CN112063532B - Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol - Google Patents

Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol Download PDF

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CN112063532B
CN112063532B CN202010801547.7A CN202010801547A CN112063532B CN 112063532 B CN112063532 B CN 112063532B CN 202010801547 A CN202010801547 A CN 202010801547A CN 112063532 B CN112063532 B CN 112063532B
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王普
熊李炉
林洪鑫
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Abstract

The invention discloses a geotrichum forestry and application thereof in preparing (S) -1- (2-trifluoromethylphenyl) ethanol, the strain has the advantages of high concentration of catalytic substrates, good stereoselectivity, high optical purity of products and the like, and expensive coenzyme is not required to be added in the conversion process, so that the cost advantage is achieved. The invention adopts the chiral biological catalysis which adopts the geotrichum linn ZJPH1811 cells as the catalyst, and adds a novel natural eutectic solvent, namely choline acetate: the buffer solution with cysteine as a cosolvent is a reaction medium system, and when the concentration of a substrate is 20mmol/L, the e.e. value of the target product (S) -1- (2-trifluoromethylphenyl) ethanol is 99.2 percent, and the yield reaches 78.9 percent.

Description

Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol
(I) technical field
The invention relates to a new microbial strain Geotrichum silvicola ZJPH1811 and application thereof in catalyzing asymmetric reduction of 2' -trifluoromethylacetophenone in a medium system taking a natural eutectic solvent as a cosolvent to prepare (S) -1- (2-trifluoromethylphenyl) ethanol.
(II) background of the invention
The chemical structural formula of the (S) -1- (2-trifluoromethylphenyl) ethanol is as follows:
Figure BDA0002627569050000011
(S) -1- (2-trifluoromethylphenyl) ethanol is an important chiral intermediate for synthesizing a NEK2 target inhibitor and a clinical medicine GSK461364 for treating malignant tumor cell proliferation. 5- [6- [ (4-methyl-1-piperazinyl) methyl group]-1H-benzimidazol-1-yl]-3- [ (1R) -1- [2- (trifluoromethyl) phenyl]Ethoxy radical]-2-thiophenecarboxamide (GSK461364, molecular formula: C)27H28F3N5O2S) is a Plk1 kinase inhibitor, can regulate cell mitosis and treat malignant proliferation of tumor cells, and has better selectivity, tolerance and clinical application advantages compared with the traditional tubulin inhibitor anticancer drugs. The synthesis method of (S) -1- (2-trifluoromethylphenyl) ethanol mainly comprises a chemical method and a biological method. When the chemical method is adopted to prepare the (S) -1- (2-trifluoromethylphenyl) ethanol, chemical catalysts are needed, and the catalysts contain toxic substances which are not easy to degrade and cause pollution to the environment. Cezmi and the like adopt a metal ruthenium catalyst to react for 30min at the high temperature of 82 ℃, and can convert 100 mmol/L2' -trifluoromethyl acetophenone substrate into (S) -1- (2-trifluoromethyl phenyl) ethanol, the conversion rate is 98 percent, and the e.e. value is 87 percent. Nakamura et al added coenzyme NAD using crude enzyme solution from Geotrichum candidum as catalyst, 2' -trifluoromethylacetophenone of 26.7mmol/L as substrate +And isopropanol, and converting for 20h at 30 ℃ to obtain the product of (S) -1- (2-trifluoromethylphenyl) ethanol with the yield and the e.e. value of 13 percent and 97 percent respectively.
Disclosure of the invention
The invention aims to provide a new microbial strain Geotrichum silvicola ZJPH1811 and application of the strain in catalyzing asymmetric reduction of 2' -trifluoromethylacetophenone to prepare (S) -1- (2-trifluoromethylphenyl) ethanol. The invention provides a novel method for preparing (S) -1- (2-trifluoromethyl phenyl) ethanol by bioconversion of a strain ZJPH1811 of the Geotrichum linnei, and compared with a chemical method, the route has the characteristics of high stereoselectivity, low catalyst preparation cost, environment-friendly conversion process and the like. Compared with the reported enzyme method preparation, the method for preparing (S) -1- (2-trifluoromethylphenyl) ethanol by taking the whole cells of the Geotrichum linum as the catalyst has higher catalytic yield and product e.e. value, obtains better catalytic effect and does not need to add expensive coenzyme. In addition, the mass transfer in the reaction process can be effectively improved by constructing a novel reaction medium system containing the eutectic solvent, the permeability of the cell membrane of the thallus is improved, and the yield of the product is further improved.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-Geotrichum silvicola ZJPH1811, which is preserved in China center for type culture Collection with the preservation date of 2020, 7 and 6 days, and the preservation number is as follows: CCTCC NO: m2020281, deposit address: china, wuhan university, zip code 430072.
