Carbonyl reductase with high stereoselectivity, mutant and application of carbonyl reductase in asymmetric synthesis of (S)-glassine

Abstract

The invention discloses a carbonyl reductase with high stereoselectivity, a mutant and application of the carbonyl reductase in asymmetric synthesis of (S)-glass.The carbonyl reductase and the mutant can both catalyze asymmetric reduction of 50-1000 mM beta-acetone xyloside to prepare (S)-glassine, the substrate conversion rate is larger than 99%, the product is optically pure (S)-glassine, and the carbonyl reductase and the mutant can be used for preparing the (S)-glassine. The chiral purity is greater than 99%, and the method has relatively high industrial application potential.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a highly stereoselective carbonyl reductase, a mutant and an application thereof in asymmetric synthesis of (S)-bosone. (II) Background technology

[0002] Pro-Xylane, chemical name is hydroxypropyl tetrahydropyrantriol, molecular formula C8H 18 O5, molecular weight 192.21, has a wide range of biological activities. Bosylamine was first discovered by L'Oreal from beech trees. It is a xylose derivative. Studies have shown that bosylamine can promote the synthesis of glycosaminoglycans (GAGs), increase the production of dermal collagen, promote dermal repair, improve skin elasticity, reduce wrinkles and increase skin firmness, thereby achieving the effect of resisting aging. Bosylamine has a variety of stereo configurations. According to literature reports (Bioorg. Med. Chem. Lett., 2009, 19 (3), 845-849), (S)-bosylamine has the best biological activity.

[0003] At present, the synthesis methods of (S)-bosylamine are divided into chemical method and biological method. The chemical synthesis method was developed by L'Oreal Company. It uses xylose and acetylacetone as raw materials. Under alkaline conditions, the condensation reaction is carried out for 12 hours to generate β-acetone xyloside. The carbonyl group of β-acetone xyloside is reacted with sodium borohydride for 12 hours, and the reduction reaction is used to generate bosylamine. Pure chemical synthesis is not only difficult to control stereoselectivity, but also faces problems such as high energy consumption, environmental pollution, harsh reaction conditions and difficulty in subsequent separation and purification.

[0004] The bioenzymatic method uses β-acetone xyloside as substrate, glucose or isopropanol or ammonium formate and oxidized coenzyme as auxiliary materials, and synthesizes (S)-Bosone under the catalysis of different carbonyl reductases. Compared with the chemical method, the bioenzymatic method has the advantages of high stereoselectivity, mild reaction conditions, environmental friendliness, and no metal residue. Despite its many advantages, the recently reported enzymatic method for synthesizing (S)-Bosone still has some shortcomings, such as low substrate concentration (CN118345057A), difficulty in product separation due to sugar byproducts (CN118291410A, CN117737149B), long catalytic time, low efficiency (CN115896199A), etc., and it is still necessary to develop industrial enzyme preparations for the efficient synthesis of high-concentration (S)-Bosone to meet the needs of industrial production. (III) Summary of the invention

[0005] In view of the problems existing in the process of preparing (S)-Bosone in the prior art, the present invention provides a carbonyl reductase and mutant with high stereoselectivity and application in the asymmetric synthesis of (S)-Bosone. The carbonyl reductase and mutant screened and obtained by the present invention have high enzyme activity and stereoselectivity, can catalyze high-concentration substrates to efficiently synthesize high-concentration (S)-Bosone in a relatively short time, and solve the problems of low substrate concentration, long reaction time and low efficiency in the prior enzymatic synthesis of (S)-Bosone.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a highly stereoselective carbonyl reductase, wherein the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.1.

[0008] The present invention also relates to a gene encoding the carbonyl reductase, the nucleotide sequence of which is shown in SEQ ID NO.2; a recombinant expression vector containing the encoding gene, and a recombinant genetically engineered bacterium prepared by transforming the recombinant expression vector; the base vector of the recombinant expression vector is preferably pET28a; and the host bacterium of the recombinant genetically engineered bacterium is preferably E. coli BL21 (DE3).

