Aldehyde ketone reductase mutants and uses thereof

Abstract

The application provides an aldehyde-ketone reductase yhdN mutant, and constructs a recombinant whole cell containing the aldehyde-ketone reductase yhdN mutant and glucose dehydrogenase (GDH). The recombinant whole cell can be used as a catalyst to catalyze the efficient preparation of N-ethyl methylcarbamoyl-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE) from a substrate N-ethyl-methylcarbamoyl acetophenone (NEMCA). The reaction conversion rate and ee can reach 99.9%. The preparation method is simple, fast, low in cost, and suitable for industrial application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to multiple mutants of an aldehyde-ketone reductase and their uses. The aldehyde-ketone reductase mutants are obtained by mutation of wild-type Bacillus subtilis aldehyde-ketone reductase (yhdN). In particular, it relates to multiple mutants of an aldehyde-ketone reductase and their preparation methods, as well as their use in preparing the key chiral intermediate (S)-NEMCA-HEPE of (S)-rivastigmine. Background Technology

[0002] (S)-Ravistigine is a first-line chiral drug approved by the US FDA for the treatment of mild to moderate Alzheimer's disease. It contains a carbamate structure, which allows it to rapidly and persistently inhibit the activity of both acetylcholine and butyrylcholinesterase. Compared to similar drugs, (S)-Ravistigine has a higher therapeutic index, fewer adverse reactions, significant clinical efficacy, and good tolerability. Currently, (S)-Ravistigine is widely used both domestically and internationally, with over 10 million patients worldwide using the drug and sales reaching $2 billion. (S)-NEMCA-HEPE (N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]-phenyl ester) is a key chiral intermediate in the synthesis of (S)-Ravistigine. Finding a simple, effective, and low-cost method to prepare (S)-NEMCA-HEPE will directly reduce the synthesis cost of (S)-Ravistigine.

[0003] Currently, the preparation of (S)-NEMCA-HEPE mainly employs chemical methods, with three approaches: (1) first synthesizing a racemic compound and then obtaining it through chemical resolution, but the product's optical purity is not high; (2) using a chiral source and preparing it through a semi-synthetic method, but chiral sources are not easy to obtain, resulting in limited practical application value; (3) directly synthesizing it using a metal-catalyzed asymmetric reduction reaction, but the catalysts are mostly heavy metals, which are relatively expensive, leading to high industrialization costs. Furthermore, chemical synthesis often involves multiple steps, requiring a large amount of organic solvents, and the high temperatures required for the reaction may lead to side reactions such as racemization and degradation of the product, resulting in low yield and optical purity of (S)-NEMCA-HEPE, which is not conducive to industrial production.

[0004] Compared with chemical catalysis, biocatalysis has advantages such as high catalytic activity, good enantioselectivity, fewer byproducts, mild reaction conditions, and environmental friendliness. In recent years, lipases and ketone reductases have been reported to be used to prepare (S)-NEMCA-HEPE. For example, *Candida antarctica* lipase B, in the presence of diimine and a transition metal complex, can catalyze the resolution of (R,S)-NEMCA-HEPE to obtain (S)-NEMCA-HEPE, with a separation yield and ee% of 84.0% and 99.0%, respectively. This method can yield (S)-NEMCA-HEPE with high optical purity. However, the reaction requires the additional addition of diimine and a transition metal complex, and its operation... The process is complex and prone to environmental pollution. Some commercially available ketone reductases can be used to catalyze the asymmetric reduction of N-ethylmethylcarbamoyl acetophenone (NEMCA) to prepare (S)-NEMCA-HEPE with a conversion rate and ee% of up to 99%. However, commercially available ketone reductases are relatively expensive and require the addition of expensive cofactors, and the reaction time is as long as 24 hours. It can be seen that the currently reported enzyme-catalyzed methods for preparing (S)-NEMCA-HEPE still have drawbacks such as high reaction cost and long reaction time.

[0005] Therefore, further research is needed on the preparation method of (S)-NEMCA-HEPE to improve its conversion rate and ee while reducing costs, and to further improve the process to adapt it to industrial production. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an aldehyde-ketone reductase yhdN mutant, which forms a recombinant whole cell with glucose dehydrogenase (GDH). The resulting wet bacterial cells are used as a catalyst to efficiently catalyze the preparation of N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]phenyl ester ((S)-NEMCA-HEPE) from the substrate N-ethyl-methylcarbamate (NEMCA). The reaction takes only 35-45 minutes, and the conversion rate and ee can both reach 99.9%. This chiral preparation method is simple, rapid, low-cost, and suitable for industrial application, thus completing this invention.

[0007] The first aspect of the present invention is to provide an aldehyde-ketone reductase yhdN mutant having the amino acid sequence shown in SEQ ID NO.1, and having a mutation at position 19.

[0008] A second aspect of the present invention aims to provide a nucleic acid capable of encoding the aldehyde-ketone reductase yhdN mutant described in the first aspect, preferably having a nucleic acid sequence as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.

[0009] A third aspect of this invention aims to provide a recombinant expression vector comprising the nucleic acid described in the second aspect. Preferably, the recombinant expression vector is selected from plasmids, granules, bacteriophages, or viral vectors.

[0010] The fourth aspect of the present invention aims to provide a recombinant expression transformant of the recombinant expression vector described in the third aspect, preferably Escherichia coli (E. coli), more preferably E. coli BL21(DE3).

[0011] The fifth aspect of this invention aims to provide a method for preparing an aldehyde-ketone reductase yhdN mutant, culturing the recombinant transformant, and obtaining the recombinant aldehyde-ketone reductase yhdN mutant from the culture.

