CN112176019B - Method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by biological catalysis - Google Patents
Method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by biological catalysis Download PDFInfo
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Abstract
The invention discloses a method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by biocatalysis, which comprises the steps of taking wet thalli obtained by fermenting and culturing recombinant escherichia coli as a catalyst, taking m-trifluoromethylacetophenone as a substrate, adding an auxiliary substrate, taking a phosphate buffer solution with the pH value of 6.0-8.0 as a reaction medium to form a conversion system, carrying out biocatalysis asymmetric reduction reaction at the temperature of 20-40 ℃ and the speed of 200rpm, and separating and purifying reaction liquid after the reaction is finished to obtain (R) -1- (3-trifluoromethylphenyl) ethanol; according to the invention, when the concentration of the substrate is 400mM, the yield of the product (R) -1- (3-trifluoromethylphenyl) ethanol is 74.2%, and the e.e. value is more than 99.9%, so that the catalytic efficiency is better.
Description
(I) technical field
The invention relates to a preparation method of (R) -1- (3-trifluoromethylphenyl) ethanol, in particular to a method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by catalyzing m-trifluoromethylacetophenone asymmetric reduction through recombinant escherichia coli with high selectivity.
(II) technical background
(R) -3- (1- (3- (tris (trifluoromethyl) phenyl) ethoxy) azetidine-1-carboxamide is a neuroprotective compound useful in the treatment of cerebral ischemia, central nervous system injury or ocular diseases in a mouse model of permanent MCAO, the compound showed significant neuroprotective effect at a dose of 60mg/kg when administered 30 minutes prior to occlusion (R) -1- (3-trifluoromethylphenyl) ethanol ((R) -MTF-PEL), formula C9H9F3O, CAS number: 127852-24-8, is a key chiral intermediate for preparing the neuroprotective agent.
The method comprises the steps of taking a complex generated by the reaction of chiral furazamine and zinc acetate as a catalyst, reducing m-trifluoromethyl acetophenone to obtain (R) -1- (3- (trifluoromethyl phenyl) ethanol, wherein the concentration of the m-trifluoromethyl acetophenone as a substrate is 500mM, the reaction time is 45h, the yield is 81%, the optical purity is 60%, the enantioselectivity of the product obtained by the method is relatively low, the reaction time is relatively long, screening the duckweed and the like to obtain a Microbacterium oxydans C3(Microbacterium oxydans C3), asymmetrically reducing the m-trifluoromethyl acetophenone as the (R) -1- (3- (trifluoromethyl phenyl) ethanol, obtaining a substrate with low concentration of the reaction, only 5mM, 24h conversion at 37 ℃, the substrate conversion rate and the e.e. value are respectively 88% and 99%, and the method is applied by the same and the like to constructing the recombinant escherichia coli cell derived from heat-resistant Kluyveromyces CGMCC 2.1492 carbonyl reductase, and (3) carrying out biological reduction on the m-trifluoromethyl acetophenone substrate, wherein the substrate concentration is 10mM, the reaction is carried out for 12 hours at 30 ℃, the substrate conversion rate is 99%, and the e.e. value is greater than 99%. The currently reported method for preparing (R) -1- (3- (trifluoromethylphenyl) ethanol by utilizing microbial cell catalysis has the problems of low concentration of catalytic substrates, unsatisfactory catalytic efficiency and the like.
The eutectic solvent is a transparent and uniform liquid eutectic salt formed by hydrogen bonding between a Hydrogen Bond Acceptor (HBA) and a Hydrogen Bond Donor (HBD) through hydrogen bonding. It has more excellent properties than conventional organic solvents and ionic liquids, such as low melting point, designability, good biocompatibility, easy preparation, high stability and environmental friendliness. Natural eutectic solvents are mainly composed of natural primary metabolites such as sugars, sugar alcohols, organic acids, amino acids and amines, and usually also contain a certain molar ratio of water, which has better biocompatibility and sustainability than general eutectic solvents.