In addition, the invention also provides an application of the Geotrichum linum ZJPH1811 in catalyzing asymmetric reduction of 2' -trifluoromethylacetophenone to prepare (S) -1- (2-trifluoromethylphenyl) ethanol, wherein the application comprises the following steps: taking wet thalli obtained by fermenting and culturing Geotrichum linum ZJPH1811 as an enzyme source, taking 2' -trifluoromethylacetophenone as a substrate, adding an auxiliary substrate, forming a conversion system in a phosphate buffer solution with the pH of 6.0-8.0, carrying out reaction under the conditions of 25-40 ℃, 100 and 200rpm (preferably 30-40 ℃ and 200rpm), obtaining a conversion solution containing a product (S) -1- (2-trifluoromethylphenyl) ethanol after the reaction is finished, and separating and purifying the conversion solution to obtain a (S) -1- (2-trifluoromethylphenyl) ethanol product; the auxiliary substrate is one of glucose, isopropanol, glycerol or ethanol.
Figure BDA0002627569050000021
Further, the dosage of the wet bacteria is 100-600g/L (preferably 160-500g/L) by volume of the buffer solution, and the dosage of the substrate is 5-50mmol/L (preferably 10-30mmol/L) by volume of the buffer solution; when the cosubstrate is glucose or glycerol, the addition amount is 10 to 250g/L (preferably 20 to 200g/L, most preferably 100g/L) based on the volume of the buffer solution, and when the cosubstrate is isopropanol or ethanol, the addition amount is 5 to 30% (preferably 10%) based on the volume of the buffer solution.
Further, the transformation system also contains a natural eutectic solvent as a cosolvent, wherein the natural eutectic solvent consists of a hydrogen bond acceptor and a hydrogen bond donor, and comprises the following components: choline chloride: urea, betaine: isopropanol, betaine: cysteine, betaine: trehalose, choline chloride: glutamic acid, choline acetate: cysteine and the like, wherein the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the natural eutectic solvent is 1: 1; the mass addition amount of the natural eutectic solvent is 2-20g/L, preferably 10g/L in terms of the volume of the buffer solution.
The enzyme source is obtained by the following method:
1) plate culture: selecting a single colony of Geotrichum linnei ZJPH1811, inoculating the single colony into a plate culture medium, performing activation culture at 30 ℃ for 2d, selecting the colony from the first activated plate according to the same steps, performing activation culture again, and storing the obtained plate in a refrigerator at 4 ℃; the final concentration composition of the plate 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，MgSO4·7H20.5g/L of O, 20g/L of agar, water as a solvent and pH of 6.5;
2) seed culture: selecting a ring of thalli from a mature culture plate, inoculating the ring of thalli into a seed culture medium, and culturing at 30 ℃ and 200rpm for 12h to obtain a seed solution; the final concentration 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 water, and the pH value is 6.5;
3) fermentation culture: inoculating the seed solution into fermentation culture medium at volume concentration of 8%, culturing at 30 deg.C and 200rpm for 24 hr to obtain fermentation broth, centrifuging the fermentation broth, and collecting precipitate with K of 0.1M and pH 7.02HPO4-KH2PO4Washing with buffer solution, centrifuging to obtain wet thallus, namely enzyme source; the final concentration of the fermentation medium is as follows: glucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO421.39g/L of anhydrous CaCl21mM, solvent water, pH 6.5.
The analytical detection method of the transformation liquid comprises the following steps: after the reaction is finished, the conversion solution is stopped reacting by using ethyl acetate with the same volume and is extracted for 30min, the concentration of the product and the unreacted residual substrate in the reaction extraction liquid is analyzed and detected by adopting gas chromatography, and the yield and the enantiomeric excess value (e.e. value) of the target product are calculated.
According to the invention, the auxiliary substrate is preferably glycerol, and the natural eutectic solvent is preferably selected from the following classes: cysteine, under the condition, when the concentration of the substrate is 20mmol/L, the e.e. value and the yield of the obtained product are high.
