Alcohol dehydrogenase mutant and application thereof in production of R-3-ethyl hydroxybutyrate

Abstract

The invention provides an alcohol dehydrogenase mutant and application of the alcohol dehydrogenase mutant in production of R-3-ethyl hydroxybutyrate. The alcohol dehydrogenase mutant is obtained by performing single-point or multi-point simultaneous mutation on wild type alcohol dehydrogenase with a sequence as shown in SEQ ID NO.1; according to the obtained S143T / E145L / K160R / Y190F mutant, the 24-hour conversion rate can reach 98% or above under the condition that the substrate concentration is 350 g / L; after the immobilized cell of the alcohol dehydrogenase mutant is repeatedly used for five times, the catalytic performance does not decline obviously; meanwhile, glucose dehydrogenase is matched for regenerating coenzyme NADH, so that D-gluconic acid-1, 5-lactone can be co-produced under the condition of not reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, and the production cost is reduced; the mutant alcohol dehydrogenase expressed by the alcohol dehydrogenase mutant strain has high tolerance to organic solvents and substrate concentration, and has the advantage of high conversion rate.

Description

Technical Field

[0001] The invention relates to the technical fields of enzyme catalysis engineering and protein modification engineering, and in particular to an alcohol dehydrogenase mutant and application thereof in the production of R-3-hydroxybutyric acid ethyl ester. Background Art

[0002] Ethyl 3-hydroxybutyrate is an important pharmaceutical intermediate, and R-3-hydroxybutyrate is one of its single chiral enantiomers. It can be used to synthesize penicillin antibiotics such as L-carnitine and selinexetine, and can also be used in the production of R-3-hydroxybutyrate.

[0003] The traditional chemical method of reducing ethyl acetoacetate to obtain 3-hydroxybutyric acid ethyl ester is an R / S racemic compound. The separation conditions for treating the racemic compound to obtain a single chiral compound are difficult and complicated, and the yield is extremely low. Chemical asymmetric synthesis reactions have complicated steps, harsh conditions, low yields, and difficult preparation of chiral catalysts. They are expensive and difficult to recycle. At the same time, most organic solvents used in the reaction are toxic, which puts great pressure on environmental protection, greatly limiting the application of this method in production.

[0004] Enzyme catalysis has the advantages of high chirality and high conversion rate, mild reaction conditions, safe and non-toxic reagents, and environmental friendliness. In 2006, Zhejiang University of Technology published patent CN101210258A, which introduced a method based on Pichia pastoris ( Pichia membranaef aciens Hansen ) A method for using fermentation products as an enzyme source to catalyze the asymmetric reduction of ethyl acetoacetate to produce ethyl R-3-hydroxybutyrate, wherein the yield of the method reaches 95% and the ee value reaches 99%, but the application does not disclose the amino acid sequence or nucleic acid sequence of the enzyme; Patent CN107083406A published by Fan Wenchao et al. in 2017 introduced a method for producing R-3-hydroxybutyric acid using genetically engineered Corynebacterium glutamicum, but the application did not introduce the construction information of the engineered bacteria; Patent CN110035991A published by Fan Wenchao et al. in 2018 describes in detail the construction method of genetically engineered Corynebacterium glutamicum for producing R-3-hydroxybutyric acid, which involves 3-HB dehydrogenase, but does not disclose its amino acid sequence or nucleic acid sequence; In 2019, the patent CN109852593A published by Fan Wenchao et al. introduced a novel Candida magnoliae ) alcohol dehydrogenase, and discloses its directed evolution results, the enzyme can catalyze ethyl acetoacetate and methyl acetoacetate, wherein the ee value of R-3-hydroxybutyrate ethyl ester can reach 99.9%, and the substrate concentration is within 150g / L; Patent CN110904161A disclosed by Zhejiang Yingmate Biotechnology in 2019, a method for producing R-3-hydroxybutyric acid by PHB depolymerase, the substrate of which is different from that of this application; Patent CN 111304140 A published by Tsinghua University in 2020 introduced a genetic intestinal bacteria that produces 3-hydroxybutyrate and the construction process of the genetically engineered bacteria, but did not disclose the amino acid sequence or nucleic acid sequence of the acetyl-CoA reductase mentioned therein; in the same year, patent CN111705068A published by Ningbo Enzyme Biotechnology introduced a 3-hydroxybutyrate-producing enteric bacterium. Leifsonia sp. strain S749 ketoreductase, which can convert ethyl acetoacetate to R-3-hydroxybutyrate at a concentration of more than 400 g / L in a micro-water environment, with a conversion rate of up to 99.5% and an ee value of 99.5%; In 2021, Yikelai Biotechnology published patent CN116410944A, which fully synthesized a dehydrogenase with PDB number 5X8H. The enzyme is derived from Chryseobacterium sp. CA49, after targeted modification, has a substrate concentration and conversion rate comparable to the ketoreductase disclosed in the Ningbo Enzyme patent, but an ee value of 99.8%, which is better than the Ningbo Enzyme patent; The paper The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency discloses an R-specific alcohol dehydrogenase and reports the crystal structure of the enzyme (PDB: 1NXQ), analyzing the key amino acid sites that affect the enzyme. However, the paper did not study the substrate spectrum of the enzyme or the key amino acid sites.