[0009] In a second aspect, the present invention also provides an application of the carbonyl reductase in the asymmetric reduction of β-acetone xyloside to prepare (S)-bosone, wherein the application method is: using a crude enzyme liquid obtained by fermentation and culturing a recombinant genetically engineered bacterium containing the carbonyl reductase after crushing and centrifugation of wet bacteria or a crude enzyme powder obtained by freeze-drying the crude enzyme liquid as a catalyst, using β-acetone xyloside as a substrate, and using NADP + The reaction system is formed by using isopropanol as a coenzyme, a buffer solution of pH 6-9 as a reaction medium, and an asymmetric hydrogenation reaction is carried out at 20-40° C. and 100-300 rpm to obtain a reaction solution containing (S)-bosone, which is separated and purified to obtain (S)-bosone.

[0010] Furthermore, in the reaction system, the substrate is added at a concentration of 50-1000 mM (preferably 1000 mM); the catalyst is added at an amount of 0.1-2 g / L (preferably 1 g / L) based on the protein content of the crude enzyme solution, and at an amount of 1-10 g / L (preferably 10 g / L) based on the weight of the crude enzyme powder; NADP + The added concentration was 0.1 mM, and the volume concentration of isopropanol added was 10% (v / v).

[0011] Furthermore, the reaction medium was 100 mM PBS buffer with a pH value of 7.0.

[0012] In a third aspect, the present invention provides a mutant of the carbonyl reductase, wherein the mutant is obtained by performing single-site mutation or combined mutation at positions 87, 130, and 161 of the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the carbonyl reductase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: the serine at position 87 is mutated into phenylalanine, the alanine at position 130 is mutated into serine, and the leucine at position 161 is mutated into phenylalanine; or the serine at position 87 is mutated into phenylalanine + the alanine at position 130 is mutated into serine + the leucine at position 161 is mutated into phenylalanine.

[0014] Furthermore, the amino acid sequence of the carbonyl reductase mutant is shown in SEQ ID NO.3.

[0015] In a fourth aspect, the present invention also provides a coding gene for the carbonyl reductase mutant, preferably as shown in SEQ ID NO.4; a recombinant expression vector containing the coding gene, and a recombinant genetically engineered bacterium prepared by transforming the recombinant expression vector; the basic vector of the recombinant expression vector is preferably pET28a; the host bacteria of the recombinant genetically engineered bacterium is preferably E. coli BL21 (DE3).

[0016] In a fifth aspect, the present invention further provides an application of the carbonyl reductase mutant in the asymmetric reduction of β-acetone xyloside to prepare (S)-bosone, wherein the application method comprises: using a crude enzyme liquid obtained by fermentation and culturing a recombinant genetically engineered bacterium containing the carbonyl reductase mutant and crushing and centrifuging wet cells or a crude enzyme powder obtained by freeze-drying the crude enzyme liquid as a catalyst, using β-acetone xyloside as a substrate, and using NADP + The reaction system is formed by using isopropanol as a coenzyme, a buffer solution of pH 6-9 as a reaction medium, and an asymmetric hydrogenation reaction is carried out at 20-40° C. and 100-300 rpm to obtain a reaction solution containing (S)-bosone, which is separated and purified to obtain (S)-bosone.

[0017] Furthermore, in the reaction system, the substrate is added at a concentration of 50-1000 mM (preferably 1000 mM); the catalyst is added at an amount of 0.1-2 g / L (preferably 1 g / L) based on the protein content of the crude enzyme solution, and at an amount of 1-10 g / L (preferably 10 g / L) based on the weight of the crude enzyme powder; NADP + The added concentration was 0.1 mM, and the volume concentration of isopropanol added was 10% (v / v).

[0018] Furthermore, the reaction medium was 100 mM PBS buffer with a pH value of 7.0.

[0019] Further, the catalyst is prepared as follows:

[0020] The recombinant genetically engineered bacteria were inoculated into LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 180 rpm; the culture solution was inoculated into LB medium containing 50 μg / mL kanamycin at a volume concentration of 1% (v / v), and cultured on a shaker at 37°C and 180 rpm. When the OD of the culture solution reached 600 When the pH value reaches 0.6, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM is added as an inducer. After induction at 16°C for 24 hours, the culture solution is centrifuged to collect the wet bacteria, which are then washed twice with physiological saline. The wet bacteria are suspended in PBS buffer (100 mM, pH 7) at an amount of 10 mL / g, and ultrasonically disrupted with an ultrasonic disruptor under an ice-water bath (at 300 W power, working for 3 seconds, resting for 6 seconds, and working for 15 minutes), followed by centrifugation at 4°C, 12000 rpm in a low-temperature high-speed centrifuge for 30 minutes, and the supernatant is collected to obtain the crude enzyme solution; the crude enzyme solution is frozen at -80°C, and then freeze-dried at -40°C for 24 hours using a vacuum freeze dryer to obtain the crude enzyme powder.

