WWYNJERNGUHSAO-XUDSTZEESA-N 0.000 title claims abstract description 19
- 229960004400 levonorgestrel Drugs 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 62
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims abstract description 54
- 235000017388 Geotrichum candidum Nutrition 0.000 claims abstract description 49
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- 238000009833 condensation Methods 0.000 claims abstract description 9
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- 239000012429 reaction media Substances 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 123
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- 244000168141 Geotrichum candidum Species 0.000 claims description 47
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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Abstract
The invention discloses a biological preparation method of a key chiral intermediate of levonorgestrel, which comprises the steps of taking wet thalli obtained by fermenting and culturing geotrichum candidum ZJPH1704 as an enzyme source, taking an ethyl condensation compound as a substrate, taking a phosphate buffer solution with the pH value of 5.8-8.0 as a reaction medium to form a reaction system, carrying out reaction at the temperature of 30-40 ℃ and the speed of 150-250 rpm, and after the reaction is finished, separating and purifying the reaction solution to obtain the key chiral intermediate of levonorgestrel; the intermediate is prepared by adopting the geotrichum candidum ZJPH1704 strain for catalytic reduction, and the method has the advantages of high substrate concentration for catalytic reduction, good stereoselectivity and the like. When the substrate concentration was 7.0g/L (22.4mM), the yield was 73%, and the ee value was 100%.
Description
(I) technical field
The invention relates to a method for preparing a key chiral intermediate of levonorgestrel by utilizing Geotrichum candidum ZJPH1704 strain biological reduction.
(II) background of the invention
The structural formula of an ethyl condensate (chemical name: 2- [2- (3, 4-dihydro-6-methoxy-1 (2H) -naphthylidene) ethyl ] -2-ethylcyclopentane-1, 3-dione, CAS number: 850-92-0):
the structural formula of ethyl hydroxylate (chemical name: 2-ethyl-3-hydroxy-2- [2- (6-methoxy-3, 4-dihydro-1 (2H) -naphthylidene) ethyl ] cyclopentanone, CAS number: 51773-49-0):
the ethyl hydroxylate is a key chiral intermediate for synthesizing a progestational drug levonorgestrel (chemical name: D (-) -17 alpha-ethynyl-17 beta-hydroxy-18-methylestra-4-en-3-one), and the drug mainly acts on hypothalamus and pituitary, has good progestational activity and stronger capacity of combining with androgen receptors. The oral contraceptive can be used as a short-acting oral contraceptive and a long-acting oral contraceptive by being combined with estrogen, and is the second generation oral contraceptive which is most widely applied at present. It can also be used for treating menoxenia, functional uterine bleeding, endometriosis, etc.
Levonorgestrel is the biologically active levorotatory form of the racemate norgestrel. The existing chemical synthesis method adopts sodium borohydride as a reducing agent, an ethyl condensation compound is reduced into an ethyl hydroxide, 4 isomers are generated in the reaction process, the stereoselectivity is not ideal, and the catalyst pollutes the environment; martina et al used oxazaborolidine as a catalyst and added 6.5mM of an ethyl condensate substrate at 25% to 30% yield with an e.e. value of 75%.
The intermediate is prepared by adopting a biological reduction method of microbial whole-cell catalysis, has the characteristics of mild reaction conditions, high stereoselectivity, environmental friendliness and the like, and in addition, the biocatalyst is microbial thallus, can be produced by self fermentation, and has stable quality and low cost.
Federica et al (Catalysis Communications,2017(93):29-32) used a continuous packed bed flow reactor to immobilize ketoreductase from Pichia pastoris (KDE1-Pglu) and glucose dehydrogenase from Bacillus megaterium (BmGDH) with aldehyde activated agarose for co-expression, added 6.5mM ethyl condensate as substrate, with a conversion rate of 62-63%. Martina et al (Eur.J.org.chem.2016,1260-1263) used recombinant KRED1-Pglu with 6.5mM ethyl condensate substrate, ethanol and glucose as cosubstrates, with high stereoselectivity (> 98% e.e.) and 65% yield after 6h reaction. When the saccharomyces cerevisiae is used as a catalyst, 10mM of an ethyl condensation product substrate is added, and after 24 hours of reaction, the yield is 87%.
Disclosure of the invention
The invention aims to provide a method for preparing a key chiral intermediate of levonorgestrel by utilizing Geotrichum candidum ZJPH1704 strain biological asymmetric reduction. The method has the advantages of high stereoselectivity, low cost, mild reaction conditions, environmental friendliness and easiness in industrialization.
The technical scheme adopted by the invention is as follows:
the invention provides a biological preparation method of a key chiral intermediate of levonorgestrel, which comprises the steps of taking wet thalli obtained by fermenting and culturing Geotrichum candidum ZJPH1704 as an enzyme source, taking an ethyl condensation compound shown as a formula (III) as a substrate, taking a phosphate buffer solution with pH of 5.8-8.0 (preferably 7.0) and 0.05-0.2M (preferably 0.1M) as a reaction medium to form a reaction system, reacting for 12-72 hours (preferably 72 hours) under the conditions of 30-40 ℃ and 150-250 rpm (preferably 30 ℃ and 200rpm), and separating and purifying the reaction solution after the reaction is finished to obtain the key chiral intermediate of levonorgestrel shown as a formula (IV); the Geotrichum candidum (Geotrichum candidum) ZJPH1704 strain is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2017380, deposit date: 26 months 6 and 2017, wherein the preservation address is Wuhan, Wuhan university, postcode 430072;
furthermore, the dosage of the substrate is 4-7.5 g/L (preferably 7g/L) by volume of the buffer solution, and the dosage of the wet bacteria is 200-500 g/L (preferably 300g/L) by volume of the buffer solution.
Further, the reaction system also comprises an auxiliary substrate, and the auxiliary substrate is one of the following substances: glucose, maltose, sucrose, lactose, methanol, isopropanol, glycerol, L-alanine, or L-cysteine, preferably glycerol.