The invention uses recombinant Escherichia coli engineering bacteria BL21(DE3)/pET28a (+) -LXCAR resting cells constructed by short-chain dehydrogenase derived from Leifsonia sp as a catalyst, and the recombinant Escherichia coli engineering bacteria BL 21/pET 28a (+) -LXCAR resting cells are prepared by reacting the following components in a reaction system: (R) -1- (3-trifluoromethylphenyl) ethanol ((R) -MTF-PEL) was prepared by conversion of m-trifluoromethylacetophenone in a lysine (ChCl: Lys) eutectic solvent-buffer medium system. Because the eutectic solvent can change the permeability of the microbial cell membrane as a catalyst, the inhibition effect of a substrate and a product is reduced, and the reduction reaction efficiency can be further improved.
Disclosure of the invention
The invention aims to provide a novel method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by catalyzing asymmetric reduction of m-trifluoromethylacetophenone by utilizing recombinant escherichia coli. Compared with a chemical reduction method, the method has the advantages of high stereoselectivity, low catalyst preparation cost, mild reaction conditions, simplicity in operation, environmental friendliness and the like, and the concentration of a catalytic substrate, the yield of a product and the e.e. value are high.
The technical scheme adopted by the invention is as follows:
the invention provides a method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol ((R) -MTF-PEL) by biocatalysis, which comprises the following steps: taking wet thalli obtained by fermentation culture of recombinant escherichia coli constructed by short-chain dehydrogenase (LXCAR) derived from Leifsonia as a catalyst, taking m-trifluoromethyl acetophenone as a substrate, adding an auxiliary substrate, taking a phosphate buffer solution with the pH of 6.0-8.0 (preferably pH of 7.5) as a reaction medium to form a conversion system, carrying out biocatalytic asymmetric reduction reaction at the temperature of 20-40 ℃ and at 200rpm, and after the reaction is finished, separating and purifying the reaction solution to obtain an (R) -1- (3-trifluoromethylphenyl) ethanol product; the auxiliary substrate is one of glucose, glycerol, maltose, lactose, isopropanol, ethanol or n-butanol, preferably isopropanol; the recombinant Escherichia coli is Escherichia coli BL21(DE3)/pET28a (+) -LXCAR.
Further, the adding amount of the catalyst is 20-160 g/L (preferably 160g/L) based on the total volume of the conversion system; the addition amount of the substrate is 50-400 mmol/L (preferably 400mmol/L) based on the total volume of the conversion system; when the auxiliary substrates are glucose, maltose and lactose, the adding amount is 100g/L based on the total volume of the conversion system; when the auxiliary substrate is glycerol, the adding amount is 60g/L based on the total volume of the conversion system; and when the auxiliary substrate is isopropanol, ethanol or n-butanol, the volume addition amount is 5-30% (preferably 10%) based on the total volume of the conversion system.
Furthermore, the conversion system is composed of a catalyst, a substrate, an auxiliary substrate, a eutectic solvent and a phosphate buffer, wherein the addition amount of the eutectic solvent is 1-10% (preferably 4%) of the total volume of the conversion system.
The molar ratio of a Hydrogen Bond Acceptor (HBA) to a Hydrogen Bond Donor (HBD) in the eutectic solvent composition is 1: 1. The HBA in the eutectic solvent is one of choline chloride, betaine, L-carnitine and L-proline, and the HBD is one of natural amino acid, isopropanol, urea, fructose, trehalose, glucose and glycerol.
The eutectic solvent of the present invention is preferably one of the following: choline chloride: lysine (ChCl: Lys), choline chloride: urea (ChCl: U), choline chloride: isopropyl alcohol (ChCl: IPA), choline chloride: glycine (ChCl: Gly), choline chloride: alanine (ChCl: Ala), Choline chloride: tryptophan (ChCl: Trp), choline chloride: tyrosine (ChCl: Tyr), choline chloride: glutamic acid (ChCl: Glu), choline chloride: glutathione (ChCl: GSH), choline chloride: fructose (ChCl: Flu), choline chloride: trehalose (ChCl: Myc), betaine: isopropyl alcohol (B: IPA), betaine: cysteine (B: Cys), betaine: alanine (B: Ala), betaine: lysine (B: Lys), betaine: glutamic acid (B: Glu), L-carnitine: lysine (C: Lys), L-proline: lysine (P: Lys), L-carnitine: glucose: glycerol (C: Glc: G) in a molar ratio of 1: 1. Preferred classes of eutectic solvents are choline chloride: lysine (ChCl: Lys).