[0012] The sixth aspect of this invention aims to provide a catalyst for preparing the chiral intermediate N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE), wherein the catalyst is a bacterial cell obtained by inducing culture of recombinant whole cells of aldehyde-ketone reductase yhdN mutant and glucose dehydrogenase (GDH).

[0013] The seventh aspect of this invention aims to provide the application of the aldehyde-ketone reductase yhdN mutant in the asymmetric reduction reaction of the pre-chiral carbonyl compound NEMCA to form the chiral hydroxyl compound (S)-NEMCA-HEPE.

[0014] The object of the eighth aspect of this invention is to provide a method for preparing the N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE). In this method, bacterial cells obtained by inducing culture of recombinant whole cells containing aldehyde-ketone reductase yhdN mutant glucose dehydrogenase (GDH) are used as a catalyst; N-ethyl-methylcarbamoyl acetophenone (NEMCA) is used as a substrate; glucose is used as a co-substrate; and a mixed solution of pH 7.0, 100 mM potassium phosphate buffer, and DMSO (wherein the volume fraction of DMSO is 2% v / v) is used as the reaction medium. The reaction is carried out at 25-35°C and 170-210 rpm. After the reaction is completed, (S)-NEMCA-HEPE is obtained by separation and purification.

[0015] The yhdN mutant aldehyde-ketone reductase provided by this invention has the following beneficial effects:

[0016] (1) The aldehyde-ketone reductase yhdN mutant provided by the present invention and glucose dehydrogenase (GDH) were used to construct recombinant whole cells. The wet cells were cultured and used as catalysts to catalyze the efficient preparation of N-ethylmethylcarbamoyl acetophenone (NEMCA) to N-ethylmethylcarbamoyl-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE).

[0017] (2) In this invention, (S)-NEMCA-HEPE is prepared by wet cell catalysis of aldehyde-ketone reductase yhdN mutant and glucose dehydrogenase (GDH) recombinant whole cells. The reaction conversion rate and ee can reach 99.9%, and the obtained (S)-NEMCA-HEPE has high optical purity, which can meet the requirements of use.

[0018] (3) In the preparation of (S)-NEMCA-HEPE by wet cell catalysis of the aldehyde-ketone reductase yhdN mutant and glucose dehydrogenase (GDH) recombinant whole cells in this invention, the reduction process can be completed in only 35-45 minutes while ensuring high conversion rate and ee value, which greatly shortens the preparation cycle.

[0019] (4) The (S)-NEMCA-HEPE preparation method provided in this invention is a simple, fast, low-cost chiral preparation process suitable for industrial application. Attached Figure Description

[0020] Figure 1 The image shows SDS-PAGE electrophoresis diagrams of the alanine scanning mutants yhdN(G19A), yhdN(W21A), yhdN(W28A), yhdN(L87A), yhdN(W126A), and yhdN(M323A) in Example 1 of the present invention.

[0021] Figure 2 The image shows SDS-PAGE electrophoresis diagrams of the protein expression of the aldehyde-ketone reductase yhdN 19th position saturation mutants G19V, G19C, G19P, G19F, G19S, G19L, and G19I in Example 1 of the present invention.

[0022] Figure 3 The image shows SDS-PAGE electrophoresis diagrams of recombinant whole cells of wild-type yhdN-GDH, yhdN(G19A)-GDH, yhdN(G19V)-GDH and yhdN(G19C)-GDH in Example 2 of the present invention.

[0023] Figure 4 The HPLC chromatogram of (R,S)-NEMCA-HEPE standard is shown.

[0024] Figure 5The liquid chromatogram of (S)-NEMCA-HEPE prepared by using yhdN(G19A)-GDH recombinant whole-cell bacterial culture as a catalyst in Example 3 of the present invention is shown.

[0025] Figure 6 The image shows the purified electrophoresis diagrams of the mutant yhdN(G19A) and yhdN(G19V) proteins in Example 4 of this invention;

[0026] Figure 7 The time progression curve of the whole-cell catalytic preparation of (S)-NEMCA-HEPE by yhdN(G19A)-GDH recombinant cells is shown in Example 5 of the present invention.

[0027] Figure 8 The time progression curve of the whole-cell catalytic preparation of (S)-NEMCA-HEPE by yhdN(G19V)-GDH recombinant cells is shown in Example 6 of the present invention.

[0028] Figure 9 The time progression curve of the whole-cell catalytic preparation of (S)-NEMCA-HEPE by yhdN(G19C)-GDH recombinant cells in Example 7 of the present invention is shown. Detailed Implementation

[0029] The present invention will now be described in detail through specific embodiments, and the features and advantages of the present invention will become clearer and more explicit with these descriptions.

[0030] The first aspect of the present invention provides an aldehyde-ketone reductase yhdN mutant having the amino acid sequence shown in SEQ ID NO.1, and having a mutation at position 19.

[0031] Preferably, the aldehyde-ketone reductase yhdN mutant has the amino acid sequence shown in SEQ ID NO.1, and the glycine at position 19 is mutated to alanine, valine, or cysteine.

[0032] More preferably, the amino acid sequences of the aldehyde-ketone reductase yhdN mutant are shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, respectively.

[0033] A second aspect of the present invention provides a nucleic acid capable of encoding the aldehyde-ketone reductase yhdN mutant described in the first aspect, preferably having a nucleic acid sequence as shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7.

