An alcohol dehydrogenase mutant and its application in production of r-3-hydroxybutyric acid ethyl ester

Abstract

The application provides an alcohol dehydrogenase mutant and application thereof in R-3-hydroxybutyric acid ethyl ester production, the alcohol dehydroportation mutant is obtained by simultaneously mutating a single point or multiple points of a wild-type alcohol dehydrogenase shown in the sequence of SEQ ID NO.1;The conversion rate of the obtained S143T / E145L / K160R / Y190F mutant can reach more than 98% under the condition of substrate concentration 350g / L for 24 hours;The catalytic performance of the immobilized cell of the alcohol dehydrogenase mutant does not obviously decline after being used repeatedly for 5 times;Meanwhile, the alcohol dehydrogenase mutant is used for coenzyme NADH regeneration with glucose dehydrogenase, and D-gluconic acid-1,5-lactone can be co-produced without reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, thereby reducing the production cost;The mutant alcohol dehydrogenase expressed by the alcohol dehydrogenase mutant strain has the advantages of high tolerance to organic solvents and substrate concentration and high conversion rate.

Description

Technical Field

[0001] This invention relates to the fields of enzyme catalysis engineering and protein modification engineering, specifically to an alcohol dehydrogenase mutant and its application in the production of ethyl R-3-hydroxybutyrate. Background Technology

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

[0003] Ethyl 3-hydroxybutyrate, obtained by the reduction of ethyl acetoacetate using traditional chemical methods, is a reciprocal (R / S) compound. Separating this reciprocal into a single chiral compound is difficult and complex, resulting in extremely low yields. Chemical asymmetric synthesis involves cumbersome reaction steps, demanding conditions, and low yields. Chiral catalysts are difficult to prepare, expensive, and difficult to recycle. Furthermore, most of the organic solvents used in the reaction are toxic, posing significant environmental pressures and severely limiting the application of this method in production.

[0004] Enzyme catalysis has the advantages of high chirality and high conversion rate, and the reaction conditions are mild, the reagents used are safe and non-toxic, and it is environmentally friendly. Patent CN101210258A, published by Zhejiang University of Technology in 2006, describes a method using Pichia pastoris (a type of yeast)... Pichia membranaefaciens Hansen The application discloses a method for using fermentation products as an enzyme source to catalyze the asymmetric reduction of ethyl acetoacetate to produce ethyl R-3-hydroxybutyrate. The method has a yield of 95% and an ee value of 99%, but the application does not disclose the amino acid sequence or nucleic acid sequence of the enzyme.

[0005] In 2017, Fan Wenchao et al. published patent CN107083406A, which introduced a method for producing R-3-hydroxybutyric acid using genetically engineered Corynebacterium glutamicum, but the application did not describe the construction information of the engineered bacteria.

[0006] The patent CN110035991A published by Fan Wenchao et al. in 2018 describes in detail the construction method of genetically engineered Corynebacterium glutamicum for the production of R-3-hydroxybutyric acid, which involves 3-HB dehydrogenase, but does not disclose its amino acid sequence or nucleic acid sequence.

[0007] In 2019, Fan Wenchao et al. published patent CN109852593A, which introduced a yeast derived from *Candida magnoliae* (…). Candida magnoliae The alcohol dehydrogenase of R-3-hydroxybutyrate was developed and its directed evolution results were disclosed. The enzyme can catalyze ethyl acetoacetate and methyl acetoacetate, with the ee value of R-3-hydroxybutyrate reaching 99.9% and the substrate concentration being within 150 g / L.