[0005] NAD-dependent alcohol dehydrogenase derived from Lactobacillus breve Levilactobacillus brevis , which can catalyze ethyl acetoacetate to produce ethyl R-3-hydroxybutyrate with an ee value of >99.95%, but has poor tolerance to substrate concentration and solvent, resulting in a long conversion time and a low conversion rate; therefore, if the effect of high conversion rate can be achieved in a short time through modification, it will be of great significance for the preparation of ethyl R-3-hydroxybutyrate. Summary of the invention

[0006] In view of this, the present invention provides an alcohol dehydrogenase mutant and its application in the production of R-3-hydroxybutyrate ethyl ester, wherein the alcohol dehydrogenase mutant is obtained by mutating a wild-type alcohol dehydrogenase, and the sequence shown in SEQ ID NO.1 is subjected to single-point or multi-point simultaneous mutation to obtain the alcohol dehydrogenase mutant; the mutation site is one or more of S143T, E145L, K160R, and Y190F; Preferably, the mutations comprise the following: S143T, E145L, K160R, Y190F, S143T / E145L, S143T / K160R, S143T / Y190F, E145L / K160R, E145L / Y190F, K160R / Y190F, S143T / E145L / K160R, S143T / E145L / Y190F, S143T / K160R / Y190F, E145L / K160R / Y190F, S143T / E145L / K160R / Y190F.

[0007] Among them, the obtained S143T / E145L / K160R / Y190F mutant has a 24-hour conversion rate of more than 98% under the condition of a substrate concentration of 350 g / L; its immobilized cells are reused 5 times without showing any obvious decline in catalytic performance; at the same time, glucose dehydrogenase is used in conjunction with coenzyme regeneration to co-produce D-glucono-1,5-lactone without reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, thereby reducing production costs.

[0008] The technical solution of the present invention is as follows: A stereoselective wild-type alcohol dehydrogenase from Lactobacillus breve Levilactobacillus brevis ATCC 367; the amino acid sequence is shown in SEQ ID NO.1, NCBI Reference Sequence: WP_011667141.1; the nucleic acid sequence is shown in SEQ ID NO.3, GenBank: CP000416.1 REGION: complement (370044..370802).

[0009] An alcohol dehydrogenase mutant is obtained by mutating a wild-type alcohol dehydrogenase. The alcohol dehydrogenase mutant is obtained by subjecting the sequence shown in SEQ ID NO.1 to single-point or multi-point mutation; the mutation site is one or more of S143T, E145L, K160R, and Y190F; the sequence shown in SEQ ID NO.1 is subjected to multi-point mutation of S143T, E145L, K160R, and Y190F, and the amino acid sequence of the obtained S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant is shown in SEQ ID NO.2, and the nucleic acid sequence is shown in SEQ ID NO.4.

[0010] An alcohol dehydrogenase mutant expression strain, the preparation process is: connecting the nucleic acid sequence of the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant to a pET vector through NdeI / XhoI restriction sites to obtain a recombinant vector; transforming the recombinant vector into an expression system through a heat shock method to obtain a mutant LbADH expression strain; The expression system includes but is not limited to the Escherichia coli expression system.

[0011] The constructed wild-type and mutant LbADH expression strains were induced to express and the wild enzyme and mutant enzyme were obtained. The target protein was purified using the standard 6×His tag method. The protein was purified by 100 mM Tris-HCl, 2 mM MgCl2, 20 mM isopropanol, 1 mM NAD + , pH 7.5 was used as the enzyme activity assay system, and the enzyme activity of the expressed enzyme was assayed at 30°C. Under this condition, the amount of enzyme required to generate 1µmol NADH per minute was 1U; after testing, the purified enzyme, the specific activity of the wild-type LbADH was 10.5U / mg, and the specific activity of the mutant LbADH was 3.3U / mg, and the specific activity was increased by about 3.2 times.