[0034] In this invention, the full-length base sequences of aldehyde-ketone reductase yhdN and its mutant are both 996 bp, with the start codon being ATG and the stop codon being TAA, from the first base to the 996th base.

[0035] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid described in the second aspect. The recombinant expression vector is constructed by ligating the nucleic acid sequence of the aldehyde-ketone reductase yhdN mutant of the present invention onto the recombinant expression vector using conventional methods in the art.

[0036] This invention does not specifically limit the recombinant expression vector. Various vectors conventional in the art can be used, such as those selected from plasmids, granules, bacteriophages or viral vectors, preferably plasmids, more preferably pET plasmids, such as pET28a, pET21a, etc.

[0037] The fourth aspect of the present invention provides a recombinant expression transformant of the recombinant expression vector described in the third aspect, preferably Escherichia coli (E. coli), more preferably E. coli BL21(DE3).

[0038] The fifth aspect of the present invention provides a method for preparing an aldehyde-ketone reductase yhdN mutant, culturing the recombinant transformant, and obtaining the recombinant aldehyde-ketone reductase yhdN mutant from the culture.

[0039] The aldehyde-ketone reductase yhdN mutant of this invention was obtained by site-directed saturation mutagenesis, which mutated wild-type aldehyde-ketone reductase yhdN. The amino acid sequence of wild-type aldehyde-ketone reductase yhdN is SEQ ID NO.1.

[0040] The recombinant plasmids containing the aldehyde-ketone reductase yhdN mutant and the recombinant plasmid containing the GDH gene were heat-shocked into E. coli BL21(DE3) competent cells to obtain recombinant bacteria. These were then inoculated, transferred, induced, and the cells recovered. Preferably, the cells were cultured at 35-39℃ for 10-14 h. Clones were then selected and transferred to LB broth containing 25-35 μg / mL kanamycin and 45-55 μg / mL ampicillin for further culture. Recombinant whole cells containing the yhdN mutant and GDH were constructed and positively screened to obtain mutants with optimal catalytic performance, such as yhdN(G19A), yhdN(G19V), and yhdN(G19C).

[0041] The sixth aspect of the present invention provides a catalyst for preparing the chiral intermediate N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE), wherein the catalyst is a bacterial cell obtained by inducing culture of recombinant whole cells of aldehyde-ketone reductase yhdN mutant and glucose dehydrogenase (GDH).

[0042] Engineered bacteria containing the aldehyde-ketone reductase yhdN mutant and GDH were inoculated into LB liquid medium containing kanamycin at a final concentration of 25-35 μg / mL and ampicillin at 45-55 μg / mL, and cultured at 35-39℃ until OD. 600 When the concentration reaches 0.4-0.6, add 0.1mM IPTG (isopropyl thiogalactoside) to induce the co-expression of aldehyde-ketone reductase yhdN mutant and GDH. The induction temperature is 20-28℃. After induction for 1 h, centrifuge at 3-8℃ to obtain wet bacterial cells containing aldehyde-ketone reductase yhdN mutant and GDH, respectively.

[0043] The seventh aspect of this invention provides the application of the aldehyde-ketone reductase yhdN mutant in the asymmetric reduction reaction of the pre-chiral carbonyl compound NEMCA to form the chiral hydroxyl compound (S)-NEMCA-HEPE.

[0044] Using N-ethyl-methylcarbamoylacetophenone (NEMCA) as the substrate and glucose as the co-substrate, and a mixed solution of pH 7.0, 100 mM potassium phosphate buffer, and DMSO (with DMSO at a volume concentration of 2% v / v) as the reaction medium, the reaction was carried out at 25-35℃ and 170-210 rpm. After the reaction was completed, (S)-NEMCA-HEPE was obtained by separation and purification.

[0045] The substrate concentration is 2-100 mM, preferably 3-50 mM, and more preferably 4-10 mM. The co-substrate concentration is 100-500 mM, preferably 150-450 mM, and more preferably 200-400 mM.

[0046] The amount of catalyst used is 0.05-0.5 g / mL based on the wet weight of the bacterial cells, preferably 0.08-0.4 g / mL, and more preferably 0.1-0.3 g / mL.

[0047] The eighth aspect of this invention provides a method for preparing the aforementioned N-ethylmethylcarbamate-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE). In this method, bacterial cells obtained by inducing culture of recombinant whole cells containing the aldehyde-ketone reductase yhdN mutant glucose dehydrogenase (GDH) are used as a catalyst. N-ethyl-methylcarbamoyl acetophenone (NEMCA) is used as the substrate, glucose as the co-substrate, and a mixed solution of pH 7.0, 100 mM potassium phosphate buffer, and DMSO (dimethyl sulfoxide) (DMSO volume concentration 2% v / v) is used as the reaction medium. The reaction is carried out at 25-35°C and 170-210 rpm. After the reaction is completed, (S)-NEMCA-HEPE is obtained by separation and purification.

[0048] The yhdN mutant aldehyde-ketone reductase provided in this invention forms a recombinant whole cell with glucose dehydrogenase (GDH) as a catalyst to efficiently prepare (S)-NEMCA-HEPE from the substrate NEMCA. The reaction process is significantly shortened, and the conversion rate and ee can both reach 99.9%. The preparation method is simple, low-cost, and suitable for industrial applications.

[0049] Example

[0050] Enantiomer excess is defined as the percentage of a mixture of enantiomers where one isomer A is more abundant than another isomer B, abbreviated as ee. The formula is (AB) / (A+B)×100%. Enantiomer excess values ​​are used to represent the optical purity of a chiral compound. A higher ee value indicates higher optical purity.