Further, when the auxiliary substrate is glucose, maltose, sucrose, lactose, glycerol, L-alanine or L-cysteine, the volume of the auxiliary substrate is 10-300 g/L calculated by the volume of the buffer solution, and when the auxiliary substrate is methanol or isopropanol, the volume of the auxiliary substrate is 10-50% calculated by the volume of the buffer solution. When the auxiliary substrate is glucose, sucrose, maltose and lactose, the addition amount is 100g/L buffer solution; when the auxiliary substrate is glycerol, 60g/L buffer solution is added; and when the auxiliary substrate is methanol and isopropanol, the volume concentration is 10-30%. When the auxiliary substrates are L-cysteine and L-alanine, 10g/L buffer solution is added. Preferably, the co-substrate is glycerol in an amount of 60g/L buffer.
Further, a cosolvent is also added into the reaction medium, and the cosolvent is one of the following solvents: (1) preheating ethanol (the ethanol is ethanol reagent with the volume content of 95 percent) at 37 ℃; (2) dimethyl sulfoxide (DMSO).
Furthermore, the volume addition amount of the cosolvent is 0.5-20% (preferably 7%) by volume of the buffer solution. Further, the enzyme source is prepared as follows: (1) slant culture: inoculating geotrichum candidum ZJPH1704 to a slant culture medium, and culturing for 1-2 days at 25-30 ℃ (preferably 30 ℃) to obtain slant strains; the final concentration composition of the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO4 1.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 15-20 g/L (preferably 20g/L) of agar powder, distilled water as a solvent and pH of 6.5;
(2) seed culture: inoculating slant strains into a seed culture medium, wherein the liquid loading amount is 100ml of the seed culture medium per 250ml of shake flask, and culturing at 25-30 ℃ and 150-250 rpm for 10-24 h (preferably culturing at 30 ℃ and 200rpm for 12h) to obtain a seed solution; the final concentration composition of the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O is 0.5g/L, the solvent is distilled water, and the pH value is 6.5;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount of 4-10% (preferably 10%) by volume concentration, wherein the liquid loading amount is 70mL/250mL shake flask, culturing at 25-30 ℃ and 150-250 rpm for 24-28 h (preferably at 30 ℃ and 200rpm for 24h), centrifuging the fermentation liquid after the fermentation is finished, washing the obtained precipitate with 0.1M, pH 6.5.5 buffer solution, and collecting wet thalli, namely an enzyme source; the final concentration of the fermentation medium is as follows: grapeGlucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO4 21.39g/L，CaCl21mmol/L, solvent distilled water, pH 6.5.
After the end of the conversion reaction, the conversion solution was extracted twice with ethyl acetate of equal volume, the extracts were combined and the solvent was evaporated off with a rotary evaporator. The resulting concentrate was dissolved in anhydrous methanol and the contents of the product and the residual substrate were determined by thin-plate chromatography (TLC) or High Performance Liquid Chromatography (HPLC).
And (3) thin plate chromatography detection: respectively sucking a certain amount of samples to be detected, an ethyl condensate substrate standard substance and an ethyl hydroxylate product standard substance by using a capillary tube, respectively spotting the samples on a pre-activated silica gel thin plate, developing at 254nm by using ethyl acetate/petroleum ether (3/7(v/v)) to observe whether a conversion product exists in the samples.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a method for preparing levonorgestrel by microorganism catalytic asymmetric reduction, which adopts geotrichum candidum ZJPH1704 strain to prepare the intermediate by catalytic reduction and has the advantages of high substrate concentration of catalytic reduction, good stereoselectivity and the like. The yield was 73% at a substrate concentration of 7.0g/L (22.4mM), and the e.e. value was 100%.
(IV) description of the drawings
FIG. 1 is a plate colony morphology of Geotrichum candidum ZJPH1704 according to example 1.
FIG. 2 is a scanning electron micrograph of Geotrichum candidum ZJPH1704, 500 times that of example 1.
FIG. 3 is a scanning electron micrograph of Geotrichum candidum ZJPH1704, example 1, at 10000 times.
FIG. 4 is a GC chromatogram of (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol (I-a), the product of example 1, containing an internal standard n-dodecane.
FIG. 5 is a GC chromatogram detection spectrum (containing internal standard n-dodecane) of 2, 6-dichloro-3-fluoroacetophenone (II) as a substrate in example 1.
FIG. 6 is a GC chromatography chromatogram (containing internal standard n-dodecane) of a mixture (1:1) of the racemate of example 1, i.e., (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol (I-a) and (R) -1- (2, 6-dichloro-3-fluorophenyl) ethanol (I-b).
FIG. 7 is a GC chromatogram detection spectrum (containing internal standard n-dodecane) of a sample (i.e., an extract) of a transformation of the Geotrichum candidum ZJPH1704 strain for the bioreduction of 2, 6-dichloro-3-fluoroacetophenone in example 1.
FIG. 8 is a HPLC chromatogram of a standard ethyl condensate (III) as a substrate in example 3.
FIG. 9 is a HPLC chromatogram of the product ethyl hydroxylate (IV) standard of example 3.
FIG. 10 is a HPLC chromatogram of a sample of the sample transformed with bioreductive ethyl condensate of Geotrichum candidum ZJPH1704 strain of example 3.
(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 ethanol used in the examples of the present invention is an ethanol reagent (available from national pharmaceutical group chemical reagent limited) with a volume content of 95%.
Example 1: screening of microbial strains for catalytic reduction of 2, 6-dichloro-3-fluorophenethyl alcohol
1. Strain screening
Enrichment culture: adding 1g fresh soil sample (soil sample collected in campus of Zhejiang university of industry (Hangzhou Zhejiang)) into a 250mL shake flask filled with 50mL enrichment medium, culturing at 30 deg.C and 200rpm for 5d, transferring 1mL culture solution into fresh enrichment medium, culturing for 5d, and repeating the enrichment for 2 times. The enrichment medium consists of: (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5g/L, the compound of formula (II) (2g/L) as the sole carbon source, distilled water as the solvent, pH 6.5.