[0012] The wild-type and mutant LbADH expression strains were fermented at high density by a common method to obtain wet cells. The enzyme activity of the wild-type alcohol dehydrogenase was 120 U / g wet cells, and the enzyme activity of the mutant alcohol dehydrogenase was 440 U / g wet cells. The invention discloses an application of an alcohol dehydrogenase mutant in the production of R-3-hydroxybutyric acid ethyl ester, using crude enzyme solution prepared by an Escherichia coli expression strain, wet bacteria, and immobilized whole cells as the enzyme catalysis system, ethyl acetoacetate as the substrate, and reacting for 24 hours with a conversion rate of >98%.

[0013] Preferably, in the above application, a coenzyme needs to be added; the coenzyme is NAD + Or NADH, the substrate for coenzyme regeneration is isopropanol or glucose.

[0014] Preferably, when isopropanol is used as the substrate for coenzyme regeneration, no additional enzyme needs to be added, and the generated acetone needs to be removed by vacuuming or ventilation; when glucose is used as the substrate for coenzyme regeneration, glucose dehydrogenase needs to be additionally added.

[0015] Preferably, the preparation method of the immobilized whole cells of wild-type and mutant alcohol dehydrogenase is as follows: 100 g of wet bacteria frozen after high-density fermentation is taken, dissolved in 900 mL of pH 8.0-9.0 buffer, stirred at room temperature until completely dissolved, 6 g of diatomaceous earth is added, 30 g of 5% polyethyleneimine is added, the pH is adjusted to 8.0-9.0 with acid, stirred for 1 hour, and then 5 g of 50% glutaraldehyde is added, the pH is controlled to be greater than 7.0, and stirring is continued for 1 hour, filtered, and then washed with purified water 2-3 times, and the immobilized whole cells are obtained after filtration.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The R-specific alcohol dehydrogenase mutant LbADH has a higher specific activity and can generate higher chiral products compared to existing patents. The ee value of the obtained product R-3-hydroxybutyric acid ethyl ester is greater than 99.95%. Using the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant, the conversion rate can reach more than 98% in 24 hours under the condition of substrate concentration of 350g / L. 2. Use immobilized whole cells for catalysis. The immobilized whole cells can be reused, which reduces the catalytic cost and the difficulty of purification. After being reused for 5 times, there is no obvious decline in catalytic performance. 3. Compared with the use of isopropanol as a substrate for the regeneration of coenzyme NADH, the use of glucose as a substrate has no acetone inhibition, does not require vacuum or ventilation, has a higher reaction rate, and can co-produce D-glucono-1,5-lactone, thereby reducing production costs.

[0017] In conclusion, the production method of R-3-hydroxybutyric acid ethyl ester provided by the present invention is particularly suitable for large-scale industrial production and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a homology comparison between three alcohol dehydrogenases with published sequences and the mutant alcohol dehydrogenase of the present invention.