[0051] The abbreviations for the 20 amino acids are as follows:

[0052]

[0053]

[0054] Example 1

[0055] The complex of aldehyde-ketone reductase yhdN and substrate NEMCA was constructed using molecular docking software. Based on the catalytic asymmetric reduction reaction mechanism of aldehyde-ketone reductase yhdN, the amino acid sites with potential influence on the enzyme catalytic site were evaluated. Finally, six amino acid sites were selected for alanine scanning, namely G19, W21, and W28 in the large catalytic pocket and L87, W126, and M323 in the small catalytic pocket of aldehyde-ketone reductase yhdN. The primer design is shown in Table 1.

[0056] Table 1 Primer sequences of the yhdN mutant

[0057]

[0058] Using the full plasmid yhdN-pET28a from E.coli BL21(DE3) / pET28a(+)-yhdN (construction details can be found in the previous article Journal of Biotechnology, 289(2019):64–70.) as a template, and with the mutation primer sequences listed in Table 1, PCR (polymerase chain reaction) was used to mutate G19, W21, W28, L87, W126 and M323 in the amino acid sequence of aldehyde-ketone reductase yhdN to alanine (A). The recombinant plasmid containing the yhdN mutant gene was transformed into E. coli BL21(DE3) competent cells via heat shock. Positive clones were screened and seeded into LB liquid medium with a final concentration of 30 μg / mL kanamycin. The cells were cultured at 37°C until the OD600 reached 0.4–0.6. IPTG (final concentration 0.1 mM) was added to induce yhdN mutant expression. Following the above transformation, yhdN(G19A), yhdN(W21A), yhdN(W28A), yhdN(L87A), yhdN(W126A), and yhdN(M323A) aldehyde reductase mutants were obtained. Protein expression is shown in the figure. Figure 1 As shown, the obtained mutant was applied to the reaction system for the asymmetric reduction of NEMCA to prepare (S)-NEMCA-HEPE, and the catalytic effect of the mutant was evaluated. The results showed that the catalytic activity of the mutant yhdN(G19A) was significantly improved (results are shown in Table 2).

[0059] Table 2 Catalytic results of alanine scanning mutants

[0060] yhdN mutant Conversion (%) ee(%) G19A 99.9 99.9(S) W21A 89.0 99.9(S) W28A 84.6 99.9(S) L87A 83.6 99.9(S) W126A 79.6 99.9(S) M323A 94.0 99.9(S)

[0061] Subsequently, using the full-length plasmid yhdN-pET28a as a template, a saturation mutation was performed on the glycine (G) site at position 19 of the aldehyde-ketone reductase yhdN to further screen for mutants with superior catalytic performance. Using the saturation mutation primer sequences in Table 1, PCR amplification, transformation, and screening of dominant strains were performed. Successfully expressed proteins are shown in [Table 1]. Figure 2 As shown, the catalytic performance of the mutants was compared with that of the original aldehyde-ketone reductase yhdN, and two other mutants with significantly enhanced catalytic activity were obtained (see Table 3). They were identified by sequencing as aldehyde-ketone reductase yhdN (G19V) and yhdN (G19C), that is, the 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 was mutated to valine (V) and cysteine ​​(C), respectively.

[0062] Table 3. Catalytic results of the 19th position saturated mutant of aldehyde-ketone reductase yhdN.

[0063] yhdN mutant Conversion rate (%) ee(%) G19V 99.9 99.9(S) G19C 99.9 99.9(S) G19P 86.3 99.9(S) G19F 94.1 99.9(S) G19S 85.5 99.9(S) G19L 84.4 99.9(S) G19I 2.3 99.9(S)

[0064] PCR reaction system (20 μL): 1 μL forward primer (100 μM), 1 μL reverse primer (100 μM), 10 μL RimeSTAR Max DNA premix (2×) enzyme, 1 μL pET28a(+)-yhdN plasmid template, and 7 μL deionized water. The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual was as follows: 98℃ pre-denaturation for 3 min, followed by 30 cycles: 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 6 min 30 s. Finally, a final extension at 72℃ for 10 min was performed, followed by incubation at 4℃. The resulting recombinant plasmid was transferred to E. coli BL21(DE3) competent cells to form recombinant transformants, and cultured at 37℃ for 12 h. Clones were then picked and transferred to 10 mL of LB broth containing 30 μg / mL kanamycin, and cultured at 37℃ and 180 rpm for 10 h. Recombinant whole cells of yhdN mutant and GDH were constructed and positive screening was performed. The mutants yhdN(G19A), yhdN(G19V) and yhdN(G19C) with the best catalytic performance were obtained and sent to Changchun Kumei Biotechnology Co., Ltd. for sequencing and stored in a -80℃ freezer.

[0065] Example 2

[0066] GDH cell preparation: The GDH gene of Bacillus subtilis 168 (GenBank NO. KM817194.1) was inserted into pET21a(+) to construct a recombinant expression vector, and this expression vector was transformed into E. coli BL21(DE3) to obtain E coli BL21(DE3) / pET21a-GDH.

