CN101319236A - Biocatalytic asymmetric reduction of carbonyl compounds in water/ionic liquid two-phase system - Google Patents

Abstract

The invention relates to a method for biocatalytic asymmetric reduction of carbonyl compounds in a water/ionic liquid two-phase system, belonging to the technical field of biochemical industry. In the present invention, selective carbonyl reductase-producing bacteria are used as the starting strain, and in the water/ionic liquid two-phase system, the corresponding chiral alcohol is prepared by reducing the latent chiral ketone as the substrate: using Aureobasidium pullulans CGM CCNo.1244 to catalyze 4 Preparation of (S)-4-chloro-3-hydroxybutyrate ethyl by asymmetric reduction of ethyl chloroacetoacetate, and preparation of 4,4,4-trifluoroacetoacetate by asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate ( R)-Ethyl 4,4,4-trifluoro-3-hydroxybutyrate. The method of the invention shows the advantages of the ionic liquid being non-toxic, tasteless, non-volatile, good biocompatibility, no environmental pollution, simple product separation, easy recovery and reusable use. At the same time, the method of the invention improves the conversion rate, concentration and enantiomer excess value of the reaction product, and accelerates the reaction process.

Description

Biocatalytic asymmetric reduction of carbonyl compounds in water/ionic liquid two-phase system

technical field

The invention relates to a method for biocatalytic asymmetric reduction of carbonyl compounds in a water/ionic liquid two-phase system, belonging to the technical field of biochemical industry. The present invention relates to a new reaction system to effectively improve the preparation of chiral alcohols by catalytic asymmetric reduction reaction of microbial cells, especially the method for catalytic preparation of halogenated hydroxybutyrate, specifically, in the water/ionic liquid two-phase system Preparation of ethyl (S)-4-chloro-3-hydroxybutyrate by asymmetric reduction of ethyl 4-chloroacetoacetate catalyzed by Aureobasidium pullulans CGMCCNo.1244, and grape juice yeast (Saccharomyces uvarum) ATCC 26602 Catalyzed asymmetric reduction reaction of ethyl 4,4,4-trifluoroacetoacetate to prepare (R)-4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester.

Background technique

Ionic liquids (ILs) are composed of positively charged ions and negatively charged ions, and are in a liquid state between -100°C and 200°C, so they are also commonly called room temperature ionic liquids. Ionic liquids have attracted widespread interest as solvents and catalysts. Unlike typical organic solvents, ionic liquids generally do not become steam, so no harmful gases that pollute the atmosphere will be produced during the experiment, and most ionic liquids are stable to water and can be easily prepared in the water phase. Convenient, the ionic liquid can be used repeatedly. The use of ILs in the chemical production process can reduce the use of highly volatile organic solvents, reduce environmental pollution, and reduce waste generation. The potential value of ILs research has been generally recognized by chemists from all over the world.

Organic-phase microbial cell catalysis can use effective methods such as media engineering and catalyst engineering to achieve optimal control of catalytic reactions. But there are problems: 1) organic solvents are easy to cause environmental pollution; 2) enzymes are easily inactivated in polar organic solvents; 3) polar substrates have very little solubility in non-polar organic solvents. As an excellent solvent to replace organic solvents, ionic liquid has the following advantages: 1) it helps to improve stereoselectivity; 2) it has strong stability in operation; 3) it can be used repeatedly, which is beneficial to environmental protection and cost saving; 4 ) products are easily separated. Most studies have shown that biocatalysts have high activity, (stereo, regio) selectivity and stability in ionic liquids.

The use of ionic liquids to replace traditional organic solvents as green reaction media for biocatalysis and biotransformation has only attracted people's attention in recent years. Most studies have shown that biocatalysts have high activity, selectivity and stability in ionic liquids. The application of ionic liquids in biocatalysis is a brand-new research field, and the research is getting deeper and wider, and there are more and more related reports. However, there are few patents and articles on biocatalytic asymmetric reduction in ionic liquids.