The construction process of the recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR is described in the previous patent application (CN104212841A, published 2014, 12 months and 17 days) of the applicant. The preparation method of the catalyst comprises the following steps: recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, cultured with shaking at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin at an inoculum size of 1% by volume concentration, cultured at 37 ℃ and 200rpm until the cell density OD600After 0.6 to 0.9, isopropyl-. beta. -D-thiogalactoside (IPTG) was added to the culture medium at a final concentration of 0.1mM, and induced at 30 ℃ and 200rpm for 10 hours. After the culture is finished, centrifuging at 4 ℃ and 10000rpm for 10min, collecting thalli, washing the thalli twice by using physiological saline, and collecting wet thalli, namely the recombinant escherichia coli BL21(DE3)/pET28a (+) -LXCAR wet thalli.
Compared with the prior art, the invention has the following beneficial effects: the invention uses the recombinant Escherichia coli whole cell as a catalyst to prepare (R) -1- (3-trifluoromethylphenyl) ethanol by biological asymmetric reduction of m-trifluoromethylacetophenone. The method has the advantages of high product stereoselectivity, simple process, environmental protection, high reaction yield and the like, and can realize the in-situ regeneration of the coenzyme by adding cheap auxiliary substrates in the biological reduction process. Simultaneously adding a eutectic solvent choline chloride into a reaction medium system: lysine (ChCl: Lys), significantly improved the substrate concentration and yield of the reaction. The carbonyl reductase from heat-resistant Kluyveromyces is utilized to construct recombinant engineering bacteria to biologically catalyze a m-trifluoromethyl acetophenone substrate, the concentration of the substrate is lower and is 10mM, the reaction is carried out for 12 hours at 30 ℃, the substrate conversion rate is 99%, and the e.e. value is greater than 99%. The invention provides a novel method for preparing (R) -1- (3-trifluoromethylphenyl) ethanol by using recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR whole-cell biological reduction m-trifluoromethylacetophenone, wherein when the substrate concentration is 100mM, the yield of the product (R) -1- (3-trifluoromethylphenyl) ethanol is 93.8%, and the e.e. value is more than 99.9%. When the eutectic solvent choline chloride is added into the reaction medium system: lysine (ChCl: Lys), the yield of the product (R) -1- (3-trifluoromethylphenyl) ethanol is 74.2% when the substrate concentration is increased to 400mM, and the e.e. value is more than 99.9%, thus showing more excellent catalytic efficiency. Compared with the reported transformation strains, the recombinant escherichia coli has higher substrate concentration and product yield in catalyzing m-trifluoromethyl acetophenone.
(IV) description of the drawings
FIG. 1 is gas phase detection spectra of a substrate m-trifluoromethylacetophenone standard and a product 1- (3-trifluoromethylphenyl) ethanol standard.
FIG. 2 is a gas phase detection chromatogram (containing internal standard dodecane) of an extract liquid of a reduction reaction of m-trifluoromethyl acetophenone catalyzed by recombinant engineering bacteria BL21(DE3)/pET28a (+) -LXCAR.
(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 adding amounts of the wet bacteria, the m-trifluoromethyl acetophenone substrate, the auxiliary substrate, the surfactant and the eutectic solvent are all measured by the total volume of the conversion system. The amounts of the catalysts used according to the invention are based on the wet weight, unless otherwise specified.
The eutectic solvent is purchased from Shanghai Fujie chemical Co.
Example 1: different kinds of microbial cells are taken as biocatalysts to carry out asymmetric reduction reaction of the m-trifluoromethyl acetophenone.
1. Catalyst and process for preparing same
(1) Recombinant escherichia coli BL21(DE3)/pET28a (+) -LXCAR wet thallus
The construction process of the recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR is described in example 1 of the previous patent application (publication No.: CN104212841A, published 2014, 12 months and 17 days) by the applicant.
The recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR obtained by the construction was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, cultured with shaking at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin at an inoculum size of 1% by volume concentration, and cultured at 37 ℃ and 200rpm until the cell density OD600After 0.6 to 0.9, isopropyl-. beta. -D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.1mM, and the mixture was subjected to induction culture at 30 ℃ and 200rpm for 10 hours. After the culture is finished, centrifuging at 4 ℃ and 10000rpm for 10min, collecting thalli, washing the thalli twice by using physiological saline, and collecting wet thalli, namely the recombinant escherichia coli BL21(DE3)/pET28a (+) -LXCAR wet thalli. The LB liquid medium consists of: 10g/L of tryptone, 5g/L of yeast extract, 9g/L of NaCl and water as a solvent, wherein the pH is natural.