[0008] The patent CN110904161A disclosed by Zhejiang Yingmate Biology in 2019, a method for producing R-3-hydroxybutyric acid by PHB depolymerase, the substrate of this method is different from the present application;

[0009] The patent CN111304140A disclosed by Tsinghua University in 2020, introduced a 3-hydroxybutyric acid-producing intestinal bacteria, and introduced 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; In the same year, Ningbo Enzyme Biology disclosed the patent CN111705068A, which introduced the ketoreductase from Leifsonia sp. strain S749, which can convert ethyl acetoacetate to R-3-hydroxybutyric acid ethyl ester in a micro-water environment with a concentration of more than 400g / L, with a conversion rate of up to 99.5% and an ee value of 99.5%;

[0010] In 2021, the patent CN116410944A disclosed by Yikelay Biological Technology, synthesized the dehydrogenase with PDB number 5X8H, which was derived from Chryseobacterium sp. CA49, which was subjected to directed evolution, and the substrate concentration and conversion rate of the enzyme were comparable to those of the ketoreductase disclosed in the Ningbo Enzyme Biology patent, but the ee value was 99.8%, superior to the Ningbo Enzyme patent;

[0011] The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency disclosed an R-specific alcohol dehydrogenase and reported the crystal structure of the enzyme (PDB:1NXQ), analyzed the key amino acid sites affecting the enzyme, but the substrate spectrum of the enzyme was not studied, nor was the key amino acid site.

[0012] The NAD-dependent alcohol dehydrogenase is derived from Lactobacillus brevis Levilactobacillus brevis , which can catalyze the conversion of ethyl acetoacetate to R-3-hydroxybutyric acid ethyl ester with an ee value of >99.95%, but its substrate concentration and solvent tolerance are poor, resulting in long conversion time and low conversion rate; Therefore, if the effect of achieving high conversion rate in a short time can be achieved through modification, it is of great significance for the preparation of R-3-hydroxybutyric acid ethyl ester. SUMMARY

[0013] In view of this, the present application provides an alcohol dehydrogenase mutant and its application in R-3-hydroxybutyric acid ethyl ester production, the alcohol dehydrogenase mutant is obtained by mutating the wild-type alcohol dehydrogenase, and the alcohol dehydrogenase mutant is obtained by simultaneously mutating the sequence shown in SEQ ID NO. 1 at one or more points; the mutation sites are one or more of S143T, E145L, K160R, Y190F;

[0014] Preferably, the mutation comprises 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.

[0015] Among them, the S143T / E145L / K160R / Y190F mutant obtained can reach more than 98% conversion rate under the condition of substrate concentration 350 g / L for 24 hours; the immobilized cells are repeatedly used for 5 times, and the catalytic performance does not show obvious decline; at the same time, the glucose dehydrogenase is used for coenzyme regeneration, without reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, co-producing D-gluconic acid-1,5-lactone, reducing the production cost.

[0016] The technical scheme of the present application is as follows:

[0017] A wild-type alcohol dehydrogenase with stereoselectivity, derived from Bifidobacterium 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).

[0018] An alcohol dehydrogenase mutant, which is obtained by mutating a wild-type alcohol dehydrogenase, and is obtained by mutating one or more sites of the sequence shown in SEQ ID NO. 1; the mutation sites are S143T, E145L, K160R, Y190F; the amino acid sequence of the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant obtained by mutating the S143T, E145L, K160R, Y190F sites of the sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2, and the nucleic acid sequence is shown in SEQ ID NO. 4.

[0019] An alcohol dehydrogenase mutant expression strain, the preparation process is as follows: the nucleic acid sequence of the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant is connected to the pET vector through NdeI / XhoI enzyme cutting sites to obtain a recombinant vector; the recombinant vector is transformed into an expression system through a heat shock method to obtain a mutant LbADH expression strain;

[0020] The expression system includes but is not limited to an Escherichia coli expression system.

[0021] The constructed wild-type and mutant LbADH expression strains are induced for expression to obtain wild-type and mutant enzymes; the standard 6×His tag method is used to purify the target protein; 100mM Tris-HCl, 2mM MgCl2, 20mM isopropyl alcohol, 1mM NAD + , pH 7.5 are used as an enzyme activity determination system, and the enzyme activity of the expressed enzyme is determined at 30°C; under this condition, 1U of enzyme is required to generate 1µmol of NADH per minute; after detection, the specific activity of the purified wild-type LbADH is 10.5U / mg, and the specific activity of the mutant LbADH is 3.3U / mg, which is about 3.2 times higher.