[0067] To construct recombinant whole cells containing the yhdN mutants yhdN(G19A), yhdN(G19V), and yhdN(G19C) and GDH as described in Example 1, the dual plasmids pET28a-yhdN(G19A) / pET21a-GDH, pET28a-yhdN(G19V) / pET21a-GDH, and pET28a-yhdN(G19C) / pET21a-GDH were transformed into E. coli BL21(DE3) for expression. The resulting recombinant whole cells were cultured in LB medium containing 30 μg / mL kanamycin and 50 μg / mL ampicillin at 37°C until OD200. 600Once the pH reached 0.4-0.6, IPTG (isopropyl galactothioglycoside, final concentration 0.1 mM) was added to induce co-expression of yhdN mutants and GDH at 25℃. After 12 h of induction, the cells were centrifuged at 8000 rpm for 4 min at 4℃ to obtain recombinant whole cells of yhdN(G19A)-GDH, yhdN(G19V)-GDH, and yhdN(G19C)-GDH, which were then resuspended in potassium phosphate buffer (100 mM, pH 7.0) for later use.

[0068] To verify the successful construction of the yhdN mutant and GDH in the same cell, the protein solution after sonication of the recombinant whole cells was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results are as follows: Figure 3 As shown. It was determined that the obtained recombinant whole cells contained both yhdN or its mutant and GDH, and could be used in the asymmetric reduction of NEMCA to prepare (S)-NEMCA-HEPE.

[0069] Example 3

[0070] The recombinant whole cells of yhdN(G19A)-GDH constructed in Example 2 were resuspended in 100mM potassium phosphate buffer (pH 7.0) to obtain yhdN(G19A)-GDH bacterial culture. Using the yhdN(G19A)-GDH bacterial culture as a catalyst, NEMCA as a substrate, and glucose as a co-substrate, without the addition of NAD(P)H or NAD(P)... + By utilizing the endogenous NAD(P)H in bacteria, a bacterial coenzyme cycle system was established.

[0071] In the reaction, the yhdN(G19A)-GDH bacterial culture catalyst was 0.2 g / mL (wet weight of bacterial cells), with a final concentration of 4 mM NEMCA, a final concentration of 300 mM glucose, 1.0 mL of potassium phosphate buffer (100 mM, pH 7.0), and a DMSO mixture (DMSO volume fraction 2% v / v) as the reaction medium. After reacting at 30℃ and 190 rpm for 40 min, the reaction mixture was extracted three times with ethyl acetate to obtain the organic extract. The extract was then dried over anhydrous Na2SO4 and subjected to vacuum distillation to obtain the product (S)-NEMCA-HEPE. The yield and ee value of (S)-NEMCA-HEPE were both 99.9% as detected by high-performance liquid chromatography (HPLC). Compared with the catalytic data of 78.2% conversion and 99.9% ee of wild-type yhdN-GDH recombinant whole cells after 1 h of reaction, the yhdN mutant with significantly improved catalytic performance was screened.

[0072] Liquid chromatography detection conditions: The reaction conversion rate and ee were analyzed by HPLC equipped with a chiral OD-H column (5μm, 250×4.6mm). The detection conditions were as follows: UV wavelength 217nm, mobile phase n-hexane:isopropanol = 95:5, flow rate 1.0mL / min. The retention times of (S)- and (R)-NEMCA-HEPE were 15.7min and 19.5min, respectively. Using yhdN(G19A)-GDH recombinant whole-cell bacterial culture as a catalyst, the liquid chromatogram of the product prepared is shown below. Figure 5 As shown, the liquid chromatography (LC) chromatogram of the (R,S)-NEMCA-HEPE standard is compared with that of the standard. Figure 4 As shown in the figure, the yhdN(G19A)-GDH recombinant whole-cell bacterial culture used in this invention was used as a catalyst to prepare high-purity (S)-NEMCA-HEPE.

[0073] Example 4

[0074] The optimal mutants yhdN(G19A) and yhdN(G19V) obtained in Example 1 were centrifuged at 8000 rpm and 4°C for 10 min, and the cells were collected. They were resuspended in 100 mM potassium phosphate buffer at pH 7.0 at a concentration of 2 g / mL, and sonicated on an ice-water mixture for 15 min. The sonication conditions were: power 400 W, 1 s of sonication followed by a 1 s pause, to obtain crude enzyme solutions of yhdN(G19A) and yhdN(G19V). The solutions were centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected. After passing the supernatant through a 0.45 μm membrane microfiltration, yhdN(G19A) and yhdN(G19V) were purified using a Ni affinity column.

[0075] Purification of yhdN (G19A) or yhdN (G19V) was performed using a Ni-NTA affinity column (1.6 × 10 cm, Bio-Rad, USA). The specific procedures were as follows: 1) Pre-equilibration was performed using buffer A (pH 7.0 containing 0.3 M NaCl, 20 mM imidazole, and 100 mM potassium phosphate buffer). 2) Unbound impurities were washed away with buffer A at a flow rate of 1.0 mL / min until conductivity stabilized. 3) The target protein was then eluted with buffer B (pH 7.0 containing 0.3 M NaCl, 120 mM imidazole, and 100 mM potassium phosphate buffer). The collected eluent was dialyzed overnight with 100 mM potassium phosphate buffer (pH 7.0). All purification steps were performed at 4°C. The final purification solution was concentrated using an ultrafiltration centrifuge tube, and the purification effect was analyzed by SDS-PAGE electrophoresis. Figure 6 As shown.