(2) Tupistra yeast ZJPH1807 wet thallus
The soil star crop yeast (Cyberlindera saturnus) ZJPH1807 is preserved in China center for type culture Collection, address: china, wuhan university, accession number: CCTCC NO: m2019215, deposited on 3/29 of 2019, has been disclosed in a previous patent application of the applicant (publication No.: CN110283733A, published on 27/9 of 2019).
The preparation method of Verticillium terrestris ZJPH1807 wet cells is the same as that of example 3 of the patent application (publication No. CN 110283733A).
(3) Geotrichum galactose ZJPH1810 wet thallus
Geotrichum galactosidanum (Galactomyces geotrichum) ZJPH1810 was deposited at the chinese collection of type cultures at the address: china, wuhan university, accession number: CCTCC NO: m2019822. The date of deposit was 10/14 in 2019, and was disclosed in the applicant's prior patent application (publication No.: CN110760449A, published: 2020: 02/07).
The wet cells of Geotrichum galactaratum ZJPH1810 were prepared in the same manner as in example 2 of the prior patent application (publication No. CN 110760449A).
(4) Geotrichum candidum ZJPH1704 wet thallus
Geotrichum candidum (Geotrichum candidum) ZJPH1704 is preserved in China center for type culture Collection, with the address of Wuhan, Wuhan university, preservation number: CCTCC NO: m2017380. The date of deposit was 26/6/2017, and the date of deposit was 430072, which was disclosed in the applicant's prior patent application (publication No.: CN107746861A, published: 3/2/2018).
The method for preparing wet cells of Geotrichum candidum ZJPH1704 was the same as that described in example 2 of the prior patent application (publication No. CN 107746861A).
(5) Pseudomonas aeruginosa ZJPH1504 wet thallus
Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJPH1504 is preserved in China center for type culture Collection, with the address of Wuhan, Wuhan university, the preservation number is CCTCC NO: m2016188. Is disclosed in the patent application (application No. CN201610369158.5, 2016, 5, 27).
The preparation method of the wet pseudomonas aeruginosa ZJPH1504 wet bacterial strain is the same as that of the previous patent application (application No. 201610369158.5, 2016, 5, 27).
2. Asymmetric reduction of m-trifluoromethylacetophenone
A50 mL Erlenmeyer flask was charged with 5mL of phosphate buffer (200mM, pH 7.0), and then with wet cells (shown in Table 1) at a final concentration of 50g/L as a catalyst, glucose at a final concentration of 100g/L as an auxiliary substrate, and m-trifluoromethylacetophenone as a substrate at a final concentration of 50mM, to form 5mL of a conversion system, and the conversion was carried out at 30 ℃ and 200rpm for 12 hours.
3. Detection method
After the conversion reaction was completed, the reaction solution was extracted with ethyl acetate of equal volume, and the ethyl acetate layer was collected by centrifugation, and the yield and e.e. value of the product were measured by gas chromatography.
The detection conditions of the gas chromatography are as follows: agilent gas chromatograph, N2000 chromatographic workstation, chiralsil-Dex CB capillary column, usa. The carrier gas is nitrogen; the flow rate is 2 mL/min; sample introduction amount: 1 mu L of the solution; the split ratio is 15: 1; the detector is a hydrogen flame ion detector; the temperature of the sample inlet and the temperature of the detector are 250 ℃; column temperature is programmed temperature rise: keeping at 115 deg.C for 2min, heating to 140 deg.C at 2 deg.C/min, and keeping for 1 min. The retention time of each substance is respectively as follows: substrate m-trifluoromethylacetophenone 2.64min, dodecane 3.94min, (S) -1- (3-trifluoromethylphenyl) ethanol 6.92min, and (R) -1- (3-trifluoromethylphenyl) ethanol 6.35 min. The gas phase detection chromatogram of the substrate meta-trifluoromethylacetophenone and the optically pure product 1- (3-trifluoromethylphenyl) ethanol standard is shown in figure 1, the cell biotransformation results of different types of microorganisms are shown in table 1, and the gas phase detection chromatogram of the recombinant engineering bacteria BL21(DE3)/pET28a (+) -LXCAR catalytic meta-trifluoromethylacetophenone reduction reaction extract is shown in figure 2.