Primary screening by a flat plate: diluting the enriched culture solution with normal saline 104-106After doubling, the cells were plated on a plate screening medium and cultured at 30 ℃ for 2 days. Single colonies were picked and streaked again and incubated at 30 ℃ for 2 days. After 2 times of streak isolation culture, a purified strain ZJPH1704 is obtained and preserved in a solid slant culture medium. The final concentration composition of the plate screening medium was: (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5g/L, agar powder 20g/L, distilled water as solvent, pH 6.5. The final concentration composition of the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO41.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 20g/L of agar powder and distilled water as a solvent, wherein the pH value is 6.5.
Culturing and collecting microbial cells: selecting and inoculating the purified seeds to a seed culture medium, culturing for 10-24 h at 30 ℃ and 200rpm, transferring the seeds to an initial fermentation culture medium according to the inoculation amount of 10% (v/v), and culturing for 24-48 h at 30 ℃ and 200 rpm. The fermentation broth was centrifuged at 9000rpm at 4 ℃ for 10min, the supernatant was discarded, and the cells were washed twice with a phosphate buffer solution having a pH of 6.5, and centrifuged again to obtain resting cells. The final concentration compositions of the seed culture medium and the initial fermentation culture medium are as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO41.5g/L，NaCl 0.75g/L，MgSO4·7H2O0.5g/L, solvent distilled water, pH 6.5.
Re-screening strains: the (S) -type product (I-a) is prepared by stereoselective reduction of 2, 6-dichloro-3-fluoro acetophenone (II) by taking a resting cell of a microorganism as an enzyme source catalytic substrate, and the reaction system is as follows: 2g of resting cells (wet weight basis) were resuspended in 10mL of phosphate buffer (0.1M, pH 6.5.5), 1g/L substrate and 100g/L glycerol were added as co-substrate, and the reaction was carried out at 40 ℃ and 200rpm for 24 h. After the reaction, the reaction solution was extracted with ethyl acetate, and the converted product was analyzed and detected by Gas Chromatography (GC), and the presence of the peak of the reduced product of formula (I-a) in the biocatalytic sample was observed in comparison with the reduced product of racemate (I-a). The strain ZJPH1704 can reduce the substrate into the compound shown in the formula (I-a). The chiral GC method is adopted to detect the enantiomeric excess value (e.e. value) of the reduction product of the strain, and the detection result shows that the e.e. value is more than 99.9 percent.
Qualitative and quantitative analysis by gas chromatography: detecting the content of the product and the residual substrate after the conversion reaction, and calculating the concentration, the Yield (Yield) and the e.e. value of the related substances.
C in formula (1)i、C0The molar concentration of the product at the end of the reaction and the molar concentration of the substrate at the beginning of the reaction, respectively.
The optical purity of the product is characterized by enantiomeric excess (e.e.).
In equation (2): cSAnd CRThe molar concentrations of (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol and (R) -1- (2, 6-dichloro-3-fluorophenyl) ethanol, respectively.
Conditions for gas chromatography: the concentrations of the product and the residual substrate in the reaction extract were analyzed by gas chromatography and quantified by the internal standard method. 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 120-170 ℃; temperature rise rate: 5 ℃/min; the detector is FID. The gas chromatogram of a conversion sample (extraction liquid) of the product (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol shown in the formula (I-a), the substrate 2, 6-dichloro-3-fluoro acetophenone shown in the formula (II), the mixture of the (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol shown in the formula (I-a) and the (R) -1- (2, 6-dichloro-3-fluorophenyl) ethanol shown in the formula (I-b), namely the racemate of the product, and the biological reduction of the 2, 6-dichloro-3-fluoro acetophenone by the strain ZJPH1704 are shown in figures 4-7.
2. Identification of strains
Morphological characteristics, physiological and biochemical characteristics and ITS sequences of the ZJPH1704 strain:
morphological observation is carried out on a nutrient agar plate, the characteristics of appearance morphology, texture, color, growth speed and the like of a bacterial colony of the strain ZJPH1704 are observed, and the cell morphology is observed by a scanning electron microscope on bacterial cells. Culturing at 30 deg.C for 36 hr, wherein the colony on the nutrient agar plate is in planar diffusion, grows rapidly, and is flat, milky white, short velvet or powder-like, has concentric circles and radial lines, and has central protrusion. Generates leucogen, fluff or powder during liquid culture. Coarse larger colonies, see fig. 1; the ZJPH1704 strain can be observed to be in a dispersed and straight rod shape under a scanning electron microscope, and the cells are shown in figures 2 and 3.
The carbon sources which can be utilized by the physiological and biochemical characteristics comprise: glucose, D-xylose and galactose, sucrose, maltose, raffinose, lactose, arabinose, cellobiose and melibiose cannot be used. Can tolerate 0.01 percent and 0.1 percent of cycloheximide and can not assimilate nitrate and urea.
Thirdly, the characteristic ITS sequence can quickly, accurately and simply identify the fungus by amplifying the gene segment of the fungus ribosome ITS (internal transformed spacer) by using PCR. The ITS region of ZJPH1704 strain rDNA is amplified by adopting universal primers ITS1 and ITS4 to generate a DNA fragment with the size of 349bp, and the PCR amplification product is subjected to sequence determination. The fungal ribosomal ITS (internal transformed spacer) gene sequence (SEQ ID NO.1) of the ZJPH1704 strain was determined as follows:
CCTGCGGAAGGATCATTAAGAATTATAAATATTTGTGAAATTTACACAGCAAACAATAATTTTATAGTCAAAACAAAAATAATCAAAACTTTTAACAATGGATCTCTTGGTTCTCGTATCGATGAAGAACGCAGCGAAACGCGATATTTCTTGTGAATTGCAGAAGTGAATCATCAGTTTTTGAACGCACATTGCACTTTGGGGTATCCCCCAAAGTATACTTGTTTGAGCGTTGTTTCTCTCTTGGAATTGCATTGCTTTTCTAAAATTTCGAATCAAATTCGTTTGAAAAACAACACTATTCAACCTCAGATCAAGTAGGATTACCCGCTGAACTTAAGCATATCAA
this sequence was submitted to GenBank (GenBank accession No. MG214158), and the ITS sequence of strain ZJPH1704 was compared for homology (BLAST) at the NCBI website (http:// www.ncbi.nlm.nih.gov), indicating that: the strain ZJPH1704 has high sequence homology with part of strains of Geotrichum sp, and has 100 percent of sequence homology with the strain Geotrichum candidum CBS:11628(GenBank accession No. KY103456.1). The strain ZJPH1704 is named Geotrichum candidum (Geotrichum candidum) ZJPH1704 and is deposited in the China center for type culture Collection with the deposit number: CCTCC NO: m2017380, deposit date: 26 th 6 th 2017, wherein the preservation address is Wuhan, Wuhan university, postcode 430072.