It has been reported that the stereoselective reduction of 2-octanone to 2-octanol with dehydroreductase catalyzed in the biphasic system of ionic liquid and buffer solution, the conversion rate is close to 100%, and the enantiomeric excess value (ee value )>99%, and showed that dehydroreductase has better stability in ionic liquid (Eckstein M, et al., Chem. Commun. 2004: 1084-1085). In addition, chiral aromatic alcohols were prepared by reducing 4-chloroacetophenone in an ionic liquid/water two-phase system using Lactobacillus cells as a catalyst. The conversion rate and ee value in the ionic liquid [Bmim]Tf ₂ N reached 92.8% and 99.7% respectively (Holger Pfruender, et al. Angew. Chem. Int. Ed. 2004, 43, 4529-4531). It has also been reported in China that the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate/water two-phase reaction system was used to catalyze the reduction of 2'-chloro-acetophenone (2'-C1-AP) to S-2'-chloro-phenylethanol (S-2'-C1-PE), product S-2'-chloro-phenylethanol yield can reach 85.9%, ee value is greater than 99.9% (Hu Jiabin etc., Chemical Reaction Engineering and Process. 2007, 23: 207-211).

The invention adopts the ionic liquid as an excellent solvent to replace the traditional organic solvent, and uses the microbial whole cell to catalyze the asymmetric reduction of the carbonyl compound in the water/ionic liquid two-phase system to prepare the optically active halogenated hydroxy ester. The present invention combines the advantages of the inventor's laboratory research on non-aqueous biocatalysis, based on the original invention "Bacteria and methods for preparing (S)-4-chloro-3-hydroxybutyrate by biocatalysis" (Sun Zhihao et al. CN1778889A) and "Biological On the basis of catalytic preparation of (R)-4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester" (Sun Zhihao CN1948499A), research and development of "asymmetric microbial cell catalysis in water/ionic liquid two-phase system New methods for the reduction of ethyl 4-chloroacetoacetate" and "asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate catalyzed by microbial cells in a water/ionic liquid two-phase system".

4-chloro-3-hydroxybutanoate esters (4-chloro-3-hydroxybutanoate esters, CHBE) is an important organic intermediate, there are multifunctional groups in the molecule, and its chiral single enantiomer (R ) and (S)-CHBE are very promising and important chiral building blocks, and can also introduce other groups to generate the desired chiral drug intermediates through reactions such as chlorine group replacement and reduction. For example, (R)-CHBE can be used to synthesize L-carnitine, (-)-large lactim and (R)-γ-amino-β-butyric acid. It can also be transformed into a synthon of (+)-negamycin and chiral 2,5-cyclohexadienone. On the other hand (S)-CHBE can also be used in the synthesis of many active drugs. It is a key chiral intermediate for the enantioselective synthesis of some important chiral drugs such as statins - hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, and can also be transformed into 1,4 - dihydropyridine beta-blockers.

(R)-4,4,4-trifluoro-3-hydroxybutanoate (Ehtyl(R)-4,4,4-trifluoro-3-hydroxybutanoate, referred to as (R)-TFHBE) is a synthetic An important intermediate of fluorinated compounds, such as it can be used in the synthesis of antidepressant monoamine oxidase-A inhibitor Befloxatone (Befloxatone), can be used as the synthesis of unnatural amino acids L-trifluorothreonine and D-trifluorothreonine The chiral intermediate of acid can also be used in the synthesis of various trifluoromethylated intermediates, such as trifluoromethyl-β-butyrolactone and γ-valerolactone, and ethyl trifluoroglycerate.

Because the latent chiral substrate (4-chloroacetoacetate ethyl ester (COBE) and 4,4,4-trifluoroacetoacetate ethyl ester (TFAAE) of above-mentioned two kinds of products is easy to synthesize and cheap, carry out with it as substrate Asymmetric reduction reaction to obtain the corresponding chiral alcohol is a very effective preparation approach, and the reaction conditions are mild, and the product is not easy to be metabolized by microorganisms. It is very beneficial to prepare chiral halogenated hydroxybutyrates with microbial cell catalysis. The above two methods are currently known The successful approach of the method for preparing chiral alcohol by substrate reduction is the microbial cell catalytic asymmetric reduction method (CN1778889A, CN1948499A) in the water/organic solvent two-phase system, but it is found that the toxicity of the substrate to the microbial cell and the Instability problem in the solvent, conversion rate and product concentration are not high, and the organic dibutyl phthalate that this method selects has a high boiling point, and separation and recovery are difficult.