[0019] Figure 2 The protein expression results of the mutant expression strain BL21 (DE3) -LbADH in Example 2; in the figure, M is a molecular weight standard, lane 1 is the whole bacteria after 0.1mM IPTG induction expression, lane 2 is the supernatant after cell disruption, and lane 3 is the precipitate after cell disruption. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0021] Example 1: Construction of wild-type and mutant LbADH expression strains A stereoselective wild-type alcohol dehydrogenase from Lactobacillus breve Levilactobacillus brevis ATCC 367; the amino acid sequence is shown in SEQ ID NO.1, NCBI Reference Sequence: WP_011667141.1; the nucleic acid sequence is shown in SEQ ID NO.3, GenBank: CP000416.1 REGION: complement (370044..370802); SEQ ID NO.1 MGSSHHHHHHSSGLVPRGSMSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDG VFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ* SEQ ID NO.3 CATATGTCAAACCGGTTAGATGGAAAAGTAGCAATCGTTACAGGTGGTACGTTGGGTATCGGTTTAGCTATCGCCACGAAGTTCGTTGAAGAAGGGGCTAAGGTCATGATTACCGGCCGGCACAGCGATGTTGGTGAAAAAGCAGCTAAGAGTGTCGGCACTCCTGATCAGATTCAATTTTTCCAACATGAT TCTTCCGATGAAGACGGCTGGACGAAATTATTCGATGCAACGGAAAAAGCTTTGGCCCAGTTTCTACATTAGTTAATAACGCTGGGATCGCAGTTAACAAGAGTGTCGAAGAAACCACGACTGCTGAATGGCGTAAACTATTAGCCGTCAACCTTGATGGTGTCTTCTTCGGTACCCGATTAGGGATTCAA CGGATGAAGAACAAAGGCTTAGGGGCTTCCATCATCAACATGTCTTCGATCGAAGGCTTTGTGGGTGATCCTAGCTTAGGGGCTTACAACGCATCTAAAGGGGCCGTACGGATTATGTCCAAGTCAGCTGCCTTAGATTGTGCCCTAAAGGACTACGATGTTCGGGTAAACACTGTTCACCCTGGCTACATC AAGACACCATTGGTTGATGACCTACCAGGGCCGAAGAAGCGATGTCACAACGGACCAAGACGCCAATGGGCCATATCGGTGAACCTAACGATATTGCCTACATCTGTGTTTACTTGGCTTCTAACGAATCTAAATTTGCAACGGGTTCTGAATTTGTAGTTGATGGTGGTTATACCGTCAATAACTCGAG A S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant, wherein the sequence shown in SEQ ID NO.1 is subjected to multi-site mutations of S143T, E145L, K160R, and Y190F, and the amino acid sequence of the obtained S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant is shown in SEQ ID NO.2, and the nucleic acid sequence is shown in SEQ ID NO.4; SEQ ID NO.2 MGSSHHHHHHSSGLVPRGSHMSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSTILGFVGDPSLGAYNASRGAVRIMSKSAALDCALKDYDVRVNTVHPGFIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ* SEQ ID NO.4 CATATGTCAAACCGGTTAGATGGAAAAGTAGCAATCGTTACAGGTGGTACGTTGGGTATCGGTTTAGCTATCGCCACGAAGTTCGTTGAAGAAGGGGCTAAGGTCATGATTACCGGCCGGCACAGCGATGTTGGTGAAAAAGCAGCTAAGAGTGTCGGCACTCCTGATCAGATTCAATTTTTCCAACATGAT TCTTCCGATGAAGACGGCTGGACGAAATTATTCGATGCAACGGAAAAAGCTTTGGCCCAGTTTCTACATTAGTTAATAACGCTGGGATCGCAGTTAACAAGAGTGTCGAAGAAACCACGACTGCTGAATGGCGTAAACTATTAGCCGTCAACCTTGATGGTGTCTTCTTCGGTACCCGATTAGGGATTCAA CGGATGAAGAACAAAGGCTTAGGGGCTTCCATCATCAACATGTCTACCATCTTAGGCTTTGTGGGTGATCCTAGCTTAGGGGCTTACAACGCATCTCGAGGGGCCGTACGGATTATGTCCAAGTCAGCTGCCTTAGATTGTGCCCTAAAGGACTACGATGTTCGGGTAAACACTGTTCACCCTGGCTTCATC AAGACACCATTGGTTGATGACCTACCAGGGCCGAAGAAGCGATGTCACAACGGACCAAGACGCCAATGGGCCATATCGGTGAACCTAACGATATTGCCTACATCTGTGTTTACTTGGCTTCTAACGAATCTAAATTTGCAACGGGTTCTGAATTTGTAGTTGATGGTGGTTATACCGTCAATAACTCGAG The homology comparison results of the three alcohol dehydrogenases disclosed so far and the mutant alcohol dehydrogenase of the present invention are shown in Figure 1 , combined with Figure 1 It can be seen that the alcohol dehydrogenase of the present invention and the three disclosed protein sequences have low consistency.

[0022] The nucleic acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4 were fully synthesized by Qingke Biotechnology, and the two nucleic acid sequences were connected to two pET-28a vectors through NdeI / XhoI restriction sites, respectively, to construct wild-type pET28a-LbADH and mutant pET28a-LbADH recombinant vectors, respectively; the two recombinant vectors were transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, respectively, to construct wild-type and mutant BL21 (DE3)-LbADH expression strains, i.e., wild-type LbADH expression strain and mutant LbADH expression strain; single clones were screened, and strains with excellent expression were selected for amplification, and stored in 20% glycerol at -80°C; the specific operation method can be referred to the molecular biology experiment manual, which is not described in detail in this article.