The invention obtains a new microbial strain which is different from the previous research report and can catalyze the reduction of 2' -trifluoromethyl acetophenone substrate into (S) -1- (2-trifluoromethyl phenyl) ethanol with high selectivity from a soil sample, and the substrate concentration and the product yield of catalytic conversion are higher than the levels reported in the literature. The invention also applies the novel natural eutectic solvent as a cosolvent to the biotransformation reaction, thereby providing beneficial references for preparing the chiral intermediate (S) -1- (2-trifluoromethylphenyl) ethanol by a microbial catalysis method and the application of the natural eutectic solvent in the field of biocatalysis.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a new microbial strain for catalyzing asymmetric reduction of 2' -trifluoromethylacetophenone to prepare (S) -1- (2-trifluoromethylphenyl) ethanol, and the (S) -1- (2-trifluoromethylphenyl) ethanol prepared by the strain has the advantages of high substrate concentration, good stereoselectivity, high optical purity of products and the like. Compared with the existing reported level, the method obtains a product e.e. value (the 99.2% vs. literature value is 87%) higher than that of the product prepared by a chemical method, and has the characteristics of high stereoselectivity, low catalyst preparation cost, environment-friendly conversion process and the like. Higher e.e. values of the product (99.2% vs. 97% of the literature value) and catalytic yields were also obtained than in the enzymatic preparation reported in the literature. In addition, expensive coenzyme is not required to be added in the conversion process, and the method has the advantage of cost. The invention adopts Geotrichum silvicola ZJPH1811 cells as chiral biological catalysis of a catalyst, and adds a novel natural eutectic solvent, namely choline acetate: the buffer solution with cysteine as a cosolvent is a reaction medium system, and when the concentration of a substrate is 20mmol/L, the e.e. value of the target product (S) -1- (2-trifluoromethylphenyl) ethanol is 99.2 percent, and the yield reaches 78.9 percent.
(IV) description of the drawings
FIG. 1 is a gas phase detection chromatogram (containing internal standard dodecane) of a substrate 2' -trifluoromethylacetophenone and a product 1- (2-trifluoromethylphenyl) ethanol standard.
FIG. 2 is a gas chromatography chromatogram of the bioreduction reaction extract of the strain ZJPH1811 of Geotrichum linnei (containing internal standard dodecane).
(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:
in the embodiment of the invention, the adding amounts of the substrate, the auxiliary substrate and the natural eutectic solvent are measured by the volume of the buffer solution.
Example 1: strain screening and identification
1. The strain source is as follows: the Geotrichum silvicola ZJPH1811 is obtained by separating and screening a soil sample from an orchard of Xinyang in Henan province, and the specific screening method comprises the following steps:
weighing 1g of soil sample, adding the soil sample into 50mL of physiological saline, shaking for 1h in a shaking table at 30 ℃ and 200rpm, adding 1mL of soil sample suspension into a 250mL shaking bottle filled with 50mL of enrichment medium, culturing for 5-6d at 30 ℃ and 200rpm, taking 1mL of enrichment medium after the culture solution becomes turbid, transferring the enrichment medium into a 250mL shaking bottle filled with 50mL of enrichment medium to culture for 5-6d, and repeating the enrichment culture for 2 times. 2' -trifluoromethyl acetophenone is added into the enrichment culture medium as the only carbon source.
The final concentration composition of the enrichment medium is as follows: 2.5g/L (NH) of 2' -trifluoromethylacetophenone4)2SO4 2.0g/L，KH2PO42.0g/L，NaCl 1.0g/L，MgSO4·7H2O0.5 g/L, solvent is water, pH 6.5.
Taking the final enrichment cultureAdding sterile water, and diluting to 10 deg.C-2、10-3、10-4And coating 0.2mL of diluent on a plate culture medium with a corresponding number, performing static culture at 30 ℃ for 2-3d, selecting single colonies with different forms from the plate, transferring the single colonies to the plate culture medium with the number, performing static culture at 30 ℃ for 2-3d, taking out the plate, and storing the plate in a refrigerator at 4 ℃ for later use. The plate culture medium consists of enrichment medium added with 20g/L agar.