Example 2: catalyst preparation for the biological reduction of ethyl condensates
1) Slant culture: geotrichum candidum (Geotrichum candidum) ZJPH1704 is inoculated to a slant culture medium, cultured for 1-2 days at 30 ℃, and stored in a refrigerator at 4 ℃. The formula of the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO4 1.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 20g/L of agar and distilled water as a solvent, and the pH value is 6.5.
2) Seed culture: a ring of thalli is picked from a mature culture inclined plane and inoculated into a 250ml shake flask filled with 100ml of seed culture medium, and the thalli is cultured for 12 hours at 30 ℃ and 200rpm to obtain seed liquid. The seed culture medium formula comprises: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5g/L, solvent distilled water, pH 6.5.
3) Fermentation culture: transferring the seed solution into a 250ml shake flask filled with 70ml of fermentation medium by an inoculation amount with a volume concentration of 7%, carrying out shake culture at 30 ℃ and 200rpm for 24h, centrifuging the fermentation liquid after the fermentation is finished, washing the obtained precipitate with 0.1M, pH 6.5.5 phosphoric acid buffer solution, and collecting wet thalli. The final concentration of the fermentation medium is as follows: glucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO4 21.39g/L，CaCl21mmol/L, solvent distilled water, pH 6.5.
EXAMPLE 3 Geotrichum candidum ZJPH1704 Bioreduction for the preparation of Ethyl hydroxide
3g of wet Geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 6.5.5 phosphoric acid buffer solution, the final concentration of the wet cells was 300g/L in terms of wet weight, an ethyl condensate represented by the formula (III) was added to the wet cells to give a substrate, the wet cells were subjected to shaking culture at 30 ℃ and 200rpm for 72 hours, followed by detection by thin layer chromatography, after completion of the reaction, the reaction mixture was centrifuged, the supernatant was extracted twice with an equal volume of ethyl acetate, the extracts were combined, and the solvent was evaporated off by a rotary evaporator. The resulting concentrate was dissolved in anhydrous methanol and then subjected to HPLC, as shown in FIG. 10. The liquid chromatogram of the substrate ethyl condensate III (FIG. 8) and the product ethyl hydroxylate IV standard (FIG. 9) were examined simultaneously, and the calculated yield was 11%.
Thin layer chromatography detection: a certain amount of the sample of the bioreduction conversion, the ethyl condensate substrate III standard substance and the ethyl hydroxylate product IV standard substance are sucked by a capillary sample application tube and respectively applied to a pre-activated silica gel thin layer plate, and a developing agent adopts ethyl acetate/petroleum ether (3/7(v/v)) to develop color at 254 nm. The sample was observed for the presence of conversion products.
High performance liquid chromatography analysis: all samples were filtered through a 0.45 μm microporous membrane before being subjected to sample injection detection, and then the concentrations of the product and the residual substrate in the reaction solution were measured. Liquid chromatography conditions: an Agilent 1260 liquid chromatograph, an Agilent liquid chromatography workstation, and a chromatographic column of Luna 5u C18(2)100A (250X 4.6mm), flow rate: 1.0ml/min, sample size: 20 μ L, column temperature: room temperature, detection wavelength: 266nm, mobile phase: acetonitrile-water (v/v), gradient elution, elution conditions as shown in table 1.
The product yield was calculated as: yield ═ Ci/C0×100％
In the formula C0、CiRespectively, the molar concentration of the starting substrate of the reaction and the molar concentration of the product at the end of the reaction.
TABLE 1HPLC gradient elution conditions
Example 4 Effect of auxiliary substrate species on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of wet geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 6.5.5 phosphate buffer, the final concentration of the wet cells was 300g/L on a wet basis, an ethyl condensate was added to the cells to give a substrate (first dissolved in hot ethanol at 37 ℃ C., the final concentration of ethanol was 7% by volume, i.e., the amount of addition was 0.7ml), 100g/L of glucose, 100g/L of sucrose, 100g/L of maltose, 100g/L of lactose, 60g/L of glycerol, 10% by volume of methanol, 30% by volume of isopropanol, 10g/L of L-cysteine and 10g/L of L-alanine were added to give an auxiliary substrate, the mixture was subjected to shaking culture at 30 ℃ C., 200rpm for 72 hours, followed by thin layer chromatography, and centrifuged after the reaction, taking supernatant, adding equal volume of ethyl acetate for extraction twice, combining extracts, and evaporating the solvent by using a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 2.
TABLE 2 Effect of cosubstrate on Ethyl hydroxylate product yield
| Serial number | Cosubstrate and concentration | Product yield (%) |
| 1 | 100g/L glucose | 14.9 |
| 2 | 100g/L sucrose | 12.1 |
| 3 | Maltose 100g/L | 13.2 |
| 4 | 100g/L lactose | 13.8 |
| 5 | 60g/L glycerol | 48.0 |
| 6 | 10% methanol | 14.5 |
| 7 | 30% isopropyl alcohol | 5.8 |
| 8 | 10g/L L-cysteine | 13.5 |
| 9 | 10g/L L-alanine | 12.9 |
| 10 | Control (without additional co-substrate) | 20.5 |
And (4) conclusion: as can be seen from Table 2, the preferred co-substrate is glycerol, with a product yield of 48%.