[0023] The constructed wild-type LbADH expression strain and mutant LbADH expression strain were induced to express, respectively, to obtain wild enzyme and mutant enzyme; the target protein was purified using the standard 6×His tag method; 100mM Tris-HCl, 2mM MgCl2, 20mM isopropanol, 1mM NAD + , pH 7.5 was used as the enzyme activity assay system, and the enzyme activity of the expressed enzyme was assayed at 30°C. Under this condition, the amount of enzyme required to generate 1µmol NADH per minute was 1U; after testing, the purified enzyme, the specific activity of the wild-type LbADH was 10.5U / mg, and the specific activity of the mutant LbADH was 3.3U / mg, and the specific activity was increased by about 3.2 times.

[0024] Example 2: Shake flask fermentation of wild-type and mutant LbADH expression strains The wild-type and mutant LbADH wet cells were prepared as follows: The expression strain screened in Example 1 was inoculated into LB medium containing 25 μg / L kanamycin, and cultured in a shaking incubator at 37°C and 220 rpm. 600 When the OD value of the culture medium reached 2.0, the culture medium was inoculated with TB medium at a rate of 5% (v / v) and cultured in a shaking incubator at 37°C and 200 rpm. 600 When the concentration reached 3.0, IPTG was added as an inducer with a final concentration of 0.1 mM, and induced at 25°C for 15 h. The culture solution was centrifuged, the supernatant was discarded, and the cells were collected to obtain wet cells, which were stored at -20°C. The wild-type and mutant LbADH expression strains were fermented at high density using a common method to obtain wet cells. The enzyme activity of the wet cells was determined. The wild-type alcohol dehydrogenase activity was 120 U / g wet cells, and the mutant alcohol dehydrogenase activity was 440 U / g wet cells. The obtained wild-type and mutant LbADH wet cells were treated, and buffer was added to the wet cells to dilute the wet cells to a concentration of 20 g / L. The broken cells were separated by ultrasonic wave, and the broken cells were centrifuged and the supernatant was collected. The protein electrophoresis of the precipitate after centrifugation was performed. The results of mutant alcohol dehydrogenase protein electrophoresis are shown in Figure 2 .

[0025] Example 3: Preparation of wild-type and mutant LbADH crude enzyme solutions The crude enzyme solutions of wild-type and mutant LbADH were prepared as follows: Take 100 g of wet bacteria stored at -20°C in Example 2, dissolve it in 400 ml of 20 mM pH 7.5 PBS solution, use a high-pressure homogenizer to disrupt the cells, add 10 g of 5% polyethyleneimine to the enzyme solution after disruption, stir for 30 min, centrifuge at 1000 rpm for 45 min, discard the precipitate, and the supernatant is the required crude enzyme solution.

[0026] Example 4: Preparation of wild-type and mutant LbADH immobilized whole cells Wild-type and mutant LbADH immobilized whole cells were prepared as follows: Take 100 g of wet cells stored at -20 ° C in Example 2, dissolve in 900 mL of pH 8.5 buffer, stir at room temperature until completely dissolved, add 6 g of diatomaceous earth and 30 g of 5% polyethyleneimine in sequence, adjust the pH to 8.5 with acid, stir for 1 hour, then add 50% glutaraldehyde 5 g, control the pH during the stirring process to be greater than 7.0, continue stirring for 1 hour, filter, and wash 3 times with purified water. The filter cake after filtration is the required immobilized whole cells.