[0021] LB medium composition: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, pH 7.0, prepared with deionized water.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) Compared with the existing microbial enzymatic method for synthesizing (S)-bosone, the crude wild-type carbonyl reductase powder screened by the present invention can catalyze 50-1000mM β-acetone xyloside to synthesize (S)-bosone at 10-100g / L and 12-48h, with a substrate conversion rate of more than 99% and an ee value greater than 99%, which has certain industrial application potential.

[0024] (2) The enzyme activity of the carbonyl reductase mutant further screened by the present invention is increased by 10-20 times compared with the wild type. The crude enzyme powder can catalyze 50-1000mM β-acetone xyloside to synthesize (S)-bosone at 1-10g / L and 6-24h. The substrate conversion rate can reach more than 99%, and the ee value is greater than 99%, which has a high potential for industrial application.

[0025] (3) The reaction system of the present invention using wild-type carbonyl reductase and mutants as catalysts for asymmetric reduction of β-acetone xyloside to prepare (S)-bosone does not require the additional addition of glucose dehydrogenase to participate in the coenzyme cycle. The coenzyme self-circulation can be completed by adding the cosubstrate isopropanol, which greatly reduces the amount of catalyst and coenzyme used in the reaction; the reaction solution only needs to be desalted and decompressed to remove the solvent, and the purity is greater than 99%. (IV) Description of the drawings

[0026] Figure 1 , the reaction formula for preparing (S)-bosone by asymmetric reduction of β-acetone xyloside by the carbonyl reductase and mutant of the present invention.

[0027] Figure 2 , HPLC chromatogram; A represents substrate standard, B represents (S)-bosone and (R)-bosone standard; C represents the supernatant of Example 8. (V) Specific implementation methods

[0028] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto: The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product instructions. The room temperature is the conventional room temperature in the art, and the room temperature range is 20-40°C.

[0029] Example 1: Construction of wild-type carbonyl reductase ZmCR recombinant plasmid

[0030] The base sequence of carbonyl reductase (ZmCR) from Zygofabospora marxiana (GenBank: BAP72132.1) was optimized for Escherichia coli codon preference, and a recombinant gene ZmCR sequence with a length of 1032 bp was synthesized by BGI Co., Ltd. (Hangzhou) (the base sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1). The recombinant gene ZmCR was inserted into the vector pET-28a (+), and the NdeI restriction site and XhoI restriction site were selected to construct the pET-28a-ZmCR-WT recombinant plasmid.

[0031] Example 2: Construction of wild-type carbonyl reductase ZmCR recombinant genetic engineering bacteria

[0032] The recombinant expression vector pET-28a-ZmCR-WT was transformed into Escherichia coli BL21 (DE3) competent cells using the heat shock method familiar to those skilled in the art to obtain recombinant Escherichia coli BL21 (DE3) / pET-28a-ZmCR-WT, and a single colony was picked for the next experiment.

[0033] Example 3: Protein expression of recombinant genetically engineered bacteria

[0034] The recombinant E. coli BL21 (DE3) / pET-28a-ZmCR-WT constructed in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37 ° C, 180 rpm. The inoculum was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculum and cultured in a shaker at 37 ° C, 180 rpm. When the OD of the culture solution reached 600 When it reaches 0.6, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2mM is added as an inducer. After induction at 16°C for 24h, the culture solution is centrifuged to collect the wet bacteria, and then washed twice with physiological saline. The wet bacteria are suspended in PBS buffer (100mM, pH 7.0) at an amount of 10mL / g, and ultrasonically disrupted with an ultrasonic disruptor under an ice-water bath (at 300W power, working for 3s, resting for 6s, working for 15min), followed by centrifugation at 4°C, 12000rpm for 30min in a low-temperature high-speed centrifuge, and the supernatant is collected to obtain the crude enzyme solution of the wild-type carbonyl reductase ZmCR-WT.