Example 5 Effect of glucose and Glycerol Dual cosubstrates on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxylate
3g of Geotrichum candidum (Geotrichum candidum) ZJPH1704 wet cells obtained by the method of example 2 were suspended in 10ml of 0.1M, pH 6.5.5 phosphoric acid buffer solution, the final concentration of the wet cells was 300g/L in terms of wet weight, an ethyl condensate with a final concentration of 7.5g/L was added as a substrate (first dissolved in hot ethanol at 37 ℃ C., wherein the volume of ethanol added was 7%, i.e., the amount added was 0.7ml), glycerol with a final concentration of 60g/L and glucose with a final concentration of 100g/L were added as cosubstrates, shaking-cultured at 30 ℃ C., 200rpm for 72 hours, after the reaction was completed, the reaction solution was centrifuged, and the supernatant was taken, extracted twice with equal volume of ethyl acetate, the extracts were combined, and the solvent was distilled off with a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, with a yield of 15%. Thus, the conversion of glycerol and glucose as dual co-substrates is not suitable for this substrate.
Example 6 Effect of additional substrate addition on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of wet Geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 6.5.5 phosphate buffer, the final concentration of the wet cells was 300g/L on a wet basis, an ethyl condensate was added as a substrate (first dissolved in hot ethanol at 37 ℃ C., wherein the volume of ethanol added was 7%, that is, the amount added was 0.7ml), glycerol was added as an auxiliary substrate at 30g/L, 40g/L, 50g/L, 60g/L, 70g/L and 80g/L, the mixture was subjected to shaking culture at 30 ℃ C., 200rpm for 72 hours, after the reaction was completed, the reaction mixture was centrifuged, the supernatant was extracted twice with an equal volume of ethyl acetate, the extracts were combined, and the solvent was distilled off with a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 3.
TABLE 3 Effect of Glycerol addition as co-substrate on Ethyl hydroxylate product yield
| Serial number | Glycerol addition amount (g/L) | Product yield (%) |
| 1 | 30 | 10.0 |
| 2 | 40 | 25.0 |
| 3 | 50 | 33.0 |
| 4 | 60 | 48.0 |
| 5 | 70 | 43.0 |
| 6 | 80 | 39.0 |
And (4) conclusion: as can be seen from Table 3, the preferred glycerol co-substrate addition was 60g/L, with a product yield of 48%.
Example 7 Effect of transformation temperature on Geotrichum candidum ZJPH1704 bioreduction to Ethyl hydroxylate
3g of wet Geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 6.5.5 phosphate buffer, the final concentration of the wet cells was 300g/L on a wet basis, an ethyl condensate having a final concentration of 7.5g/L was added as a substrate (first dissolved in hot ethanol at 37 ℃ C., wherein the volume of ethanol added was 7%, that is, the amount added was 0.7ml), glycerol having a final concentration of 60g/L was added as an auxiliary substrate, the mixture was cultured with shaking at 30 ℃, 34 ℃, 37 ℃, 40 ℃ C., and 200rpm for 72 hours, after the reaction was completed, the reaction solution was centrifuged, the supernatant was taken, an equal volume of ethyl acetate was added and extracted twice, the extracts were combined, and the solvent was distilled off using a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 4.
TABLE 4 influence of temperature on Ethyl hydroxylate product yield
| Serial number | Conversion temperature (. degree.C.) | Product yield (%) |
| 1 | 30 | 48.0 |
| 2 | 34 | 33.0 |
| 3 | 37 | 29.0 |
| 4 | 40 | 18.0 |
And (4) conclusion: as can be seen from Table 4, the preferred conversion temperature is 30 ℃ under which the product yield is 48%.
Example 8 Effect of buffer pH on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of wet Geotrichum candidum ZJPH1704 thalli obtained by the method of example 2 are respectively suspended in purified water and 0.1M and 10ml of phosphoric acid buffer solution with the pH values of 5.8, 6.0, 6.5, 7.0, 7.5 and 8.0, the final concentration of the wet thalli is 300g/L in terms of wet weight, a 7.5/L ethyl condensation compound with the final concentration is added as a substrate (firstly, the wet thalli is dissolved by hot ethanol with the temperature of 37 ℃, wherein the volume addition amount of the ethanol is 7 percent, namely, the addition amount is 0.7ml), glycerol with the final concentration of 60g/L is added as an auxiliary substrate, the oscillating culture is carried out for 72h at the temperature of 30 ℃ and 200rpm, after the reaction is finished, the reaction solution is centrifuged, the supernatant is taken, the equal volume of ethyl acetate is added for extraction twice, the extraction. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 5.
TABLE 5 influence of buffer pH on Ethyl hydroxylate product yield
| Serial number | pH value of buffer solution | Product yield (%) |
| 1 | 5.8 | 10.0 |
| 2 | 6.0 | 15.0 |
| 3 | 6.5 | 50.0 |
| 4 | 7.0 | 60.0 |
| 5 | 7.5 | 45.0 |
| 6 | 8.0 | 40.0 |
| 7 | Water (W) | 5.0 |
And (4) conclusion: as can be seen from Table 5, the pH of the buffer is preferably 7.0, and the yield of the product is 60%.
Example 9 Effect of buffer Ionic Strength on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of Geotrichum candidum ZJPH1704 wet cells obtained by the method of example 2 were suspended in 10ml of a phosphate buffer solution having ionic strengths of 0.05M, 0.1M, 0.15M, 0.2M and pH7.0, the wet cells were suspended at a final concentration of 300g/L in terms of wet weight, an ethyl condensate having a final concentration of 7.5g/L was added as a substrate (first dissolved in hot ethanol at 37 ℃ C., wherein the volume of ethanol added is 7%, i.e., the amount added is 0.7ml), glycerol having a final concentration of 60g/L was added as an auxiliary substrate, shaking-cultured at 30 ℃ C., 200rpm for 72 hours, after the reaction was completed, the reaction solution was centrifuged, and the supernatant was taken, extracted twice with an equal volume of ethyl acetate, and the extracts were combined and the solvent was distilled off with a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 6.
TABLE 6 influence of buffer ionic strength on Ethyl hydroxylate product yield
| Serial number | Ionic strength of buffer (M) | Product yield (%) |
| 1 | 0.05 | 50.0 |
| 2 | 0.1 | 61.0 |
| 3 | 0.15 | 57.0 |
| 4 | 0.2 | 52.0 |
And (4) conclusion: as can be seen from Table 6, the preferred buffer ionic strength is 0.1M, under which the yield of the product is 61%.