[0027] Example 5: Synthesis of R-3-hydroxybutyrate catalyzed by wild-type and mutant LbADH enzymes in isopropanol system Reaction 1: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 15 mL of pH 7.5 0.1 M PBS, and NAD + 10 mg, reaction temperature is 25°C, add 10 ml of crude enzyme solution of wild-type LbADH in Example 3 and start the reaction; Reaction 2: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 15 mL of pH 7.5 0.1 M PBS, and NAD + 10 mg, reaction temperature is 25°C, add 10 ml of crude enzyme solution of mutant LbADH of Example 3 and start the reaction; Reaction 3: In a 200 mL beaker, add 35 mL of methyl acetoacetate, 40 mL of isopropanol, 15 mL of pH 7.5 0.1 M PBS, and NAD +10 mg, reaction temperature is 25°C, add 10 ml of crude enzyme solution of wild-type LbADH in Example 3 and start the reaction; Reaction 4: In a 200 mL beaker, add 35 mL of methyl acetoacetate, 40 mL of isopropanol, 15 mL of pH 7.5 0.1 M PBS, and NAD + 10 mg, reaction temperature is 25°C, add 10 ml of crude enzyme solution of mutant LbADH of Example 3 and start the reaction; Reaction 5: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 23 mL of pH 7.5 0.1 M PBS, and NAD + 10 mg, the reaction temperature is 25°C, 2.0 g of wild-type LbADH immobilized whole cells from Example 4 are added and the reaction is started; Reaction 6: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 23 mL of pH 7.5 0.1 M PBS, and NAD + 10 mg, the reaction temperature is 25°C, 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 are added and the reaction is started; Reaction 7: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 10 mL of pH 7.5 0.1 M PBS, 13 mL of purified water, and NAD + 10 mg, the reaction temperature is 25°C, 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 are added and the reaction is started; Reaction 8: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 5 mL of pH 7.5 0.1 M PBS, 18 mL of purified water, and NAD + 10 mg, the reaction temperature is 25°C, add 2.0 g of the mutant LbADH immobilized whole cells of Example 4 and start the reaction; Reaction 9: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 23 mL of purified water, and NAD + 10 mg, the reaction temperature is 25°C, 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 are added and the reaction is started; Reaction 10: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 5 mL of pH 7.5 0.1 M PBS, 18 mL of purified water, and NAD + 5 mg, the reaction temperature is 25°C, 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 are added and the reaction is started; Reaction 11: In a 200 mL beaker, add 35 mL of ethyl acetoacetate, 40 mL of isopropanol, 5 mL of pH 7.5 0.1 M PBS, 18 mL of purified water, and NAD + 2 mg, the reaction temperature is 25°C, 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 are added and the reaction is started; Reaction 12: In a 200 mL beaker, 35 ml of ethyl acetoacetate, 40 ml of isopropanol, 5 ml of pH 7.5 0.1 M PBS, and 18 ml of purified water were added in sequence with magnetic stirring. The reaction temperature was 25° C. 2.0 g of the immobilized whole cells of mutant LbADH of Example 4 was added and the reaction was started.

[0028] Reactions 1-12 were sampled and tested at different time points, and the conversion rates were calculated. The results are shown in Table 1; Table 1 Reaction results of different systems

[0029] It can be seen from the data in Table 1 that, under the same conditions, with ethyl acetoacetate as the substrate, the conversion rate of the wild-type LbADH is lower than that of the mutant LbADH, and the chirality of the mutant enzyme product is significantly higher than that of the wild enzyme. This may be due to the change in the catalytic pocket of the mutant enzyme compared with the wild type, or it may be due to the increase in the specific activity of the mutant enzyme, which causes the relative proportion of the miscellaneous enzymes that have other effects on ethyl acetoacetate to decrease. The reaction results show that the mutant enzyme is significantly better than the wild-type enzyme in terms of ethyl acetoacetate conversion rate and product ee value.

[0030] Using methyl acetoacetate as substrate, the ee value of the product is no more than 98%, and the application value is not great.

[0031] When immobilized whole cells are used as the enzyme source, the effect of phosphate buffer as low as 0 mM on catalysis is not particularly obvious. However, considering the possible influence of residual phosphate in the immobilized cells, 5 mM phosphate buffer should be used when the immobilized cells are reused.

[0032] Without coenzyme NAD + When the product conversion rate is significantly reduced; coenzyme NAD + When the dosage is as low as 2 mg, there is no obvious effect on substrate conversion.

[0033] Example 6: Reuse of mutant LbADH immobilized whole cells to synthesize R-3-hydroxybutyrate ethyl ester Reaction system: 1000mL beaker, magnetic stirring, ethyl acetoacetate 180ml, isopropanol 200ml, pH7.5 0.1M PBS 25ml, purified water 95ml, NAD +10 mg, room temperature 25 ° C, add 10.0 g of the mutant LbADH immobilized whole cells of Example 4, and start the reaction by timing; after reacting for 24 hours, filter with a double-layer filter paper using a Buchner funnel, and filter until no liquid drips, and the filter cake after filtration, i.e., the immobilized whole cells, is continuously put into the next reaction solution for the next reaction; the results of the number of times the mutant LbADH immobilized whole cells are used and the conversion rate are shown in Table 2.