And (3) selecting a single colony growing on a plate culture medium, inoculating the single colony to a seed culture medium, culturing at 30 ℃ and 200rpm for 12h, transferring the cultured seed solution into a fermentation culture medium by the inoculation amount with the volume concentration of 8%, culturing at 30 ℃ and 200rpm for 24h, and centrifuging the fermentation liquor to obtain wet thalli. The seed culture medium and the fermentation culture medium have the same composition, and the specific composition is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H2O0.5 g/L, water as solvent, pH 6.5.
In 0.1M phosphate buffer solution with pH 7.0, 6.7 mmol/L2' -trifluoromethyl acetophenone (calculated by the volume of the buffer solution) is taken as a substrate, 100g/L glucose is taken as an auxiliary substrate, and 100g/L microbial cells (namely wet bacteria after fermentation culture) are taken as a catalyst, and the biotransformation is carried out for 24h at 30 ℃. The conversion solution is quenched by equal volume of ethyl acetate and extracted for 30min, supernatant is taken after centrifugation, and the yield and the enantiomeric excess value (e.e. value) of the target product (S) -1- (2-trifluoromethylphenyl) ethanol in the conversion solution are determined by chiral gas chromatography. The gas chromatogram of the substrate 2' -trifluoromethylacetophenone and the product 1- (2-trifluoromethylphenyl) ethanol standard (containing internal standard dodecane) is shown in FIG. 1. The microbial strain ZJPH1811 with high enantiomeric excess value (e.e. value is more than 99%) and higher yield is obtained by screening, and the gas chromatogram of the biological reduction reaction extract liquid (containing internal standard dodecane) of the ZJPH1811 strain is shown in figure 2.
Gas chromatography conditions: agilent 7820A gas chromatograph, Varian CP-Chirasil-Dex CB capillary gas chromatography column (25 m. times.0.25 mm. times.0.25 μm). The FID detector, the injection port temperature and the detector temperature are both 250 ℃, and the temperature rise program is as follows: 100 deg.CKeeping for 3min, raising the temperature to 150 ℃ at 8 ℃/min, keeping for 0min, and sampling amount of 1uL, N2Is used as carrier gas, the flow rate is 2ml/min, and the split ratio is 15: 1.
And (5) calculating the yield and the e.e. value of the product in the conversion solution according to a gas chromatography detection spectrogram.
The yield calculation method comprises the following steps:
preparation method of standard curve: preparing substrate solution and product standard solution with concentration of 5, 10, 15, 20, 25mmol/L with ethyl acetate, and detecting with gas chromatography. And integrating the obtained chromatogram to obtain a peak area, drawing by taking the ratio of the peak area of the substrate or the product to the peak area of the dodecane as a horizontal ordinate, and taking the ratio of the concentration of the substrate or the product to the concentration of the dodecane as a vertical ordinate, performing linear regression fitting, and drawing a standard curve. Obtaining a substrate standard curve equation of y being 1.501x +0.0086 and R20.9998; product standard curve equation, y is 1.4557x-0.0005, R2＝0.9988。
The product yield calculation formula is as follows:
yield ═ Cp/C0
In the formula CpIs the (S) -1- (2-trifluoromethylphenyl) ethanol concentration, C 0The initial concentration of 2' -trifluoromethylacetophenone.
The optical purity of the product is characterized by enantiomeric excess (e.e.). The calculation formula is as follows:
e.e.＝(Cs-CR)/(Cs+CR)×100％
in the formula CSAnd CRThe molar concentrations of 1- (2-trifluoromethylphenyl) ethanol of S type and R type respectively.
2. The characteristics of strain ZJPH1811 are as follows:
the morphological characteristics of the strain ZJPH1811 obtained by screening are as follows: the appearance, texture, color and growth rate of the colonies of the strain ZJPH1811 were observed on nutrient agar plates, and the morphology of the cells was observed by an optical microscope. The bacterial colony on the nutrient agar plate is in a plane diffusion, grows fast, is flat, has a protruded center, is white and is in a short velvet shape, the edge of the bacterial colony is in a sawtooth shape, the liquid culture is turbid, the propagation mode is fission, and a large amount of rectangular, barrel-shaped and oval spores are formed to be single or connected into chains.