The yield calculation method comprises the following steps:
an internal standard method: and measuring a product concentration standard curve by using dodecane as an internal standard substance. During the determination, dodecane is added into the sample as an internal standard substance, and the product concentration is calculated according to the internal standard substance concentration.
The yield of the product was calculated from the following formula:
yield ═ Cp/C0
In the formula CpIs the (R) -1- (3-trifluoromethylphenyl) ethanol concentration, C0Is the initial concentration of m-trifluoromethyl acetophenone.
The optical purity of the product is characterized by enantiomeric excess (e.e.). The calculation formula is as follows:
in the formula CRAnd CSRespectively the molar concentrations of R-1- (3-trifluoromethylphenyl) ethanol and S-1- (3-trifluoromethylphenyl) ethanol.
Table 1 effect of different whole-cell biocatalysts on product yield, e.e. value and configuration
Example 2: influence of the kind of co-substrate
5mL of phosphate buffer (200mM, pH 7.0) was added to a 50mL Erlenmeyer flask, and recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells and various kinds of cosubstrates (Table 2) were added to a final concentration of 50g/L, and the substrate m-trifluoromethylacetophenone was added to a final concentration of 50mM to prepare 5mL of a conversion system, and the conversion was carried out at 30 ℃ and 200rpm for 12 hours. After the reaction was completed, the yield and e.e. value were measured by the method of example 1, and the results are shown in table 2. The results show that the highest yield, up to 86.2%, is achieved when isopropanol is used as an auxiliary substrate, and the value of the product e.e. is greater than 99.9%.
TABLE 2 Effect of different cosubstrates on the product yield and ee value
Example 3: effect of buffer pH on transformation results
A phosphate buffer (200mM) with the pH value of 6.0-8.0 is added into a 50mL triangular flask, recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR wet bacteria with the final concentration of 50g/L and 10% (v/v) isopropanol are added as auxiliary substrates, and a m-trifluoromethyl acetophenone substrate with the final concentration of 50mM form a 5mL conversion system. The transformation was carried out at 200rpm for 12h at 30 ℃. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in table 3. The results showed that the optimal buffer pH was 7.5, at which time the yield was 89.6%.
Table 3 effect of buffer pH on reaction yield and e.e. value
Example 4: effect of buffer Ionic Strength on conversion results
A50 mL Erlenmeyer flask was charged with 50-400mM phosphate buffer (pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 50g/L, 10% (v/v) isopropanol, and m-trifluoromethylacetophenone, a substrate at a final concentration of 50mM, to constitute 5mL of the conversion system. The transformation was carried out at 200rpm for 12h at 30 ℃. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in table 4. The results showed that the optimum ionic strength was 100 mM.
Table 4 influence of buffer ionic strength on the yield and e.e. value of the reduction reaction
Example 5: effect of isopropanol addition on conversion results
A50 mL Erlenmeyer flask was charged with phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 50g/L, an isopropanol co-substrate at a final concentration of 5-30% (v/v), and m-trifluoromethylacetophenone, a substrate at a final concentration of 100mM, to form a 5mL conversion system. The transformation was carried out at 200rpm for 12h at 30 ℃. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in table 5. The results show that the optimum isopropanol addition is 15%.
Table 5 effect of different isopropanol additions on the yield and e.e. value of the product
Example 6: influence of reaction temperature on the conversion result
A50 mL Erlenmeyer flask was charged with phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 50g/L, isopropanol as a co-substrate at a final concentration of 15% (v/v), and 100mM of the substrate, m-trifluoromethylacetophenone, to make up 5mL of the transformation system. The conversion is carried out for 12 hours at the temperature of 20-40 ℃ and the speed of 200 rpm. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in table 6. The results show that the optimum conversion temperature is 30 ℃.
Table 6 influence of the conversion temperature on the yield and e.e. value of the product
Example 7: effect of Wet cell addition on transformation results
A50 mL Erlenmeyer flask was charged with phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 20-100g/L, isopropanol co-substrate at a final concentration of 15% (v/v), and m-trifluoromethylacetophenone substrate at final concentrations of 100mM and 200mM, respectively, to make up 5mL of the transformation system. The transformation was carried out at 200rpm for 12h at 30 ℃. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in Table 7. The results showed that the optimum amount of wet cells added was 40g/L at a substrate concentration of 100 mM; the optimum amount of wet cells added was 80g/L at a substrate concentration of 200 mM.