Example 10 Effect of cell concentration on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of wet geotrichum candidum ZJPH1704 thalli obtained by the method of example 2 are suspended in 10ml of 0.1M, pH 7.0.0 phosphate buffer solution, the wet thalli are respectively 200g/L, 300g/L, 400g/L and 500g/L in wet weight, ethyl condensation products with the final concentration of 7.5g/L are added as substrates (firstly, hot ethanol with the temperature of 37 ℃ is used for dissolving, the volume addition amount of the ethanol is 7 percent, namely, the addition amount is 0.7ml), glycerol with the final concentration of 60g/L is added as an auxiliary substrate, the mixture is subjected to shaking culture at the temperature of 30 ℃ and the speed of 200rpm for 72 hours, the reaction solution is centrifuged after the reaction is finished, supernate is taken, equal volume of ethyl acetate is added for extraction twice, the extraction liquid is combined, and the solvent is removed by a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 7.
TABLE 7 influence of cell concentration on the yield of the product obtained from the production of ethyl hydroxide
| Serial number | Cell concentration (g/L) | Product yield (%) |
| 1 | 200 | 45.0 |
| 2 | 300 | 62.0 |
| 3 | 400 | 53.0 |
| 4 | 500 | 39.0 |
And (4) conclusion: as is clear from Table 7, the preferable cell concentration was 300g/L, and the yield under these conditions was 69%.
EXAMPLE 11 Effect of substrate concentration on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl hydroxide
3g of wet Geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 7.0.0 phosphate buffer, the wet cells were taken up to a final concentration of 300g/L by wet weight, ethyl condensates at final concentrations of 4g/L, 4.5g/L, 5.0g/L, 5.5g/L, 6.0g/L, 6.5g/L, 7.0g/L and 7.5g/L were added as substrates (first dissolved in hot ethanol at 37 ℃ C., wherein the volume of ethanol added was 7%, i.e., the amount added was 0.7ml), glycerol at a final concentration of 60g/L was added as an auxiliary substrate, the mixture was subjected to shaking culture at 30 ℃ C. and 200rpm for 72 hours, after the reaction was completed, the reaction solution was centrifuged, the supernatant was taken up, ethyl acetate equivalent to volume was added and extracted twice, the extracts were combined, and the solvent was distilled off with a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 8.
TABLE 8 influence of substrate concentration on Ethyl hydroxylate product yield
| Serial number | Substrate concentration (g/L) | Product yield (%) |
| 1 | 4.0 | 98.0 |
| 2 | 4.5 | 95.0 |
| 3 | 5.0 | 90.0 |
| 4 | 5.5 | 80.0 |
| 5 | 6.0 | 75.0 |
| 6 | 6.5 | 74.0 |
| 7 | 7.0 | 73.0 |
| 8 | 7.5 | 60.0 |
And (4) conclusion: as can be seen from Table 8, the preferred substrate concentration is 7.0g/L, under which conditions the product yield is 73%.
Example 12 Effect of transformation time on Geotrichum candidum ZJPH1704 bioreduction to Ethyl hydroxylate
3g of wet geotrichum candidum ZJPH1704 cells obtained by the method of example 2 are suspended in 10ml of 0.1M, pH 7.0.0 phosphate buffer solution, the wet cells are dissolved by hot ethanol at 37 ℃ with the volume of 7 percent of ethanol added (the added amount is 0.7ml) and added with ethyl condensation compound with the final concentration of 7.0g/L as a substrate, glycerol with the final concentration of 60g/L is added as an auxiliary substrate, the mixture is subjected to shaking culture at 30 ℃ and 200rpm for 12h, 22h, 32h, 42h, 52h, 62h and 72h respectively, after the reaction is finished, the reaction solution is centrifuged, the supernatant is taken, equal volume of ethyl acetate is added for extraction twice, the extracts are combined, and the solvent is removed by a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 9.
TABLE 9 Effect of conversion time on Ethyl hydroxylate product yield
| Serial number | Conversion time (h) | Product yield (%) |
| 1 | 12 | 20.0 |
| 2 | 22 | 40.0 |
| 3 | 32 | 50.0 |
| 4 | 42 | 60.0 |
| 5 | 52 | 65.0 |
| 6 | 62 | 68.0 |
| 7 | 72 | 73.0 |
And (4) conclusion: as can be seen from Table 9, the preferred conversion time is 72 hours, under which the product yield is 73%.
EXAMPLE 13 Effect of Co-solvent species on the Bioreduction of Geotrichum candidum ZJPH1704 to Ethyl Hydroxide
3g of wet geotrichum candidum ZJPH1704 thalli obtained by the method of example 2 are suspended in 10ml of 0.1M, pH 7.0.0 phosphate buffer solution, the final concentration of the wet thalli is 300g/L in terms of wet weight, an ethyl condensation compound with the final concentration of 7.0g/L is added as a substrate, hot ethanol with the temperature of 37 ℃ and DMSO (dimethyl sulfoxide) are respectively added as cosolvents for dissolving the substrate, wherein the volume addition amount of the ethanol is 7 percent (namely the addition amount is 0.7ml), the volume addition amount of the DMSO is 20 percent (namely the addition amount is 2ml), glycerol with the final concentration of 60g/L is added as an auxiliary substrate, the mixture is subjected to shaking culture at the temperature of 30 ℃ and 200rpm for 72 hours, after the reaction is finished, the reaction solution is centrifuged, a supernatant is taken, equal volume of ethyl acetate is added for extraction twice, the extraction. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 10.
TABLE 10 influence of cosolvent type on Ethyl hydroxylate product yield
| Serial number | Kind and amount of cosolvent | Product yield (%) |
| 1 | 7% Hot ethanol | 73.0 |
| 2 | 20％DMSO | 40.0 |
| 3 | Control (without cosolvent) | 25.0 |
And (4) conclusion: as can be seen from Table 10, ethanol is the preferred co-solvent, and the yield of the product is 73% under these conditions.