[0034] Table 2 Transformation results of mutant LbADH immobilized whole cells using repeated use

[0035] From the experimental results in Table 2, it can be seen that the mutant LbADH immobilized whole cells were reused five times, and there was no significant change in the 24-h substrate conversion rate.

[0036] Example 7: Synthesis of R-3-hydroxybutyrate ethyl ester in a mutant LbADH immobilized whole cell 3L system Reaction system 1: 5L three-necked flask, mechanical stirring, ethyl acetoacetate 1080ml, isopropanol 1200ml, pH 7.5 0.1M PBS 150ml, purified water 570ml, NAD + 60 mg, the water bath temperature was set at 30°C, 60.0 g of the mutant LbADH immobilized whole cells of Example 4 were added, and the reaction was started; after 6 hours of reaction, the substrate conversion rate was 58.4%, and after 24 hours, the substrate conversion rate was 89.3%, which was significantly lower than the conversion rate in Example 6. The conversion rate was 91.3% after 38 hours. It is speculated that the acetone produced by the dehydrogenation of isopropanol cannot volatilize in a large system, resulting in a high concentration of acetone in the reaction system, resulting in inhibition; a vacuum pump and a reflux device were installed on the reactor. After evacuation for 2 hours, the conversion rate was 95.4%, after evacuation for 4 hours, the conversion rate was 97.4%, and after evacuation for 8 hours, the conversion rate was 98.9%; Reaction system 2: 5L three-necked flask, mechanical stirring, ethyl acetoacetate 1080ml, isopropanol 1200ml, pH 7.5 0.1M PBS 150ml, purified water 570ml, NAD + 60 mg, the water bath temperature was set to 30 ° C, a vacuum pump and reflux device were installed, 60.0 g of the mutant LbADH immobilized whole cells of Example 4 were added, and the reaction was started; the reaction time was 6 hours, the conversion rate was 75.8%, the reaction time was 12 hours, the conversion rate was 94.0%, and the reaction time was 16 hours, the conversion rate was 96.5%; the conversion rate increased slowly from 12h to 16h, it was speculated that the vacuum force was too large and the reflux water temperature was too high, resulting in a large amount of isopropanol being drawn away. After adding 50 ml of isopropanol, the vacuum reaction was continued for 4 hours, and the conversion rate was 98.8%; Reaction system 3: 5L three-necked flask, mechanical stirring, ethyl acetoacetate 1080ml, isopropanol 1200ml, pH 7.5 0.1M PBS 150ml, purified water 570ml, NAD + 60 mg, the water bath temperature was set at 30°C, bottom aeration (1.5 L / min) and reflux device (cooling water temperature was controlled at 5-10°C) were installed, 60.0 g of the immobilized whole cells of mutant LbADH of Example 4 were added and the reaction was started; the reaction lasted for 6 hours, the conversion rate was 77.4%, the reaction lasted for 12 hours, the conversion rate was 94.0%, the reaction lasted for 16 hours, the conversion rate was 98.3%; From the above experimental results, it can be seen that when the reaction system is enlarged, the acetone produced by the reaction will be seriously inhibited if it is not discharged in time. This problem can be solved by vacuuming (at the same time, isopropanol needs to be added in batches during the reaction) or bottom ventilation and a reflux device. However, the temperature of the cooling water in the reflux device needs to be strictly controlled at 5-10°C to achieve the effect of discharging acetone but not isopropanol and substrate.

[0037] Example 8: Mutant LbADH and glucose dehydrogenase cooperate to convert and synthesize R-3-hydroxybutyrate ethyl ester In this embodiment, the glucose dehydrogenase used is derived from Bacillus megaterium IWG3, which is constructed and prepared by our company with reference to the document Crystal Structure of Glucose Dehydrogenase from Bacillus megaterium IWG3. It is not described in detail in the present invention, and glucose dehydrogenase from other sources may also be used; Reaction system: 5L three-necked flask, mechanical stirring, ethyl acetoacetate 450ml, purified water 1800ml, anhydrous glucose 720g, NAD + 60 mg, the water bath temperature was set at 30°C, 300 ml of the crude enzyme solution of mutant LbADH in Example 3 and 300 ml of the crude enzyme solution of glucose dehydrogenase were added, and the reaction was started; the reaction timer was set; the conversion rate was 69.8% after 2 hours, 89.6% after 4 hours, and 99.3% after 6 hours.