26S rDNA sequence characteristics: using total cell DNA extracted by an Ezup column type yeast genome DNA extraction kit as a template, amplifying 26S rDNA D1/D2 region sequence genes of a strain by using universal primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'), wherein a PCR reaction system is 25 mu l, and the PCR reaction system comprises 20-25 ng of genome DNA, 0.5 mu l and 10 xbuffer (containing Mg) 2+) 2.5. mu.l, 0.2. mu.l of DNA polymerase, 0.5. mu.l each of forward and reverse primers 10. mu.M, 1. mu.l of dNTP (2.5 mM each), and double distilled water to 25. mu.l; and (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 4min, 30 cycles at 94 ℃ for 45s, 55 ℃ for 45s, and 72 ℃ for 1min, final extension at 72 ℃ for 10min, and termination at 4 ℃. After extracting genome DNA and amplifying 26S rDNA fragment by PCR, DNA fragment with size of 560bp is obtained. The 26S rDNA gene sequence of the strain obtained by purifying the PCR product and sequencing the PCR product by biological engineering (Shanghai) corporation is shown as SEQ ID NO. 1. The 26S rDNA sequence (SEQ ID NO.1) of strain ZJPH1811 was subjected to homology alignment (BLAST) at the NCBI website (http:// www.ncbi.nlm.nih.gov), and the results showed that: the sequence homology of strain ZJPH1811 with Geotrichum silvicola strain VTT C-04559 strain (GenBank accession No. DQ377646.1) reached 97%.
Based on the morphological characteristics of the strain and combined with molecular biological identification, the strain ZJPH1811 is identified as Geotrichum linum (Geotrichum silvicola), named as Geotrichum linum (Geotrichum silvicola) ZJPH1811, deposited in China center for type culture Collection with a date of 7/6 (2020), accession number: CCTCC NO: m2020281, deposit address: china, wuhan university, zip code 430072.
Example 2: obtaining of Wet cells
1) Plate culture: picking out single colony of Geotrichum linnei ZJPH1811, inoculating to plate culture medium, activating and culturing at 30 deg.C for 2d, picking out colony from the first activated plate according to the same steps, and activating again. Storing the obtained flat plate in a refrigerator at 4 ℃; the final concentration composition of the plate culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO42g/L，NaCl 1g/L，MgSO4·7H20.5g/L of O, 20g/L of agar and water as a solvent, and the pH value is 6.5.
2) Seed culture: selecting a ring of thalli from a mature culture plate, inoculating the ring of thalli into a seed culture medium, and culturing at 30 ℃ and 200rpm for 12h to obtain a seed solution; the final concentration 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，MgSO4·7H2O0.5 g/L, solvent is water, pH 6.5.
3) Fermentation culture: inoculating the seed solution into a fermentation culture medium at a volume concentration of 8%, culturing at 30 deg.C and 200rpm for 24 hr to obtain a fermentation broth, centrifuging the fermentation broth, and collecting the precipitate with K with pH of 7.02HPO4-KH2PO4Washing with buffer solution, centrifuging to obtain wet thallus, namely enzyme source; the final concentration of the fermentation medium is as follows: glucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO421.39g/L of anhydrous CaCl21mM, solvent water, pH 6.5.
Example 3:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 100g/L glucose as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 42.9% as determined by analysis using the detection method of example 1.
Example 4:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 60.9% as determined by analysis according to the detection method of example 1.
Example 5:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 10% ethanol by volume concentration (v/v) as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 9.3% as determined by analysis using the detection method of example 1.
Example 6:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 6.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 53.4% as determined by analysis using the detection method of example 1.
Example 7:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 62.5% as determined by analysis using the detection method of example 1.
Example 8:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 100g/L glycerol as auxiliary substrate, and reacting in a shaker at 25 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 54.8% as determined by analysis using the detection method of example 1.
Example 9:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 200g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 100g/L glycerol as auxiliary substrate, and reacting in a shaking table at 40 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 42.5% as determined by analysis using the detection method of example 1.
Example 10:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 160g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 48.8% as determined by analysis using the detection method of example 1.
Example 11:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 500g/L buffer; adding 15 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 86.9% as determined by analysis according to the detection method of example 1.
Example 12:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 10 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 95.7% as determined by analysis using the detection method of example 1.
Example 13:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 100g/L glycerol as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 62.3% as determined by analysis using the detection method of example 1.
Example 14:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 30 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glycerin as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 48.9% as determined by analysis using the detection method of example 1.