[0076] Example 5

[0077] The yhdN(G19A)-GDH recombinant whole-cell bacterial cells obtained in Example 2 were resuspended in potassium phosphate buffer (100 mM, pH 7.0). In the reaction solution, 0.2 g / mL of yhdN(G19A)-GDH recombinant whole-cell wet bacterial cells were added, with a final NEMCA concentration of 4.0 mM, a final glucose concentration of 300 mM, and a final potassium phosphate buffer concentration of 100 mM. The pH after adding the potassium phosphate buffer was 7.0, and the DMSO volume concentration after addition was 2% v / v. A 1 mL reaction system was constructed as the reaction medium. The reaction was carried out at 30°C and 190 rpm. The reaction progress curve is shown below. Figure 7 As shown, the substrate can be completely converted into the product (S)-NEMCA-HEPE within 40 min, with a conversion rate of 99.9% and an ee value of 99.9%.

[0078] Example 6

[0079] The product (S)-NEMCA-HEPE was prepared according to the method of Example 5, except that the yhdN(G19V)-GDH recombinant whole-cell bacteria obtained in Example 2 were resuspended in potassium phosphate buffer (100 mM, pH 7.0). The reaction progress curve is shown below. Figure 8 As shown, the substrate can be completely converted into the product (S)-NEMCA-HEPE within 40 min, with a conversion rate of 99.9% and an ee value of 99.9%.

[0080] Example 7

[0081] The product (S)-NEMCA-HEPE was prepared according to the method of Example 5, except that the yhdN(G19C)-GDH recombinant whole-cell bacteria obtained in Example 2 were resuspended in potassium phosphate buffer (100 mM, pH 7.0). The reaction progress curve is shown below. Figure 9 As shown, the substrate can be completely converted into the product (S)-NEMCA-HEPE within 40 min, with a conversion rate of 99.9% and an ee value of 99.9%.

[0082] Example 8

[0083] The reaction of yhdN(G19A)-GDH whole-cell catalytic asymmetric reduction of NEMCA was scaled up according to the method in Example 6. Whole-cell yhdN(G19A)-GDH cells were resuspended in potassium phosphate buffer (100 mM, pH 7.0). The wet cells were added to the reaction mixture at a final concentration of 4.0 mM NEMCA (0.28 g / mL), 300 mM glucose, and 100 mM potassium phosphate buffer. The pH after adding the potassium phosphate buffer was 7.0. The volume concentration of DMSO after adding the DMSO was 2% v / v. A 50 mL reaction system was constructed. The reaction was carried out at 30°C and 190 rpm for 40 min. After extraction of the reaction mixture three times with ethyl acetate, an organic extract was obtained. This extract was then dried over anhydrous Na2SO4 and subjected to vacuum distillation to obtain the product (S)-NEMCA-HEPE, with a conversion rate of 99.9%, an ee value of 99.9%, and a space-time yield of 0.89 g / L h.