[0035] Example 4: Construction of carbonyl reductase ZmCR mutant

[0036] 1. Selection of mutation sites

[0037] The structural model of the wild-type carbonyl reductase ZmCR-WT was constructed using AlphaFold2. After the substrate β-acetone xyloside was connected to the active center, the interaction between ZmCR-WT and the substrate was analyzed. The sites that were screened to be closer to the substrate were positions 87, 130, and 161. By introducing mutations at positions 87, 130, and 161, the interaction between the enzyme and the substrate was strengthened to enhance the enzyme activity.

[0038] 2. Single-site directed saturation mutagenesis

[0039] (1) Primer design

[0040] According to the full-length gene sequence of carbonyl reductase ZmCR obtained in Example 1 (SEQ ID NO. 2) and the mutation sites screened in step 1, site-directed saturation mutagenesis primers at positions 87, 130, and 161 were designed, respectively, as shown in Table 1, and the mutation sites are underlined.

[0041] Table 1. Site-directed saturation mutagenesis primers

[0042]

[0043] In Table 1, N represents A / C / G / T, M represents A / C, and K represents G / T.

[0044] (2) Site-directed saturation mutagenesis

[0045] The pET-28a-ZmCR-WT plasmid DNA of E.coli BL21(DE3) / pET-28a-ZmCR-WT in Example 2 was extracted as a template, and mutations were introduced by PCR. The PCR reaction procedure was as follows: 98°C pre-denaturation for 3min; 98°C denaturation for 10s, 55°C annealing for 5s, 72°C extension for 3min, repeated 25 cycles; 72°C continued extension for 10min. The PCR product was treated with DpnI at 37°C for 3h, then transformed into the E.coli BL21(DE3) host bacteria, coated on an LB solid plate containing a final concentration of 50mg / L kanamycin resistance, and cultured at 37°C for 12h. Single colonies were randomly picked for sequencing analysis to obtain a mutant library.

[0046] (3) Single-site mutant screening

[0047] The enzyme activity of the mutant library in step (2) was tested by the method of Example 5, and three groups were set up in parallel. The results are shown in Table 2. The dominant mutants obtained by screening were: S mutated to F at position 87, A mutated to S at position 130, and L mutated to F at position 161.

[0048] Table 2. Enzyme activities of mutants

[0049]

[0050] 3. Combination mutation

[0051] On the basis of step 2, primers in Table 1 were used for combined mutation, and the enzyme activity was tested using the method in Example 5. Three groups were set up in parallel. The results are shown in Table 3. The optimal mutant ZmCR was screened and obtained. S87F / A130S / L161F (S at position 87 was mutated to F, A at position 130 was mutated to S, and L at position 161 was mutated to F), and the amino acid sequence is shown in SEQ ID NO. 3. The crude enzyme solution of the mutant was obtained in the same manner as in Example 3.

[0052] Table 3. Enzyme activity of combined mutants

[0053]

[0054] Example 5: Determination of the activity of carbonyl reductase ZmCR and its mutants

[0055] The crude enzyme solutions obtained in Examples 3 and 4 were respectively subjected to activity assays. Activity assay reaction system 1 mL: final concentration 10 mM β-acetone xyloside, 0.1 mM NADP +, 1g / L (based on protein content) crude enzyme solution, 10% (v / v) isopropanol, 100mM potassium phosphate buffer solution (pH 7.0) to 1mL. React at 30℃, 200rpm for 5min. After the reaction, take 100μL of the reaction solution and add 10μL 2M NaOH solution to terminate the reaction. Centrifuge at 12000rpm for 1min, take the supernatant and pass it through a 0.22μm membrane. Detect the characteristic peak area by high performance liquid chromatography (HPLC). Calculate the content of (S)-bosine based on the standard curve of (S)-bosine standard concentration and peak area. HPLC detection conditions: instrument Agilent 1260InfinityⅡ (Agilent Technologies, USA), chromatographic column Xtimate C18 (4.6×250mm, 5μm), detector is differential detector, mobile phase is ultrapure water, 10μL injection volume, 1mL / min flow rate, column temperature 30℃. The HPLC test results of substrate and product standards are shown in Figure 2 As shown in A and B.

[0056] Enzyme activity definition: Under the above conditions, the amount of enzyme required to produce 1 μmol (S)-bosone per minute is one enzyme activity unit U.

[0057] Specific activity: enzyme activity per unit mass of sample, U / mg.

[0058] The results of enzyme activity assay showed that the enzyme activity of wild-type ZmCR-WT was 0.12 U / mg based on protein content, and the carbonyl reductase mutant ZmCR S87F / A130S / L161F The enzyme activity was 6.1 U / mg.