Contents of the invention

The purpose of the present invention is to provide a new reaction system to effectively improve the preparation of chiral alcohols by catalytic asymmetric reduction reaction of microbial cells, especially the method for catalytic preparation of halogenated hydroxybutyrates, specifically, in the water/ionic liquid two (S)-4-Chloro-3-hydroxybutyrate ethyl ester, or grape Saccharomyces uvarum (Saccharomyces uvarum) ATCC 26602 catalyzes the method for preparing (R)-4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester by asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate. In this way, high optical purity, high reaction yield, and high product concentration can be obtained, and the product separation and extraction method can be simplified at the same time.

Technical scheme of the present invention: the method for the biocatalytic asymmetric reduction of carbonyl compounds in the water/ionic liquid two-phase system, using the selective carbonyl reductase producing bacteria as the starting strain, in the water/ionic liquid two-phase system, the subchiral Ketones are used to reduce substrates to prepare corresponding chiral alcohols. The steps are as follows:

Step (1): Aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.1244 or grape juice yeast (Saccharomyces uvarum) ATCC 26602 were conventionally cultured and fermented respectively, and the fermentation broth was filtered to obtain bacteria, which were used as whole-cell biocatalysts;

Step (2): Use the whole cell biocatalyst of Aureobasidium pullulans CGMCC No.1244 as substrate with 4-chloroacetoacetate (COBE), or the whole cell of grape juice yeast (Saccharomycesuvarum) ATCC 26602 The biocatalyst uses 4,4,4-trifluoroacetoacetate (TFAAE) as a substrate, and uses phosphate buffer solution and ionic liquid as a two-phase system for conversion reaction. The ionic liquid used is 1-butyl-3- Methylimidazolium hexafluorophosphate ([bmim]PF ₆ ), the volume ratio of ([bmim]PF ₆ ) to buffer solution is 0.2:1～2:1, and the substrate concentration is 10～100g/L; the phosphoric acid The salt buffer is 0.1mol/L potassium phosphate buffer solution with pH 6.6-7.0 containing 5% glucose;

Step (3): Enzyme conversion reaction: add wet bacteria to the reaction system, the amount of wet bacteria added is 50-200g/L, the enzyme reaction temperature is 20-35°C, the enzyme conversion time is 1-30 hours, pH5. 5～7.5;

Step (4): Feed-batch conversion reaction: In step (3), add 10-100 g/L of substrate once every two hours for conversion, 4-6 times in a row, and online control of pH 5.5-7.5;

Step (5): The product is obtained after extraction and precipitation: the transformed liquid is centrifuged to separate the bacterial cells, the water phase is removed, the ionic liquid phase is extracted with isopropanol, the product is extracted three times with isopropanol, the combined extracts are collected, and the solvent isopropanol is recovered by evaporation. Propanol, the residue is the corresponding product (S)-4-chloro-3-hydroxybutyric acid ethyl ester, or (R)-4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester;

Step (6): Recovery and use of the ionic liquid: the transformation liquid is centrifuged to separate the bacteria, the water phase is removed, the ionic liquid phase is extracted with isopropanol, the ionic liquid is separated and collected, the pH is adjusted by adding alkali, washed with water until neutral, and the activated carbon is decolorized. The pure ionic liquid recovered after washing with water and drying is reused in step (2).

Microbial strain: Aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.1244, screened for the inventor and preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (www.im.ac.cn), Zhongguancun, Beijing, China, and has been published , Publication No. CN1778889A, on May 31, 2006. Grape juice yeast (Saccharomyces uvarum) ATCC 26602, deposited with the American Type Culture Collection, Rockefeller, MD, USA, (www.atcc.org), publicly available.

The cultivation of step (1) microbial thalline:

A) The medium and culture conditions of Aureobasidium pullulans CGMCC No.1244 are:

Maltose 5-50g/L, yeast extract 10-50g/L, peptone 5-20g/L, ammonium sulfate 1-10g/L, potassium dihydrogen phosphate 1-5g/L, magnesium sulfate 0.1-1g/L, pH 4-9, temperature 20-40°C, culture for 1-5 days.