TABLE 7 influence of the amount of added wet cells on the yield and e.e. value of the product
Example 8: effect of transformation time on transformation results
A5 mL transformation system was constructed by adding phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at final concentrations of 40g/L and 80g/L, respectively, isopropanol as a co-substrate at a final concentration of 15% (v/v), and m-trifluoromethylacetophenone as a substrate at final concentrations of 100mM and 200mM, respectively, to a 50mL Erlenmeyer flask. The transformation was carried out at 200rpm at 30 ℃ for various times. After the reaction was complete, the yield and the e.e. value of the product were determined by the method of example 1 and the results are shown in Table 8. The results show that the optimum conversion time is 21 h.
TABLE 8 Effect of reaction time on reduction yield and e.e. value
Example 9: effect of adding different kinds of eutectic solvents on the conversion results
A50 mL Erlenmeyer flask was charged with 3.25mL of phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 160g/L, a different type of eutectic solvent at a final concentration of 10g/L, an isopropanol co-substrate at a final concentration of 15% (v/v), and m-trifluoromethylacetophenone, a substrate at a final concentration of 400mM, to form 5mL of the conversion system. The transformation was carried out at 200rpm for 21h at 30 ℃. After the reaction was completed, the yield and the e.e. value of the product were measured by the method of example 1, and the result is shown in Table 9 using a system without the eutectic solvent as a control under the same conditions.
Table 9 influence of addition of different kinds of eutectic solvents on yield and e.e. value of the product
As can be seen from table 9, the yields of the obtained products were all 99.9% or more in the conversion systems containing different kinds of eutectic solvents, but the different kinds of eutectic solvents had different effects on the reduction yield. Mixing choline chloride: lysine (1: 1 molar ratio) was optimal with a yield of 70.2%, while the conversion system without eutectic solvent gave a yield of 66.8%. Therefore, the preferred type of eutectic solvent is choline chloride lysine (ChCl: Lys).
Example 10: choline chloride: effect of lysine (ChCl: Lys) addition on conversion results
To a 50mL Erlenmeyer flask were added 3.25mL of phosphate buffer (100mM, pH 7.5), recombinant E.coli BL21(DE3)/pET28a (+) -LXCAR wet cells at a final concentration of 160g/L, 1-10% (v/v) choline chloride: lysine (ChCl: Lys), isopropanol co-substrate to a final concentration of 15% (v/v) and substrate m-trifluoromethylacetophenone 5mL to a final concentration of 400 mM. The conversion was carried out at 200rpm for 21h at 30 ℃ and the yield and the e.e. value of the product were determined after the reaction was complete by the method of example 1 and the results are shown in Table 10.
Table 10 choline chloride: effect of lysine (ChCl: Lys) addition on product yield and e.e. value
As can be seen from table 10, when choline chloride: lysine (ChCl: Lys) was added at 4% and the yield was 74.2% at a final substrate concentration of 400 mM.