[0059] Example 6: Preparation of crude enzyme powder

[0060] Carbonyl reductase ZmCR-WT and carbonyl reductase mutant ZmCR obtained in Examples 3 and 4 S87F / A130S / L161F The crude enzyme solution was frozen at -80°C and then freeze-dried at -40°C for 24 h using a vacuum freeze dryer to obtain carbonyl reductase ZmCR-WT and carbonyl reductase mutant ZmCR S87F / A130S / L161F Crude enzyme powder. The obtained crude enzyme powder can be stored in a refrigerator at 4°C for convenient use.

[0061] Example 7: Carbonyl reductase ZmCR and its mutants catalyze the reduction reaction of β-acetone xyloside at different concentrations

[0062] The crude enzyme powder obtained in Example 6 was used for substrate catalysis reaction, and the carbonyl reductase ZmCR and its mutant ZmCR were further investigated under different substrate concentrations. S87F / A130S / L161F The effect of catalyzing the reduction reaction of β-acetone xyloside to prepare (S)-bosone (Table 4).

[0063] Prepare the reaction system according to Table 4: final concentration 50-1000 mM β-acetone xyloside, 0.1 mM NADP + , 1-10 g / L crude enzyme powder, 10% (v / v) isopropanol, 100 mM potassium phosphate buffer solution (pH 7.0) to 1 mL. React at 30° C., 200 rpm for 6-48 h. Use the method in Example 5 to detect the substrate conversion rate and the ee value of the product (S)-bosone.

[0064] The results are shown in Table 4. ZmCR-WT can catalyze the reaction of 1000mM substrate to produce optically pure (S)-bosone. The crude enzyme powder dosage is 100g / L and the conversion rate is more than 99% after 48h reaction. It has certain industrial application potential. S87F / A130S / L161F The strict enantioselectivity consistent with ZmCR-WT is maintained, which is 99.9% (S), and the substrate concentration of 50-1000mM can also be catalyzed. When the substrate concentration increases to 1000mM, the amount of crude enzyme powder is only 10g / L, and the conversion rate after 24h of reaction is >99%. Compared with before mutation, the amount of catalyst is significantly reduced and the reaction time is significantly shortened. The results show that the carbonyl reductase ZmCR and its mutants screened by the present invention can catalyze the synthesis of (S)-bosone from high-concentration β-acetone xyloside, and have strict stereoselectivity and high substrate conversion rate; compared with the method of using glucose dehydrogenase to add glucose to participate in the coenzyme cycle, the method of the present invention uses the ability of carbonyl reductase to oxidize isopropanol to form coenzyme regeneration by adding isopropanol, thereby reducing the amount of coenzyme.

[0065] Table 4 Effect of substrate concentration on the reduction reaction of wild-type carbonyl reductase and mutants

[0066]

[0067] Example 8: Carbonyl reductase ZmCR-WT, mutant ZmCR S87F / A130S / L161F Asymmetric reduction preparation of (S)-Boseine

[0068] The ZmCR-WT and ZmCR prepared in Example 6 were tested in a 100 mL reaction system. S87F / A130S / L161F Substrate conversion by crude enzyme powder.

[0069] 1. Carbonyl reductase ZmCR-WT

[0070] Reaction system: final concentration 1000mM β-acetone xyloside, 0.1mM NADP +, 100 g / L crude enzyme powder, 10% (v / v) isopropanol, 100 mM potassium phosphate buffer solution (pH 7.0) to 100 mL. React at 30°C, 200 rpm for 48 hours. After the reaction is completed, the cells are first centrifuged to remove the cells, and then the supernatant is desalted by nanofiltration and concentrated to dryness under reduced pressure to obtain 17.1 g of (S)-bosine, with a product yield of 90% and a chemical purity of >99%.

[0071] 2. Mutant ZmCR S87F / A130S / L161F

[0072] Reaction system: final concentration 1000mM β-acetone xyloside, 0.1mM NADP + , 10g / L crude enzyme powder, 10% (v / v) isopropanol, 100mM potassium phosphate buffer solution (pH 7.0) to 100mL. React at 30℃, 200rpm for 24h. After the reaction is completed, centrifuge to remove cells. The HPLC chromatogram of the supernatant is shown in Figure 2 C, and then the supernatant was desalted by nanofiltration and concentrated to dryness under reduced pressure to obtain 17.4 g of (S)-bosine, with a product yield of 92% and a chemical purity of >99%.