B) medium and culture conditions of grape juice yeast ATCC 26602 are:

Sodium acetate 10-100g/L, corn steep liquor 10-100g/L, potassium dihydrogen phosphate 1-10g/L, magnesium sulfate 0.1-1g/L, pH 4-9, temperature 20-40°C, culture for 1-5 days ;

Ionic liquids (ILs) are used in the reaction process, and the use of ILs is used to dissolve products and substrates, so as to inhibit the toxicity of substrates and products to bacteria, and at the same time improve the conversion rate and optical purity of products. In addition to 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF ₆ ), the ionic liquid can also be 1-butyl-2,3-dimethylimidazolium hexafluorophosphate ([bmmim] ]PF ₆ ), 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF ₄ ), N-octylpyridine tetrafluoroborate ([opy]BF ₄ ), 1-butyl - One of 3-methylimidazolium triflate ([bmim]MeF ₃ ). The ionic liquid was purchased from Henan Lihua Pharmaceutical Co., Ltd. and Hangzhou Comer Chemical Co., Ltd.

The specific operation of extracting the product is as follows: after the conversion reaction is completed, centrifuge the reaction solution (8,000 rpm, 20 minutes, 4°C), take out the ionic liquid in the lower layer and extract it three times with an equal volume of isopropanol, combine the extracts, and pour 1% to 5% (w/v) of anhydrous magnesium sulfate is added therein, stirred evenly and left standing overnight to remove residual moisture. The ionic liquid was filtered to remove anhydrous magnesium sulfate. The solvent was recovered by rotary evaporation in a water bath at 80°C to obtain an oily liquid product, which was analyzed and determined by GC, and compared with the spectrum of a standard product (Sigma Co.), it was determined to be the same substance.

Determination of the concentration of substrates COBE and CHBE: using VARIAN CP WAX 52CB polar chromatographic column (30m×0.25mm×0.25μm), the analysis conditions are vaporization chamber temperature 250°C, detector temperature 250°C, column temperature 100°C for 2 minutes , the temperature was raised at 8°C/min to 240°C and maintained for 2min, the carrier gas was hydrogen, the flow rate was 2.0mL/min, and the split ratio was 1:50.

Determination of the enantiomeric excess value of the product: use a chiral chromatographic column CP-Chirasil Dex CB (0.25mm×25m×0.25μm). Product processing method: Take the supernatant and extract with ethyl acetate to obtain 0.5 mL of extract, and evaporate to remove the solvent. Then, 2 drops of acetic anhydride and 2 drops of pyridine were added dropwise, placed in a boiling water bath for 1 h, and diluted with 5 mL of ethyl acetate after cooling. The analysis conditions are as follows: gasification chamber temperature 250°C, detector temperature 250°C, column temperature 100°C for 2 minutes, temperature rise to 130°C at 2°C/min and hold for 1 minute, then heat up to 180°C at 5°C/min and hold for 2 minutes, carrier gas It is hydrogen, the flow rate is 2.0mL/min, and the split ratio is 1:50.

Concentration determination of TFAAE and TFHBE: VARIAN CP WAX 52CB polar chromatographic column (30m×0.25mm×0.25μm) was used, the analysis conditions were vaporization chamber temperature 250°C, detector temperature 250°C, column temperature 80°C for 2min, and Raise the temperature at 10°C/min to 180°C and hold for 5min, the carrier gas is hydrogen, the flow rate is 2.0mL/min, and the split ratio is 1:50.

Determination of the enantiomeric excess value of the product: separated by a chiral chromatographic column CP-Chirasil Dex CB (25m×0.25mm×0.25μm), the analysis conditions are vaporization chamber temperature 250°C, detector temperature 250°C, column temperature 100°C Hold for 2 minutes, raise the temperature at 2°C/min to 130°C and maintain for 1 minute, then raise the temperature at 5°C/min to 180°C and hold for 2 minutes. The carrier gas is hydrogen, the flow rate is 2.0mL/min, and the split ratio is 1:50.

Beneficial effects of the present invention: the present invention selects a new type of two-phase system to catalyze the asymmetric reduction of latent chiral carbonyl ester compounds with whole cells of microorganisms, and the product concentration, conversion rate and The optical purity is improved, such as (S)-CBHE concentration can reach 75.1g/L (fed-batch method), the conversion rate is 96.5%, and the enantiomeric excess value reaches up to 98.2% e.e. The highest concentration of (R)-TFHBE can reach 42.0g/L, the highest conversion rate is 90.3%, and the highest enantiomeric excess value is 85.4% e.e. Compared with other catalytic reaction systems, the present invention has the following advantages: 1) the ionic liquid system is non-toxic and tasteless, not volatile, has good biocompatibility, and does not pollute the environment; 2) the product separation in the ionic liquid system is simple, and the ionic liquid itself is easy to recycle, It can be used repeatedly without obvious influence on the conversion rate and enantiomeric excess value of the product; 3) the ionic liquid system can promote the improvement of product concentration, conversion rate and optical purity.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. However, these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific experimental conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are generally followed.