[0038] In combination with the above embodiments of the present invention, it can be seen that the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant has a 24-hour conversion rate of >98% under the condition of a substrate concentration of 350 g / L; the organic solvent tolerance, substrate concentration and conversion rate of the S143T / E145L / K160R / Y190F alcohol dehydrogenase are comparable to those of the enzymes of Ningbo Enzyme and Yikelai Biology, but the product ee value generated by the enzyme provided by the patent of the present invention is greater than 99.95%, which is significantly better than the enzymes provided by the above two applications, and the enzyme of the present invention can be used in conjunction with glucose dehydrogenase for coenzyme NADH regeneration under comparable substrate concentrations and conversion rates, and D-glucono-1,5-lactone can be co-produced without reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, thereby further reducing costs.

[0039] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all of them shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A wild-type alcohol dehydrogenase having stereoselectivity, characterized in that: The wild-type alcohol dehydrogenase is derived from Lactobacillus breve Levilactobacillus brevis ATCC 367; the amino acid sequence is shown in SEQ ID NO.1, NCBI Reference Sequence: WP_011667141.1; the nucleic acid sequence is shown in SEQ ID NO.3, GenBank: CP000416.1 REGION: complement (370044..370802).

2. An alcohol dehydrogenase mutant, characterized in that: The alcohol dehydrogenase mutant is obtained by mutating the wild-type alcohol dehydrogenase according to claim 1, and the sequence shown in SEQ ID NO.1 is mutated at a single point or multiple points at the same time to obtain the alcohol dehydrogenase mutant, wherein the mutation site is one or more of S143T, E145L, K160R, and Y190F.

3. An alcohol dehydrogenase mutant, characterized in that The wild-type alcohol dehydrogenase according to claim 2 is mutated at a single point or multiple points simultaneously, and the mutations include the following: S143T, E145L, K160R, Y190F, S143T / E145L, S143T / K160R, S143T / Y190F, E145L / K160R, E145L / Y190F, K160R / Y190F, S143T / E145L / K160R, S143T / E145L / Y190F, S143T / K160R / Y190F, E145L / K160R / Y190F, S143T / E145L / K160R / Y190F.

4. The alcohol dehydrogenase mutant according to claim 3, characterized in that The sequence shown in SEQ ID NO.1 was subjected to multi-site mutations of S143T, E145L, K160R, and Y190F, and the amino acid sequence of the obtained S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant is shown in SEQ ID NO.2, and the nucleic acid sequence is shown in SEQ ID NO.

4.

5. A mutant alcohol dehydrogenase expression strain, characterized in that: The preparation process is as follows: connecting the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant nucleic acid sequence described in claim 3 to the pET vector through the NdeI / XhoI restriction site to obtain a recombinant vector; transforming the recombinant vector into an expression system by heat shock method to obtain a mutant LbADH expression strain; The expression system includes but is not limited to the Escherichia coli expression system.

6. Use of an alcohol dehydrogenase mutant in the production of R-3-hydroxybutyrate ethyl ester, characterized in that: The wet bacteria, crude enzyme solution and immobilized whole cells prepared by high-density fermentation of the mutant LbADH expression strain according to claim 5 are used as the enzyme catalysis system, ethyl acetoacetate is used as the substrate, the reaction is carried out for 24 hours, and the conversion rate is greater than 98%.

7. The use according to claim 6, characterized in that Need to add coenzyme NAD + Or NADH; use isopropanol or glucose coenzyme as coenzyme regeneration substrate.

8. The use according to claim 7, characterized in that When isopropanol is used as the substrate for coenzyme regeneration, no additional enzyme needs to be added, and the generated acetone needs to be removed by vacuum or ventilation; when glucose is used as the substrate for coenzyme regeneration, additional glucose dehydrogenase needs to be added.

9. The use according to claim 7, characterized in that The preparation method of the mutant alcohol dehydrogenase immobilized whole cells is as follows: 100 g of wet bacteria frozen after high-density fermentation is taken, dissolved in 900 mL of pH 8.0-9.0 buffer, stirred at room temperature until completely dissolved, 6 g of diatomaceous earth is added, 30 g of 5% polyethyleneimine is added, the pH is adjusted to 8.0-9.0 with acid, stirred for 1 hour, and then 5 g of 50% glutaraldehyde is added to control the pH>7.0, and stirring is continued for 1 hour, filtered, and then washed with purified water 2-3 times, and the immobilized whole cells are obtained after filtration.

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