[0073] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A highly stereoselective carbonyl reductase, characterized in that: The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.

1.

2. Use of the carbonyl reductase according to claim 1 in the asymmetric reduction of β-acetone xyloside to prepare (S)-bosone.

3. The use according to claim 2, characterized in that The application method is: using the crude enzyme liquid obtained by fermentation and culture of the recombinant genetic engineering bacteria containing the carbonyl reductase after crushing and centrifugation of wet bacteria or the crude enzyme powder obtained by freeze-drying the crude enzyme liquid as a catalyst, using β-acetone xyloside as a substrate, and using NADP + The reaction system is formed by using isopropanol as a coenzyme, a buffer solution of pH 6-9 as a reaction medium, and an asymmetric hydrogenation reaction is carried out at 20-40° C. and 100-300 rpm to obtain a reaction solution containing (S)-bosone, which is separated and purified to obtain (S)-bosone.

4. The use according to claim 3, characterized in that In the reaction system, the substrate is added at a concentration of 50-1000 mM; the catalyst is added at an amount of 0.1-2 g / L based on the protein content of the crude enzyme solution and 1-10 g / L based on the weight of the crude enzyme powder; NADP + The added concentration was 0.1 mM, and the volume concentration of isopropanol was 10%.

5. A mutant of the carbonyl reductase according to claim 1, characterized in that: The mutant is obtained by performing single-site mutation or combined mutation at positions 87, 130 and 161 of the amino acid sequence shown in SEQ ID NO.

1.

6. The mutant according to claim 5, characterized in that The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: the serine at position 87 is mutated into phenylalanine, the alanine at position 130 is mutated into serine, and the leucine at position 161 is mutated into phenylalanine; or the serine at position 87 is mutated into phenylalanine + the alanine at position 130 is mutated into serine + the leucine at position 161 is mutated into phenylalanine.

7. Use of a mutant of the carbonyl reductase according to claim 5 in the preparation of (S)-bosone by asymmetric reduction of β-acetone xyloside.

8. The use according to claim 7, characterized in that The application method comprises: using a crude enzyme liquid obtained by fermentation and culture of a recombinant genetically engineered bacterium containing the carbonyl reductase mutant and crushing and centrifuging wet bacteria or a crude enzyme powder obtained by freeze-drying the crude enzyme liquid as a catalyst, using β-acetone xyloside as a substrate, and using NADP + The reaction system is formed by using isopropanol as a coenzyme, a buffer solution of pH 6-9 as a reaction medium, and an asymmetric hydrogenation reaction is carried out at 20-40° C. and 100-300 rpm to obtain a reaction solution containing (S)-bosone, which is separated and purified to obtain (S)-bosone.

9. The use according to claim 8, characterized in that In the reaction system, the substrate is added at a concentration of 50-1000 mM; the catalyst is added at an amount of 0.1-2 g / L based on the protein content of the crude enzyme solution and 1-10 g / L based on the weight of the crude enzyme powder; NADP + The added concentration was 0.1 mM, and the volume concentration of isopropanol was 10%.

10. The use according to claim 8, characterized in that The catalyst was prepared as follows: the recombinant genetically engineered bacteria were inoculated into LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37° C. and 180 rpm with shaking; The culture solution was inoculated into LB medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and the culture was placed in a shaking incubator at 37°C and 180 rpm. 600 When the pH value reaches 0.6, isopropyl-β-D-thiogalactoside with a final concentration of 0.2 mM is added as an inducer. After induction at 16°C for 24 hours, the culture solution is centrifuged to collect the wet bacteria, and then washed twice with physiological saline. The wet bacteria are suspended in PBS buffer at an amount of 10 mL / g, and ultrasonically crushed for 15 minutes at 300 W power with an ultrasonic crusher under an ice water bath, working for 3 seconds, and resting for 6 seconds. Subsequently, centrifuge at 4°C, 12000 rpm for 30 minutes with a low-temperature high-speed centrifuge, collect the supernatant, and obtain the crude enzyme solution; the crude enzyme solution is frozen at -80°C, and then freeze-dried at -40°C for 24 hours using a vacuum freeze dryer to obtain the crude enzyme powder.

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