Example 1

Choice of ionic liquid

A. Selection of Ionic Liquids in Aureobasidium pullulans CGMCC No.1244 Asymmetric Reduction of COBE to Form (S)-CBHE

Slant culture: the medium is 100mL wort, add 2g glucose, 2g agar, pH6.5, sterilize at 121°C for 20 minutes according to the usual method, cool down after sterilization to inoculate the slant, cultivate at 30°C for 2 days, and use it as an activated seed on the slant .

Seed cultivation and fermentation: medium: maltose 30g/L, yeast extract 20g/L, peptone 5g/L, (NH ₄ ) ₂ SO ₄ 5g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.7g/L, pH 6.0. The filling volume is 80mL of 500mL Erlenmeyer flask, sterilized at 121°C for 20 minutes, cooled and connected to slanted seeds after sterilization, shaken at 180 rpm, and cultivated at 30°C for 2 days, as seeds or fermentation enzyme liquid (whole cell catalyst).

The content of wet cells in the fermented enzyme liquid is 2.5g/100mL, centrifuge for 10 minutes (8,000 rpm) to collect the cells, wash twice with potassium phosphate buffer (0.1mol/L, pH6.6), transfer the cells to Put it into 10mL of the same buffer solution containing glucose, mix it with an equal volume of ionic liquid or organic solvent (for comparison), add 35g/L substrate COBE at the same time, and react at 30°C and 180 rpm for 8h. After the reaction is over, remove the bacteria and the water phase by centrifugation to obtain an ionic liquid phase or an organic phase (for comparison), extract the ionic liquid with isopropanol, separate and collect the extract, dry an appropriate amount of anhydrous magnesium sulfate overnight, and perform gas chromatography analysis after filtration . The results are shown in Table 1:

Table 1 Selection of Aureobasidium pullulans CGMCC No.1244 asymmetric reduction COBE ionic liquid

two-phase system Conversion rate(%) e.e.(%) Water single phase 35.9 96.1 Water/Dibutyl Phthalate 90..0 96.6 water/n-hexane 38.7 96.9 water/[bmim]PF ₆ 94.2 96.6 water/[bmmim]PF ₆ 50.1 97.3 water/[bmim]BF ₄ 32.0 97.3 water/[opy]BF ₄ - - water/[bmim]MeF ₃ - -

It can be seen from Table 1 that the asymmetric reduction of COBE by Aureobasidium pullulans CGMCC No.1244 in water/[bmim]PF ₆ to generate (S)-CBHE is better.

B. Selection of Ionic Liquids in the Reaction of (R)-TFBHE by Asymmetric Reduction of TFAAE by Grape Juice Yeast ATCC 26602

Slope culture: the medium is 100mL wort, add 2g glucose, 2g agar, pH 6.5, sterilize at 121°C for 20 minutes, cool and inoculate after sterilization, cultivate at 30°C for 2 days, and use it as slant activated seeds.

Seed cultivation and fermentation: medium: maltose 2g/L, yeast extract 2g/L, peptone 0.5g/L, sodium acetate 2g/L, ammonium sulfate 0.5g/L, potassium dihydrogen phosphate 0.2g/L, magnesium sulfate 0.01g/L, pH 6. The filling volume is 80mL of 500mL Erlenmeyer flask, sterilized at 121°C for 20 minutes, cooled and connected to slant seeds after sterilization, shaken at 180 rpm, and cultivated at 30°C for 2 days, as seeds or fermented enzyme liquid.

The wet bacteria content in the fermented enzyme liquid was 2.5g/100mL, and the bacteria were collected by centrifugation for 10 minutes (8,000 rpm), washed twice with potassium phosphate buffer (0.1mol/L, pH7.0), and the bacteria were transferred to Pour into 10mL of the same buffer solution containing glucose, mix with an equal volume of ionic liquid or organic solvent (for comparison), add 30g/L substrate TFAAE at the same time, and react at 30°C and 180 rpm for 8h. After the reaction is finished, the cells and the water phase are removed by centrifugation to obtain an ionic liquid or an organic phase (for comparison), the ionic liquid is extracted with isopropanol, the extract is separated and collected, an appropriate amount of anhydrous magnesium sulfate is dried overnight, and gas chromatographic analysis is performed after filtration. The results are shown in Table 2:

Table 2 Selection of ionic liquids in grape juice yeast ATCC 26602 asymmetric reduction of TFAAE

two-phase system Conversion rate(%) e.e.(%) Water single phase 56.7 80.4 Water/Dibutyl Phthalate 74.6 79.0 water/n-hexane 11.9 64.0 water/[bmim]PF ₆ 77.9 83.5 water/[bmmim]PF ₆ 68.9 65.3 water/[bmim]BF ₄ 62.4 67.9 water/[opy]BF ₄ - - water/[bmim]MeF ₃ - -

It can be seen from Table 2 that grape juice yeast ATCC 26602 also has a better effect of catalyzing the asymmetric reduction of TFAAE in water/[bmim]PF ₆ to generate (R)-TFBHE.

Example 2

Effect of different phase volume ratios on transformation reactions in water/ionic liquid two-phase system

After Aureobasidium pullulans CGMCC No.1244 or grape juice yeast ATCC 26602 were cultured for 24 hours to produce enzyme according to the method in Example 1, weigh 1 g of wet bacteria and add 10 mL of 0.1 mol/L, pH 6.6 to 7.0 potassium phosphate buffer in a 150 mL Erlenmeyer flask In , the substrate content is 30g/L, and the ionic liquid [bmim]PF ₆ with different phase volume ratios are added respectively, and the volume ratio of ionic liquid and buffer solution is 1:5, 5:5, 10:5, 15:5 , 20:5, reacted at 30°C, 180 rpm for 8h. After the reaction, the reaction solution was centrifuged to remove the bacteria and the water phase to obtain the ionic liquid phase, which was extracted three times with an equal volume of isopropanol, the combined extracts were dried with an appropriate amount of anhydrous magnesium sulfate, filtered and then analyzed by gas chromatography. The results are shown in Table 3 :

Table 3 Effect of phase volume ratio on conversion reaction in water/ionic liquid two-phase system

It can be seen from Table 3 that in the water/ionic liquid two-phase system, Aureobasidium pullulans CGMCC No.1244 or grape juice yeast ATCC 26602 catalyze the asymmetric reduction reaction, when the volume ratio of ionic liquid and buffer solution is 1:1 Better results were obtained for conversion and enantiomeric excess values.

Example 3

Effects of Different Temperatures on Transformation Reactions in Water/Ionic Liquid Two-Phase System

After Aureobasidium pullulans CGMCC No.1244 or grape juice yeast ATCC 26602 were cultured for 24 hours according to the method of Example 1, 1 g of wet bacteria was weighed and added to 5 mL of 0.1 mol/L, pH 6.6-7.0 potassium phosphate buffer in a 50 mL Erlenmeyer bottle , mixed with an equal volume of ionic liquid [bmim]PF ₆ , with a substrate content of 35g/L, and investigated the effects of different temperatures on the conversion reaction, at temperatures of 20°C, 25°C, 30°C, 35°C React at 40°C and 180 rpm for 8 hours. After the reaction, the reaction solution was centrifuged to remove the thallus and the water phase to obtain an ionic liquid phase, which was extracted three times with an equal volume of isopropanol, and the extracts were combined, dried with an appropriate amount of anhydrous magnesium sulfate, filtered and then analyzed by gas chromatography. The results are shown in Table 4 :

Table 4 Effects of different temperatures on transformation reactions in water/ionic liquid two-phase system

Example 4

Effects of different substrate concentrations on transformation reactions in water/ionic liquid two-phase system

After Aureobasidium pullulans CGMCC No.1244 or grape juice yeast ATCC 26602 were cultured for 24 hours according to the method of Example 1 for enzyme production, 1 g of wet bacteria was weighed and added to 5 mL of 0.1 mol/L, pH 6.6 to 7.0 potassium phosphate buffer in a 50 mL Erlenmeyer flask solution, mixed with an equal volume of ionic liquid [bmim]PF ₆ , respectively added substrates containing 25g/L, 36g/L, 49g/L, 60g/L, 72g/L, at 30°C, 180 rpm Sub-reaction 8h. After the reaction, the reaction solution was centrifuged to remove the thallus and the water phase to obtain the ionic liquid phase, which was extracted three times with an equal volume of isopropanol, and the extracts were combined, dried with an appropriate amount of anhydrous magnesium sulfate, filtered and then analyzed by gas chromatography. The results are shown in Table 5 :