[0084] SEQ ID NO.1

[0085] MEYTSIADTGIEASRIGL G TWAIGGTMWGGTDEKTSIETIRAALDQGITLIDTAPAYGFG

[0086] QSEEIVGKAIKEYGKRDQVILATKTALDWKNNQLFRHANRARIVEEVENSLKRLQTDY

[0087] IDLYQVHWPDPLVPIEETAEVMKELYDAGKIRAIGVSNFSIEQMDTFRAVAPLHTIQPPY

[0088] NLFEREMEESVLPYAKDNKITTLLYGSLCRGLLTGKMTEEYTFEGDDLRNHDPKFQKP

[0089] RFKEYLSAVNQLDKLAKTRYGKSVIHLAVRWILDQPGADIALWGARKPGQLEALSEITGWTLNSEDQKDINTIL ENTISDPVGPEFMAPPTREEI

[0090] SEQ ID NO.2

[0091] MEYTSIADTGIEASRIGL A TWAIGGTMWGGTDEKTSIETIRAALDQGITLIDTAPAYGFG

[0092] QSEEIVGKAIKEYGKRDQVILATKTALDWKNNQLFRHANRARIVEEVENSLKRLQTDY

[0093] IDLYQVHWPDPLVPIEETAEVMKELYDAGKIRAIGVSNFSIEQMDTFRAVAPLHTIQPPY

[0094] NLFEREMEESVLPYAKDNKITTLLYGSLCRGLLTGKMTEEYTFEGDDLRNHDPKFQKP

[0095] RFKEYLSAVNQLDKLAKTRYGKSVIHLAVRWILDQPGADIALWGARKPGQLEALSEITGWTLNSEDQKDINTIL ENTISDPVGP EFMAPPTREEI

[0096] SEQ ID NO.3

[0097] MEYTSIADTGIEASRIGL V TWAIGGTMWGGTDEKTSIETIRAALDQGITLIDTAPAYGFG

[0098] QSEEIVGKAIKEYGKRDQVILATKTALDWKNNQLFRHANRARIVEEVENSLKRLQTDY

[0099] IDLYQVHWPDPLVPIEETAEVMKELYDAGKIRAIGVSNFSIEQMDTFRAVAPLHTIQPPY

[0100] NLFEREMEESVLPYAKDNKITTLLYGSLCRGLLTGKMTEEYTFEGDDLRNHDPKFQKP

[0101] RFKEYLSAVNQLDKLAKTRYGKSVIHLAVRWILDQPGADIALWGARKPGQLEALSEITGWTLNSEDQKDINTIL ENTISDPVGP EFMAPPTREEI

[0102] SEQ ID NO.4

[0103] MEYTSIADTGIEASRIGL CTWAIGGTMWGGTDEKTSIETIRAALDQGITLIDTAPAYGFG

[0104] QSEEIVGKAIKEYGKRDQVILATKTALDWKNNQLFRHANRARIVEEVENSLKRLQTDY

[0105] IDLYQVHWPDPLVPIEETAEVMKELYDAGKIRAIGVSNFSIEQMDTFRAVAPLHTIQPPY

[0106] NLFEREMEESVLPYAKDNKITTLLYGSLCRGLLTGKMTEEYTFEGDDLRNHDPKFQKP

[0107] RFKEYLSAVNQLDKLAKTRYGKSVIHLAVRWILDQPGADIALWGARKPGQLEALSEITGWTLNSEDQKDINTIL ENTISDPVGP EFMAPPTREEI

[0108] SEQ ID NO.5

[0109] ATGGAATATACCAGTATAGCAGATACAGGAATAGAAGCCTCCAGAATCGGCCTCG

[0110] CT ACATGGGCCATTGGCGGAACGATGTGGGGAGGCACTGACGAAAAAACATCGAT

[0111] TGAAACAATCCGCGCCGCTCTTGATCAGGGGATTACACTGATTGACACCGCACCG

[0112] GCTTACGGCTTCGGGCAGTCCGAGGAAATTGTCGGAAAGGCAATCAAAGAGTACG

[0113] GCAAAAGAGACCAGGTGATTCTCGCAACGAAAACGGCTCTGGACTGGAAGAACA

[0114] ACCAGCTGTTCCGCCATGCGAACAGAGCGAGAATTGTAGAGGAAGTTGAGAATTC

[0115] TTTGAAGCGGCTTCAAACAGACTATATTGATCTTTATCAGGTGCATTGGCCCGATC

[0116] CGCTTGTGCCAATTGAAGAAACGGCTGAAGTCATGAAGGAATTATATGATGCGGG

[0117] AAAAATCCGGGCGATTGGCGTCAGCAATTTTTCAATTGAGCAAATGGATACATTTC

[0118] GCGCCGTCGCACCTCTCCATACGATTCAGCCTCCATATAATCTGTTTGAAAGAGAG

[0119] ATGGAAGAGAGTGTCCTTCCTTATGCGAAAGATAACAAGATAACAACATTATTAT

[0120] ACGGCAGTTTATGCAGAGGGCTGTTAACAGGCAAAATGACTGAAGAATATACATT

[0121] TGAGGGCGATGATCTGCGTAATCACGATCCAAAATTCCAGAAGCCCCGCTTTAAA

[0122] GAGTATCTTTCTGCTGTGAATCAATTGGATAAGCTGGCGAAGACACGTTATGGAAA

[0123] ATCAGTGATTCACTTGGCTGTCAGATGGATCTTAGATCAGCCGGGAGCGGATATCG

[0124] CTCTTTGGGGAGCAAGAAAGCCTGGGCAGCTTGAGGCCCTATCTGAGATTACAGG

[0125] CTGGACGCTGAACAGTGAAGATCAGAAAGATATCAATACTATATTGGAAAATACG

[0126] ATATCAGACCCTGTCGGACCGGAGTTTATGGCCCCGCCGACCAGAGAGGAAATAT

[0127] AA

[0128] SEQ ID NO.6

[0129] ATGGAATATACCAGTATAGCAGATACAGGAATAGAAGCCTCCAGAATCGGCCTCG

[0130] TT ACATGGGCCATTGGCGGAACGATGTGGGGAGGCACTGACGAAAAAACATCGAT

[0131] TGAAACAATCCGCGCCGCTCTTGATCAGGGGATTACACTGATTGACACCGCACCG

[0132] GCTTACGGCTTCGGGCAGTCCGAGGAAATTGTCGGAAAGGCAATCAAAGAGTACG

[0133] GCAAAAGAGACCAGGTGATTCTCGCAACGAAAACGGCTCTGGACTGGAAGAACA

[0134] ACCAGCTGTTCCGCCATGCGAACAGAGCGAGAATTGTAGAGGAAGTTGAGAATTC

[0135] TTTGAAGCGGCTTCAAACAGACTATATTGATCTTTATCAGGTGCATTGGCCCGATC

[0136] CGCTTGTGCCAATTGAAGAAACGGCTGAAGTCATGAAGGAATTATATGATGCGGG

[0137] AAAAATCCGGGCGATTGGCGTCAGCAATTTTTCAATTGAGCAAATGGATACATTTC

[0138] GCGCCGTCGCACCTCTCCATACGATTCAGCCTCCATATAATCTGTTTGAAAGAGAG

[0139] ATGGAAGAGAGTGTCCTTCCTTATGCGAAAGATAACAAGATAACAACATTATTAT

[0140] ACGGCAGTTTATGCAGAGGGCTGTTAACAGGCAAAATGACTGAAGAATATACATT

[0141] TGAGGGCGATGATCTGCGTAATCACGATCCAAAATTCCAGAAGCCCCGCTTTAAA

[0142] GAGTATCTTTCTGCTGTGAATCAATTGGATAAGCTGGCGAAGACACGTTATGGAAA

[0143] ATCAGTGATTCACTTGGCTGTCAGATGGATCTTAGATCAGCCGGGAGCGGATATCG

[0144] CTCTTTGGGGAGCAAGAAAGCCTGGGCAGCTTGAGGCCCTATCTGAGATTACAGG

[0145] CTGGACGCTGAACAGTGAAGATCAGAAAGATATCAATACTATATTGGAAAATACG

[0146] ATATCAGACCCTGTCGGACCGGAGTTTATGGCCCCGCCGACCAGAGAGGAAATAT

[0147] AA

[0148] SEQ ID NO.7

[0149] ATGGAATATACCAGTATAGCAGATACAGGAATAGAAGCCTCCAGAATCGGCCTCT

[0150] GT ACATGGGCCATTGGCGGAACGATGTGGGGAGGCACTGACGAAAAAACATCGAT

[0151] TGAAACAATCCGCGCCGCTCTTGATCAGGGGATTACACTGATTGACACCGCACCG

[0152] GCTTACGGCTTCGGGCAGTCCGAGGAAATTGTCGGAAAGGCAATCAAAGAGTACG

[0153] GCAAAAGAGACCAGGTGATTCTCGCAACGAAAACGGCTCTGGACTGGAAGAACA

[0154] ACCAGCTGTTCCGCCATGCGAACAGAGCGAGAATTGTAGAGGAAGTTGAGAATTC