[0022] The constructed wild-type and mutant LbADH expression strains are subjected to high-density fermentation by a general method to obtain wet bacterial cells; enzyme activity determination shows that the enzyme activity of the wild-type alcohol dehydrogenase is 120U / g of wet bacterial cells, and the enzyme activity of the mutant alcohol dehydrogenase is 440U / g of wet bacterial cells;

[0023] An application of an alcohol dehydrogenase mutant in the production of R-3-hydroxybutyric acid ethyl ester, in which a crude enzyme solution, wet bacterial cells, or immobilized whole cells prepared from an Escherichia coli expression strain are used as an enzyme catalytic system, and ethyl acetoacetate is used as a substrate, and the conversion rate is >98% after 24 hours of reaction.

[0024] Preferably, in the above application, a coenzyme needs to be added; the coenzyme is NAD + or NADH, and isopropyl alcohol or glucose is used as a substrate for coenzyme regeneration.

[0025] 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 vacuum or aeration; when glucose is used as the substrate for coenzyme regeneration, additional glucose dehydrogenase needs to be added.

[0026] Preferably, the wild-type and mutant alcohol dehydrogenase immobilized whole cells are prepared as follows: 100 g of wet cells stored after high-density fermentation are dissolved in 900 mL of a buffer with a pH of 8.0-9.0, 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, 5 g of 50% glutaraldehyde is then added, the pH is controlled to be greater than 7.0, and stirring is continued for 1 hour, then suction filtration is performed, and the immobilized whole cells are obtained after washing with purified water for 2-3 times and suction filtration.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] 1. The specific activity of the mutant LbADH of R-specific alcohol dehydrogenase is higher, and compared with existing patents, it can generate higher chiral products, and 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% under the condition of a substrate concentration of 350 g / L for 24 hours;

[0029] 2. The use of immobilized whole cells for catalysis can reduce the cost of catalysis and the difficulty of purification, and the catalytic performance does not show obvious decline after repeated use for 5 times;

[0030] 3. Compared with the use of isopropanol as the substrate for coenzyme NADH regeneration, the use of glucose as the substrate does not have acetone inhibition, does not require vacuum or aeration, has a higher reaction speed, and can co-produce D-gluconic acid-1,5-lactone, thereby reducing production costs.

[0031] In summary, the production method of R-3-hydroxybutyric acid ethyl ester provided by the present application is particularly suitable for large-scale industrial production and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Comparison of the homology of the alcohol dehydrogenases of the three published sequences with the mutant alcohol dehydrogenase of the present application.

[0033] 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 0.1 mM IPTG induction expression, lane 2 is the supernatant after cell disruption, and lane 3 is the precipitate after cell disruption. DETAILED DESCRIPTION

[0034] In order to make the technical solutions in the present application better understood by those skilled in the art, the technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0035] Example 1: Construction of wild-type and mutant LbADH expression strains

[0036] A wild-type alcohol dehydrogenase with stereoselectivity is derived from Lactobacillus brevis Levilactobacillus brevis ATCC 367; the amino acid sequence is shown as SEQ ID NO. 1, NCBI Reference Sequence: WP_011667141.1; the nucleic acid sequence is shown as SEQ ID NO. 3, GenBank: CP000416.1 REGION: complement (370044..370802);