Table 5 Effects of different substrate concentrations on conversion reactions in water/ionic liquid two-phase system

Example 5

Biocatalytic asymmetric reduction transformation time curve in water/ionic liquid two-phase system

A. Conversion time curve of Aureobasidium pullulans CGMCC No.1244 asymmetric reduction COBE to (S)-CBHE reaction in water/ionic liquid two-phase system

Aureobasidium pullulans CGMCC No.1244 was cultured according to the method of Example 1A for enzyme production, and according to the optimized conditions of Examples 2 to 4, 1 g of wet bacteria was weighed and added to a 50 mL Erlenmeyer flask containing 5 mL of 0.1 mol/L, pH 6.6 phosphoric acid In the potassium buffer, mix with an equal volume of ionic liquid [bmim]PF ₆ , add 49g/L COBE, react at 30°C, 180 rpm, and sample at different times. The reaction solution was centrifuged to remove the bacteria and the water phase to obtain the ionic liquid phase, which was extracted three times with an equal volume of isopropanol, the extracts were combined, dried with an appropriate amount of anhydrous magnesium sulfate, filtered and then analyzed by gas chromatography. The results are shown in Table 6:

Table 6 Conversion time curve of CGMCC No.1244 asymmetric reduction reaction in water/ionic liquid two-phase system

Response time (h) Conversion rate% e.e.% Concentration of (S)-CHBE (g/L) 0.25 11.8 97.5 5.8 0.5 25.4 96.8 12.4 1 36.7 97.2 18.0 1.5 55.8 96.7 27.3 2 66.8 96.6 32.7 3 87.7 97.4 43.0 4 88.7 97.0 43.5 5 95.4 96.8 46.7 6 93.1 97.4 45.6 7 92.9 97.6 45.5

B. The conversion time curve of the asymmetric reduction of TFAAE by grape juice yeast ATCC 26602 in the water/ionic liquid two-phase system to generate (R)-TFBHE

Grape juice yeast ATCC 26602 was cultured according to the method of Example 1B for enzyme production, and according to the optimized conditions of Examples 2 to 4, 1g of wet bacteria was weighed and added to 5mL of 0.1mol/L, pH7.0 potassium phosphate buffer in a 50mL Erlenmeyer flask , mixed with an equal volume of ionic liquid [bmim]PF ₆ , added 38g/L TFAAE, reacted at 30°C, 180 rpm, and samples were taken at different times. The reaction solution was centrifuged to remove the bacteria and the water phase to obtain the ionic liquid phase, which was extracted three times with an equal volume of isopropanol, the extracts were combined, dried with an appropriate amount of anhydrous magnesium sulfate, filtered and then analyzed by gas chromatography. The results are shown in Table 7:

Conversion time curve of ATCC26602 asymmetric reduction reaction in water/ionic liquid system in table 7

Reaction time Conversion rate e.e.% Concentration of (R)-TFBHE (g/L) 1 7.3 80.7 2.8 2 24.1 81.6 9.2 3 36.6 78.8 13.9 4 54.5 79.0 20.7 5 60.3 81.1 22.9 6 65.4 84.6 24.9 7 68.1 84.9 25.9 8 72.0 84.6 27.4 9 79.6 82.7 30.2 10 85.7 83.1 32.6 11 85.4 79.4 32.5 12 85.2 81.0 32.4 13 84.7 79.9 32.2 14 84.9 81.4 32.3 15 84.2 78.8 32.0

Example 6

Biocatalytic Asymmetric Reductive Transformation Reaction in Water/Ionic Liquid Two-Phase System with Substrate Addition in Batches

Aureobasidium pullulans CGMCC No.1244 was cultured for 24 hours according to the method of Example 1A for enzyme production, and transformed according to the method of Example 5A. In 0.1mol/L, 20 mL of pH6.6 potassium phosphate buffer, 4 g of wet thallus was added, and divided into four times The substrate COBE was added at 0h, 2h, 6h, and 8h respectively, adding 480mg each time (the total concentration of the substrate added was 24g/L). The pH was kept at about 5.5-7.5, and the reaction was sampled at regular intervals for gas chromatography analysis. The results are shown in Table 8:

Table 8 Water/ionic liquid two-phase system The conversion reaction results of adding substrate in batches