EXAMPLE 14 Effect of Co-solvent ethanol addition on Geotrichum candidum ZJPH1704 bioreduction for Ethyl hydroxylate production
3g of wet Geotrichum candidum ZJPH1704 cells obtained in example 2 were suspended in 10ml of 0.1M, pH 7.0.0 phosphate buffer solution to give a final concentration of 300g/L on a wet basis, and an ethyl condensate was added thereto to give a substrate having a final concentration of 7.0g/L, and the substrate was dissolved in hot ethanol at 37 ℃ in an amount of 0%, 3% (i.e., 0.3ml), 5% (i.e., 0.5ml), 7% (i.e., 0.7ml), 8% (i.e., 0.8ml), 9% (i.e., 0.9ml), 10% (i.e., 1ml) and 11% (i.e., 1.1ml), respectively. Adding glycerol with final concentration of 60g/L as auxiliary substrate, performing shake culture at 30 deg.C and 200rpm for 72h, centrifuging the reaction solution after reaction, collecting supernatant, adding equal volume of ethyl acetate, extracting twice, mixing the extractive solutions, and removing solvent by rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then examined, as shown in example 3, and the results are shown in Table 11.
TABLE 11 Effect of Co-solvent addition on Ethyl hydroxylate product yield
| Serial number | Amount of ethanol added (%) | Product yield (%) |
| 1 | 0 | 25.0 |
| 2 | 3 | 32.0 |
| 3 | 5 | 43.0 |
| 4 | 7 | 73.0 |
| 5 | 8 | 38.0 |
| 6 | 9 | 31.0 |
| 7 | 10 | 11.0 |
| 8 | 11 | 8.0 |
And (4) conclusion: as can be seen from Table 11, the preferred amount of ethanol added was 7% by volume final concentration, and the yield of the product was 73%.
Example 15 examination of Rhizopus microsporum var. rhizomorpha ZJPH1308 for bioconversion activity of Ethyl condensate
(1) Rhizopus microsporidianus variant ZJPH1308, deposited in the chinese typical culture collection at the address of china, wuhan university, zip code 430072, with the preservation number of CCTCC No. m 2014645, with the preservation date 2014, 12 and 14, which has been disclosed in a previous patent application (application No. 201510289729.X, 2015, 6 and 1).
(2) Slant culture: inoculating rhizopus microsporus fibrous variant ZJPH1308 to a slant culture medium, and culturing for 4-5 days at 30 ℃ to obtain slant thalli; the final concentration of the slant culture medium is as follows: 200g/L of potato, 20g/L of glucose and 20g/L of agar, wherein the solvent is distilled water and the pH value is natural;
(3) seed culture: selecting a ring of thalli from the inclined plane thalli, inoculating the selected ring of thalli into a seed culture medium, and culturing at 30 ℃ and 200rpm for 22h to obtain a seed solution; the final concentration of the seed culture medium is as follows: 25g/L glucose, 30g/L peptone and KH2PO4 1g/L，MgSO4·7H2O1.3g/L, pH 6.0, and the solvent is distilled water.
(4) Fermentation culture: inoculating the seed solution into a fermentation culture medium by an inoculation amount with the volume concentration of 6%, fermenting and culturing for 24 hours at 30 ℃ and 200rpm, taking out thalli, washing with distilled water for three times, and collecting wet thalli; the final concentration of the fermentation medium is as follows: dextrin 20g/L, peptone 27.5g/L, (NH)4)2SO4 3g/L，KH2PO4 1g/L，MgSO4·7H2O1.3g/L, pH 6.0, solvent is distilled water.
(5) Biological reduction of ethyl condensate:
suspending 3g of wet thallus obtained by fermentation culture of Rhizopus microsporus hairy varieties ZJPH1308 in 10ml of 0.1M phosphate buffer solution (pH 6.5), wherein the final concentration of the wet thallus is 300g/L in terms of wet weight, adding an ethyl condensation compound with the final concentration of 7.0g/L as a substrate (firstly dissolving the wet thallus in hot ethanol at 37 ℃, wherein the volume addition of the ethanol is 7%), adding 60g/L of glycerol as an auxiliary substrate, performing shaking culture at 30 ℃, 200rpm for 72h, centrifuging the reaction solution after the reaction is finished, taking supernatant, adding equal volume of ethyl acetate for extraction twice, combining the extracts, and evaporating the solvent by using a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and then subjected to HPLC detection, as shown in example 3, to obtain the product ethyl hydroxide in a yield of 7.8%.
Example 16 examination of the bioconversion Activity of Ethyl condensate by Pseudomonas putida ZJPH1606
(1) Pseudomonas putida (Pseudomonas putida) ZJPH1606, deposited at the China center for type culture Collection, address: china, wuhan university, zip code: 430072; the preservation number is: CCTCC NO: m2017110, date of deposit: year 2017, month 3 and day 9. This strain has been disclosed in a previous patent application (application No. 201710344320.2, application date 2017, 05 and 16).
(2) Slant culture: inoculating pseudomonas putida ZJPH1606 to a slant culture medium, and culturing at 30 ℃ for 1-2 days to obtain slant thalli; the final concentration composition of the slant culture medium is as follows: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 1g/L，NaCl 0.5g/L，MgSO4·7H20.5g/L of O, 20g/L of agar, pH 6.5 and distilled water as a solvent.
(3) Seed culture: selecting a ring of thalli from the inclined plane thalli, inoculating the selected ring of thalli into a seed culture medium, and culturing at 30 ℃ and 200rpm for 24 hours to obtain a seed solution; the final concentration of the seed culture medium is as follows: 10g/L glucose, 5g/L peptone, 4g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 0.5g/L，MgSO4·7H2O0.5g/L, pH 6.5, and the solvent is distilled water.