Example 15:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 200g/L glycerol as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 59.6% as determined by analysis using the detection method of example 1.
Example 16:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 60g/L glycerol as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol had an e.e. value of 99.2% and a yield of 64.0% by the detection method of example 1.
Example 17:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone by volume of buffer solution as substrate, adding 60g/L glycerol as auxiliary substrate, and reacting in a shaking table at 30 deg.C and 200rpm for 22 h. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 65.0% as determined by analysis using the detection method of example 1.
Example 18:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone as substrate, adding 60g/L glycerin as auxiliary substrate, and reacting at 30 deg.C in 200rm shaking table for 12 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 54.1% as determined by analysis using the detection method of example 1.
Example 19:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone calculated by buffer volume as a substrate, adding 60g/L glycerol as an auxiliary substrate, and adding 10g/L betaine: cysteine (molar ratio 1:1) was reacted for 22h in a shaker at 30 ℃ and 200 rpm. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 73.2% as determined by analysis using the detection method of example 1.
Example 20:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone calculated by the volume of the buffer solution as a substrate, adding 60g/L glycerol as an auxiliary substrate, and then adding 10g/L choline acetate: cysteine (molar ratio 1:1) was reacted for 22h in a shaker at 30 ℃ and 200 rpm. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 73.3% as determined by analysis using the detection method of example 1.
Example 21:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone calculated by buffer solution volume as a substrate, adding 60g/L glycerol as an auxiliary substrate, and adding 10g/L betaine: isopropanol (molar ratio 1:1) was reacted for 22 hours in a shaker at 30 ℃ and 200 rpm. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 48.1% as determined by analysis using the detection method of example 1.
Example 22:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 6.6) at a wet cell loading of 360g/L buffer based on wet weight; adding 20 mmol/L2' -trifluoromethyl acetophenone calculated by the volume of the buffer solution as a substrate, adding 60g/L glycerol as an auxiliary substrate, and then adding 10g/L choline acetate: cysteine (molar ratio 1:1) was reacted for 22h in a shaker at 30 ℃ and 200 rpm. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.3% and a yield of 63.5% as determined by analysis using the detection method of example 1.
Example 23:
the wet cells obtained according to the method of example 2 were resuspended in 5mL of phosphate buffer (0.1M, pH 7.4) at a wet cell loading of 360g/L buffer; adding 20 mmol/L2' -trifluoromethyl acetophenone calculated by the volume of the buffer solution as a substrate, adding 60g/L glycerol as an auxiliary substrate, and then adding 10g/L choline acetate: cysteine (molar ratio 1:1) was reacted for 22h in a shaker at 30 ℃ and 200 rpm. The product (S) -1- (2-trifluoromethylphenyl) ethanol exhibited an e.e. value of 99.2% and a yield of 78.9% as determined by analysis according to the detection method of example 1.
Example 24:
1) candida parapsilosis ZJPH1305 is selected and preserved in China center for type culture Collection, address: china, wuhan university, accession number: CCTCC NO: m2013559, preservation date 11/8 in 2013. This strain has been disclosed in a prior patent application (publication No. CN103849574A, published: 6/11/2014). The culture method of the strain and the preparation process of the enzyme-derived cells were carried out according to the prior patent application (publication No. CN103849574A, published: 6/11/2014).
2) The wet cells cultured as described above were resuspended in 5mL of phosphate buffer (0.1M, pH 7.0) at a wet weight concentration of 100g/L buffer; adding 6.7 mmol/L2' -trifluoromethyl acetophenone as substrate and 100g/L glucose as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 hr. The product (S) -1- (2-trifluoromethylphenyl) ethanol was examined for its e.e. value and yield using the examination method of example 1.
And (4) conclusion: candida parapsilosis ZJPH1305 is unable to convert 2' -trifluoromethylacetophenone to (S) -1- (2-trifluoromethylphenyl) ethanol.