Reaction time Conversion rate e.e.% Concentration of (S)-CHBE (g/L) 2 97.5 97.3 23.4 4 72.3 96.7 34.7 6 95.4 97.7 45.8 7 84.3 98.2 60.7 8 78.2 97.2 75.1 10 77.3 96.7 73.4 12 73.7 97.5 70.8

Example 7

Water/ionic liquid two-phase system biocatalytic asymmetric reduction reaction of ionic liquid recovery and reuse

Aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.1244 was cultured for 24 hours according to the method of Example 1A for enzyme production, and transformed according to the method of Example 5A to obtain the transformation liquid and centrifuge the thalline, remove the water phase, and use twice the volume of ionic liquid Extract the product therein with isopropanol, and extract about 2 to 3 times. Separating and taking out the ionic liquid phase, adding an alkalizing agent such as sodium hydroxide, potassium hydroxide, ammonia water, sodium carbonate, etc. to the ionic liquid or a mixture of one or more of them to make the ionic liquid pH ≥ 7; stir the mixed solution at room temperature , so that the neutralization reaction is complete; static layering, take the ionic liquid phase and add water, wash until neutral; static layering, use heating and vacuum distillation to fully dehydrate the ionic liquid, and then put it in a drying oven at 80 ° C for 24 hours above. The recovery yield of the ionic liquid can reach more than 90%. The dried ionic liquid was transformed according to the method of Example 5A, and was recycled and used 6 times in this way. The results are shown in Table 9:

Table 9 Water/ionic liquid two-phase system biocatalytic reaction ionic liquid recovery and repeated use results

usage count Conversion rate e.e.% 1 96.7 97.2 2 95.4 98.4 3 96.2 97.3 4 94.3 96.7 5 94.6 97.7 6 92.1 96.4

Claims (1)

1. A method for the biocatalytic asymmetric reduction of carbonyl compounds in a water/ionic liquid two-phase system, characterized in that a selective carbonyl reductase producing bacterium is used as a starting strain, and in a water/ionic liquid two-phase system, a latent chiral ketone To prepare the corresponding chiral alcohol for substrate reduction, the steps are as follows: Step (1): Aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.1244 or grape juice yeast (Saccharomyces uvarum) ATCC 26602 were conventionally cultured and fermented respectively, and the fermentation broth was filtered to obtain bacteria, which were used as whole-cell biocatalysts; Step (2): Use the whole cell biocatalyst of Aureobasidium pullulans CGMCC No.1244 with 4-chloroacetoacetate COBE as the substrate, or the whole cell biocatalyst of grape juice yeast (Saccharomycesuvarum) ATCC 26602 With 4,4,4-trifluoroacetoacetate ethyl TFAAE as the substrate, the transformation reaction is carried out with phosphate buffer solution and ionic liquid as a two-phase system, and the ionic liquid used is 1-butyl-3-methylimidazole hexa Fluorophosphate [bmim]PF ₆ , the volume ratio of [bmim]PF ₆ to buffer solution is 0.2:1～2:1, and the substrate concentration is 10～100g/L; the phosphate buffer solution contains 5% glucose Potassium phosphate buffer solution of 0.1mol/L, pH 6.6-7.0; Step (3): Enzyme conversion reaction: add wet bacteria to the reaction system, the amount of wet bacteria added is 50-200g/L, the enzyme reaction temperature is 20-35°C, the enzyme conversion time is 1-30 hours, pH5. 5～7.5; Step (4): Feed-batch conversion reaction: In step (3), add 10-100 g/L of substrate once every two hours for conversion, 4-6 times in a row, and online control of pH 5.5-7.5; Step (5): The product is obtained after extraction and precipitation: the transformed liquid is centrifuged to separate the bacterial cells, the water phase is removed, the ionic liquid phase is extracted with isopropanol, and the product is extracted three times with isopropanol, and the combined extracts are collected and evaporated to recover the solvent isopropanol. Propanol, the residue is the corresponding product (S)-4-chloro-3-hydroxybutyric acid ethyl ester, or (R)-4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester; Step (6): Recovery and use of the ionic liquid: the transformation liquid is centrifuged to separate the bacterial cells, the water phase is removed, the ionic liquid phase is extracted with isopropanol, the ionic liquid is separated and collected, the pH is adjusted by adding alkali, washed with water until neutral, and the activated carbon is decolorized. The pure ionic liquid recovered after washing with water and drying is reused in step (2).