[0155] TTTGAAGCGGCTTCAAACAGACTATATTGATCTTTATCAGGTGCATTGGCCCGATC

[0156] CGCTTGTGCCAATTGAAGAAACGGCTGAAGTCATGAAGGAATTATATGATGCGGG

[0157] AAAAATCCGGGCGATTGGCGTCAGCAATTTTTCAATTGAGCAAATGGATACATTTC

[0158] GCGCCGTCGCACCTCTCCATACGATTCAGCCTCCATATAATCTGTTTGAAAGAGAG

[0159] ATGGAAGAGAGTGTCCTTCCTTATGCGAAAGATAACAAGATAACAACATTATTAT

[0160] ACGGCAGTTTATGCAGAGGGCTGTTAACAGGCAAAATGACTGAAGAATATACATT

[0161] TGAGGGCGATGATCTGCGTAATCACGATCCAAAATTCCAGAAGCCCCGCTTTAAA

[0162] GAGTATCTTTCTGCTGTGAATCAATTGGATAAGCTGGCGAAGACACGTTATGGAAA

[0163] ATCAGTGATTCACTTGGCTGTCAGATGGATCTTAGATCAGCCGGGAGCGGATATCG

[0164] CTCTTTGGGGAGCAAGAAAGCCTGGGCAGCTTGAGGCCCTATCTGAGATTACAGG

[0165] CTGGACGCTGAACAGTGAAGATCAGAAAGATATCAATACTATATTGGAAAATACG

[0166] ATATCAGACCTGTCGGACCGGAGTTTATGGCCCCGCCGACCAGAGAGGAAATAT

[0167] AA

[0168] The present invention has been described in detail above with reference to specific embodiments and / or exemplary examples, as well as the accompanying drawings. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. An aldehyde-ketone reductase yhdN mutant, characterized in that, The aldehyde / ketone reductase yhdN mutant amino acid sequence is shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO.

4.

2. A nucleic acid, characterized in that, The nucleic acid sequence is shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO.

7.

3. The nucleic acid of claim 2, wherein The nucleic acid sequence is shown in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO.

7.

4. A recombinant expression vector, which is a recombinant expression vector containing the nucleic acid according to claim 2 or 3, characterized in that, The recombinant expression vector is selected from a plasmid or a viral vector.

5. The recombinant expression vector of claim 4, wherein, The recombinant expression vector is a plasmid.

6. A recombinant expression transformant, which is a recombinant expression transformant containing the recombinant expression vector according to claim 4 or 5, characterized by, The recombinant expression transformant is Escherichia coli (BL21 (DE3) pLysSRARE E. coli ).

7. The recombinant expression transformant according to claim 6, wherein, The recombinant expression transformant is E. coli BL21(DE3).

8. A method for producing the aldehyde / ketone reductase yhdN mutant according to claim 1, characterized by, The recombinant transformant according to claim 6 or 7 is cultured, and the recombinant aldehyde / ketone reductase yhdN mutant is obtained from the culture.

9. The method of claim 8, wherein, The obtained recombinant plasmid containing the aldoketoreductase yhdN mutant and the recombinant plasmid containing the GDH gene of Bacillus subtilis 168 were transformed into the competent cells of BL21 (DE3) by heat shock E. coli The competent cells of BL21 (DE3) were obtained, and the recombinant bacteria were inoculated, transferred, induced, and the bacterial cells were recovered.

10. A catalyst for the preparation of the chiral intermediate N-ethylmethylcarbamic acid-3-[(1S)-hydroxyethyl]-phenyl ester ((S)-NEMCA-HEPE), characterized in that, The catalyst is a bacterium obtained by induced culture of a recombinant whole cell containing the aldehyde / ketone reductase yhdN mutant and glucose dehydrogenase (GDH) of Bacillus subtilis 168 according to claim 1.

11. A catalyst according to claim 10 for the catalytic formation of chiral hydroxyl compounds ( S Its application in )-NEMCA-HEPE is characterized by, With N-ethyl-methylcarbamoyl acetophenone as the substrate, glucose as the co-substrate, potassium phosphate buffer and 2% v / v DMSO added as the reaction medium, the reaction was carried out at 25-35 °C, 170-210 rpm, and after separation and purification, ( S )-NEMCA-HEPE, The substrate concentration is 2-100 mM, The co-substrate concentration is 100-500 mM, The amount of the catalyst is 0.05-0.5 g / mL in terms of the wet weight of the bacterium.

12. The use according to claim 11, wherein, The substrate concentration is 3-50 mM, The co-substrate concentration is 150-450 mM, The amount of the catalyst is 0.08-0.4 g / mL in terms of the wet weight of the bacterium.

13. The use according to claim 12, wherein, The substrate concentration is 4-10 mM, The co-substrate concentration is 200-400 mM, The amount of the catalyst is 0.1-0.3 g / mL in terms of the wet weight of the bacterium.

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