Sequence listing
<110> Zhejiang industrial university
<120> Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 560
<212> DNA
<213> Geotrichum silvicola)
<400> 1
gaggaaaaga aaccaacagg gattgcctta gtaacggcga gtgaagcggc aaaagctcaa 60
atttgaaatc ggccaccagg tcgagttgta atttgtagat tgtatcttga gagcggatta 120
aagtctgttg gaacacagcg ccttagaggg tgacagcccc gtaaaatcta ttctcattgt 180
aagatacttt cgaagagtcg agttgtttgg gaatgcagct ctaagtggga ggtaaattcc 240
ttctaaagct aaatattgac gagagaccga tagcgaacaa gtactgtgaa ggaaagatga 300
aaagcacttt gaaaagagag tgaaaaagta cgtgaaattg ttaaaaggga agggtattga 360
atcagacttg gtgctgttgt tcaactgtgt ttcggcatag tgtactcagc agtactaggc 420
caaggtgggg tgtttgggag tgaaaaagaa gttggaacgt aactcttcgg agtgttatag 480
cctactttca tagctcctca ggcgcctcag gactgcgctt cggcaaggac cttggcataa 540
tgattctata ccgcccgtct 560

Claims (7)

1. Geotrichum silvicola ZJPH1811, deposited in China center for type culture Collection with a date of 7/6/2020, accession number: CCTCC NO: m2020281, deposit address: china, wuhan university, zip code 430072.
2. Use of the Geotrichum linnei ZJPH1811 of claim 1 in the preparation of (S) -1- (2-trifluoromethylphenyl) ethanol.
3. The use according to claim 2, characterized in that said use is: taking wet thalli obtained by fermenting and culturing Geotrichum linum ZJPH1811 as an enzyme source, taking 2' -trifluoromethylacetophenone as a substrate, adding an auxiliary substrate, forming a conversion system in a phosphate buffer solution with the pH of 6.0-8.0, reacting at the temperature of 25-40 ℃ and the speed of 100-200rpm, obtaining a conversion solution containing a product (S) -1- (2-trifluoromethylphenyl) ethanol after the reaction is finished, and separating and purifying the conversion solution to obtain (S) -1- (2-trifluoromethylphenyl) ethanol; the auxiliary substrate is one of glucose, isopropanol, glycerol or ethanol.
4. The use according to claim 3, wherein the amount of the wet cells is 100-600g/L based on the volume of the buffer solution, and the amount of the substrate is 5-50mmol/L based on the volume of the buffer solution; when the auxiliary substrates are glucose and glycerol, the addition amount is 10-250g/L calculated by the volume of the buffer solution, and when the auxiliary substrates are isopropanol and ethanol, the addition amount by volume is 10-30% calculated by the volume of the buffer solution.
5. The use according to claim 3, wherein the conversion system further comprises a natural eutectic solvent, said natural eutectic solvent consisting of a hydrogen bond acceptor and a hydrogen bond donor, comprising: choline chloride: urea, betaine: isopropyl alcohol, betaine: cysteine, betaine: trehalose, choline chloride: glutamic acid, choline acetate: cysteine, wherein the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the natural eutectic solvent is 1: 1.
6. The use according to claim 5, wherein the mass addition of the natural eutectic solvent is from 2 to 20g/L based on the volume of the buffer.
7. Use according to claim 3, characterized in that the enzyme source is obtained as follows:
1) plate culture: selecting a single colony of Geotrichum linnei ZJPH1811, inoculating the single colony into a plate culture medium, performing activation culture at 30 ℃ for 2d, selecting the colony from the first activated plate according to the same steps, performing activation culture again, and storing the obtained plate in a refrigerator at 4 ℃; the final concentration composition of the plate culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH) 4)2SO42g/L，KH2PO4 2g/L，NaCl 1g/L，MgSO4·7H20.5g/L of O, 20g/L of agar, water as a solvent and pH of 6.5;
2) seed culture: selecting a ring of thalli from a mature culture plate, inoculating the ring of thalli into a seed culture medium, and culturing at 30 ℃ and 200rpm for 12h to obtain a seed solution; the seed culture mediumThe concentration composition 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 water, and the pH value is 6.5;
3) fermentation culture: inoculating the seed solution into a fermentation culture medium at a volume concentration of 8%, culturing at 30 deg.C and 200rpm for 24 hr to obtain a fermentation broth, centrifuging the fermentation broth, and collecting precipitate with K of pH 7.02HPO4-KH2PO4Washing with buffer solution, centrifuging to obtain wet thallus, namely enzyme source; the final concentration of the fermentation medium is as follows: glucose 24.45g/L, peptone 15.75g/L, (NH)4)2SO421.39g/L of anhydrous CaCl21mM, solvent water, pH 6.5.
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