Claims (2)
1. Biocatalytic preparation (A)R) -1- (3-trifluoromethylphenyl) ethanol, characterized in that it is a process comprising: taking wet bacteria obtained by fermentation culture of recombinant escherichia coli as a catalyst, taking m-trifluoromethyl acetophenone as a substrate, adding an auxiliary substrate and a eutectic solvent, taking a phosphate buffer solution with the pH value of 7.0-8.0 as a reaction medium to form a conversion system, carrying out biocatalytic asymmetric reduction reaction at the temperature of 30-35 ℃ and at the speed of 200rpm, and after the reaction is finished, separating and purifying the reaction solution to obtain (A)R) -1- (3-trifluoromethylphenyl) ethanol; the cosubstrate is isopropanol; the recombinant Escherichia coli is Escherichia coli BL21(DE3)/pET28a (+) -LXCAR;
the conversion system consists of a catalyst, a substrate, an auxiliary substrate, a eutectic solvent and a phosphate buffer solution; the molar ratio of a hydrogen bond acceptor to a hydrogen bond donor in the eutectic solvent composition is 1: 1; the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is lysine;
the addition amount of the catalyst is 40-60 g/L based on the total volume of the conversion system; the adding amount of the substrate is 100 mmol/L based on the total volume of the conversion system; the volume addition of the auxiliary substrate is 15-20% of the total volume of the conversion system; the adding amount of the eutectic solvent is 1-4% of the total volume of the conversion system;
the recombinant Escherichia coli is Escherichia coli BL21(DE3)/pET28a (+) -LXCAR which is constructed according to the following method: with Leifsonia (B), (B) and (C)Leifsonia xyli) The plasmid pET28a (+) of HS0904 source short-chain dehydrogenase is used as a template, and a mutant LXCAR is obtained through error-prone PCR and site-specific mutagenesis, wherein the nucleotide sequence of the mutant is shown in SEQ ID NO. 3; the obtained mutant gene is connected with an expression plasmid pET28a (+) and then transferred into escherichia coli BL21(DE3), thereby constructing and obtaining the Leifsonia (A) containingLeifsonia xyli) Recombinant escherichia coli BL21(DE3)/pET28a (+) -LXCAR of HS0904 short-chain dehydrogenase mutant gene;
SEQ IDNO.3：
atggcgcagt acgacgtggc cggacggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgcca tcgccctcac cctcgcggcg agcggagcgg ccgtcctcgt caccgacctg 120
aacgaggaaa acgcaaatgc cgtcgtggcg gagatcagcg ccgcgggcgg caccgcgcgg 180
gcactcgccg gcgatgtgac cgacccggcc ttcgccgagg ccagcgtcgc ggccgcgaac 240
gagctggccc cgctgcgcat cgccgtcaac aacgccggca tcggcggagc ggcagcaccg 300
gtcggcgact acccgctcga ctcgtggcgc aaggtcatcg aggtcaacct caacgccgtc 360
ttctacggga tgcaggcgca gctcgacgcg atcggcgcga acggcggcgg cgcgatcgtc 420
aacatggcgt ccatcctggg cagcgtcggc ttcgccaact attcggcgta cgtcaccgcg 480
aagcacgcgc tgctcggcct gacgcagaac gcggcgctgg agtacgccgg caagaacgtc 540
cgtgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggcgtc gaacatggac 600
gcggacaccc tcgccttcct cgagggcaag cacgcgctcg gccgcctggg cgagccggag 660
gaggtcgcct cgctggtcgc gttcctcgcc tccgacgccg ccagcttcat caccggcagc 720
taccacctgg tcgacggagg ctacacagca caatag 756。
2. the method of claim 1, wherein the catalyst is prepared by: recombinant Escherichia coli BL21(DE3)/pET28a (+) -LXCAR was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, cultured with shaking at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin at an inoculum size of 1% by volume concentration, cultured at 37 ℃ and 200rpm until the cell density OD600After the concentration is 0.6-0.9, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.1mmol/L into the culture solution, and placing at 30 ℃ and 200rpm for induction culture for 10 hours; after the culture is finished, centrifuging at 4 ℃ and 10000rpm for 10min, collecting thalli, washing the thalli twice by using physiological saline, and collecting wet thalli, namely the recombinant escherichia coli BL21(DE3)/pET28a (+) -LXCAR wet thalli.
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| CN102102087A (en) * | 2010-10-28 | 2011-06-22 | 浙江工业大学 | Leifsonia xyli and use thereof in preparation of (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol |
| CN104212841A (en) * | 2014-08-22 | 2014-12-17 | 浙江工业大学 | Method for preparing (R)-3,5-bis(trifluoromethyl)phenethyl alcohol in ionic liquid-containing cosolvent medium |
| CN107746861A (en) * | 2017-11-20 | 2018-03-02 | 浙江工业大学 | A kind of biological preparation method of (R) 1 (2 trifluoromethyl) ethanol |
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| CN102102087A (en) * | 2010-10-28 | 2011-06-22 | 浙江工业大学 | Leifsonia xyli and use thereof in preparation of (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol |
| CN104212841A (en) * | 2014-08-22 | 2014-12-17 | 浙江工业大学 | Method for preparing (R)-3,5-bis(trifluoromethyl)phenethyl alcohol in ionic liquid-containing cosolvent medium |
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