(4) Fermentation culture: inoculating the seed solution into a fermentation culture medium by an inoculation amount with the volume concentration of 8%, fermenting and culturing for 36h at 30 ℃ and 200rpm, taking out thalli, washing with distilled water for three times, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 38.5g/L glucose, 32.7g/L beef extract and MgSO4·7H2O1.1 g/L, pH 6.0, and distilled water as solvent
(5) Biological reduction of ethyl condensate:
suspending 3g of wet thallus obtained by fermentation culture of pseudomonas putida ZJPH1606 in 10ml of 0.1M phosphate buffer solution (pH 6.5), wherein the final concentration of the wet thallus is 300g/L in terms of wet weight, adding an ethyl condensation compound with the final concentration of 7.0g/L as a substrate (firstly dissolving the wet thallus by hot ethanol with the temperature of 37 ℃, wherein the volume addition of the ethanol is 7%), adding glycerol with the final concentration of 60g/L as an auxiliary substrate, performing shaking culture at the temperature of 30 ℃, 200rpm for 72 hours, centrifuging the reaction solution after the reaction is finished, taking supernate, adding equal volume of ethyl acetate for extraction twice, combining the extraction solutions, and evaporating the solvent by using a rotary evaporator. The concentrated solution was dissolved in anhydrous methanol and subjected to HPLC detection in example 3, and no ethyl hydroxide product was detected.
And (4) conclusion: pseudomonas putida ZJPH1606 was not able to convert ethyl condensate to produce ethyl hydroxylates.
Sequence listing
<110> Zhejiang industrial university
<120> biological preparation method of key chiral intermediate of levonorgestrel
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 349
<212> DNA
<213> Geotrichum candidum (Geotrichum)
<400> 1
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tttatagtca aaacaaaaat aatcaaaact tttaacaatg gatctcttgg ttctcgtatc 120
gatgaagaac gcagcgaaac gcgatatttc ttgtgaattg cagaagtgaa tcatcagttt 180
ttgaacgcac attgcacttt ggggtatccc ccaaagtata cttgtttgag cgttgtttct 240
ctcttggaat tgcattgctt ttctaaaatt tcgaatcaaa ttcgtttgaa aaacaacact 300
attcaacctc agatcaagta ggattacccg ctgaacttaa gcatatcaa 349
Claims (5)
1. A biological preparation method of a key chiral intermediate of levonorgestrel is characterized in that wet thalli obtained by fermentation culture of Geotrichum candidum ZJPH1704 is used as an enzyme source, an ethyl condensation compound shown in a formula (III) is used as a substrate, an auxiliary substrate and a cosolvent are added, a phosphate buffer solution with the pH of 5.8-8.0 is used as a reaction medium to form a reaction system, the reaction is carried out at the temperature of 30-40 ℃ and the speed of 150-250 rpm, and after the reaction is finished, the reaction solution is separated and purified to obtain the key chiral intermediate of levonorgestrel shown in the formula (IV); the Geotrichum candidum (Geotrichum candidum) ZJPH1704 is preserved in the China center for type culture Collection with the preservation number: CCTCC NO: m2017380, deposit date: 26 months 6 and 2017, wherein the preservation address is Wuhan, Wuhan university, postcode 430072; the co-substrate is one of the following: glucose, maltose, sucrose, lactose, methanol, isopropanol, glycerol, L-alanine or L-cysteine; the cosolvent is one of the following: (1) preheating ethanol at 37 ℃; (2) dimethyl sulfoxide;
2. the method for biologically preparing the key chiral intermediate of levonorgestrel according to claim 1, wherein the dosage of the substrate is 4-7.5 g/L based on the volume of the buffer solution, and the dosage of the wet bacterial cells is 200-500 g/L based on the volume of the buffer solution.
3. The method for biologically preparing the key chiral intermediate of levonorgestrel according to claim 1, wherein the auxiliary substrate is glucose, maltose, sucrose, lactose, glycerol, L-alanine or L-cysteine, the dosage is 10-300 g/L based on the volume of the buffer solution, and the dosage is 10-50% based on the volume of the buffer solution when the auxiliary substrate is methanol or isopropanol.
4. The method for biologically preparing the levonorgestrel key chiral intermediate as claimed in claim 1, wherein the volume of the cosolvent added is 0.5-20% by volume of the buffer solution.
5. The process for the biological preparation of a key chiral intermediate of levonorgestrel according to claim 1, wherein the enzyme source is prepared by the following method: (1) slant culture: inoculating geotrichum candidum ZJPH1704 to a slant culture medium, and culturing for 1-2 days at 25-30 ℃ to obtain slant strains; the final concentration composition of the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO4 1.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 15-20 g/L of agar powder, distilled water as a solvent and pH of 6.5;
(2) seed culture: inoculating slant strains into a seed culture medium, and culturing at 25-30 ℃ and 150-250 rpm for 10-24 hours to obtain a seed solution; the final concentration composition of the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O is 0.5g/L, the solvent is distilled water, and the pH value is 6.5;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 4-10%, shaking the flask with the liquid loading amount of 70mL/250mL, culturing at 25-30 ℃ and 150-250 rpm for 24-28 h, centrifuging the fermentation liquid after the fermentation is finished, washing the obtained precipitate with 0.1M, pH 6.5.5 buffer solution, and collecting wet thalli, namely an enzyme source; the final concentration of the fermentation medium is as follows: glucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO4 21.39g/L，CaCl21mmol/L, solvent distilled water, pH 6.5.
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| CN1258735A (en) * | 1999-12-24 | 2000-07-05 | 中国科学院上海有机化学研究所 | Geotrichosis candium strain and its culture process and application |
| CN102146421A (en) * | 2010-02-05 | 2011-08-10 | 武汉泰祥通生物技术有限公司 | Novel fermentation reducing method for contraceptive midbody |
| CN105906656A (en) * | 2016-05-17 | 2016-08-31 | 凯莱英医药集团（天津）股份有限公司 | Synthetic method of crizotinib intermediate |
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| CN1258735A (en) * | 1999-12-24 | 2000-07-05 | 中国科学院上海有机化学研究所 | Geotrichosis candium strain and its culture process and application |
| CN102146421A (en) * | 2010-02-05 | 2011-08-10 | 武汉泰祥通生物技术有限公司 | Novel fermentation reducing method for contraceptive midbody |
| CN105906656A (en) * | 2016-05-17 | 2016-08-31 | 凯莱英医药集团（天津）股份有限公司 | Synthetic method of crizotinib intermediate |
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