[0037] SEQ ID NO. 1

[0038] MSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ*

[0039] SEQ ID NO. 3

[0040] ATGTCAAACCGGTTAGATGGAAAAGTAGCAATCGTTACAGGTGGTACGTTGGGTATCGGTTTAGCTATCGCCACGAAGTTCGTTGAAGAAGGGGCTAAGGTCATGATTACCGGCCGGCACAGCGATGTTGGTGAAAAAGCAGCTAAGAGTGTCGGCACTCCTGATCAGATTCAATTTTTCCAACATGATTCTTCCGATGAAGACGGCTGGACGAAATTATTCGATGCAACGGAAAAAGCCTTTGGCCCAGTTTCTACATTAGTTAATAACGCTGGGATCGCAGTTAACAAGAGTGTCGAAGAAACCACGACTGCTGAATGGCGTAAACTATTAGCCGTCAACCTTGATGGTGTCTTCTTCGGTACCCGATTAGGGATTCAACGGATGAAGAACAAAGGCTTAGGGGCTTCCATCATCAACATGTCTTCGATCGAAGGCTTTGTGGGTGATCCTAGCTTAGGGGCTTACAACGCATCTAAAGGGGCCGTACGGATTATGTCCAAGTCAGCTGCCTTAGATTGTGCCCTAAAGGACTACGATGTTCGGGTAAACACTGTTCACCCTGGCTACATCAAGACACCATTGGTTGATGACCTACCAGGGGCCGAAGAAGCGATGTCACAACGGACCAAGACGCCAATGGGCCATATCGGTGAACCTAACGATATTGCCTACATCTGTGTTTACTTGGCTTCTAACGAATCTAAATTTGCAACGGGTTCTGAATTTGTAGTTGATGGTGGTTATACCGCTCAATAA

[0041] A S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant, wherein the sequence shown in SEQ ID NO. 1 is subjected to S143T, E145L, K160R, and Y190F multi-site mutations, 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;

[0042] SEQ ID NO. 2

[0043] MSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSTILGFVGDPSLGAYNASRGAVRIMSKSAALDCALKDYDVRVNTVHPGFIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ*

[0044] SEQ ID NO. 4

[0045] ATGTCAAACCGGTTAGATGGAAAAGTAGCAATCGTTACAGGTGGTACGTTGGGTATCGGTTTAGCTATCGCCACGAAGTTCGTTGAAGAAGGGGCTAAGGTCATGATTACCGGCCGGCACAGCGATGTTGGTGAAAAAGCAGCTAAGAGTGTCGGCACTCCTGATCAGATTCAATTTTTCCAACATGATTCTTCCGATGAAGACGGCTGGACGAAATTATTCGATGCAACGGAAAAAGCCTTTGGCCCAGTTTCTACATTAGTTAATAACGCTGGGATCGCAGTTAACAAGAGTGTCGAAGAAACCACGACTGCTGAATGGCGTAAACTATTAGCCGTCAACCTTGATGGTGTCTTCTTCGGTACCCGATTAGGGATTCAACGGATGAAGAACAAAGGCTTAGGGGCTTCCATCATCAACATGTCTACCATCTTAGGCTTTGTGGGTGATCCTAGCTTAGGGGCTTACAACGCATCTCGAGGGGCCGTACGGATTATGTCCAAGTCAGCTGCCTTAGATTGTGCCCTAAAGGACTACGATGTTCGGGTAAACACTGTTCACCCTGGCTTCATCAAGACACCATTGGTTGATGACCTACCAGGGGCCGAAGAAGCGATGTCACAACGGACCAAGACGCCAATGGGCCATATCGGTGAACCTAACGATATTGCCTACATCTGTGTTTACTTGGCTTCTAACGAATCTAAATTTGCAACGGGTTCTGAATTTGTAGTTGATGGTGGTTATACCGCTCAATAA

[0046] The homology comparison results of the three protein sequences of alcohol dehydrogenase disclosed at present and the mutant alcohol dehydrogenase of the present application are shown in Figure 1 , and it can be known from Figure 1 that the consistency of the present application and the three protein sequences of alcohol dehydrogenase disclosed is low.

[0047] The nucleic acid sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are synthesized by GenScript, and the two nucleic acid sequences are connected to two pET-28a vectors through NdeI / XhoI enzyme cutting sites, respectively, to construct wild type pET28a-LbADH and mutant pET28a-LbADH recombinant vectors; the two recombinant vectors are transformed into E. 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; monoclones are screened, and strains with excellent expression are selected and amplified, and stored in 20% glycerol at -80°C; the specific operation method can refer to the molecular biology experiment manual, and will not be described in detail herein.

[0048] The constructed wild type LbADH expression strain and mutant LbADH expression strain are induced and expressed, respectively, to obtain wild enzymes and mutant enzymes; the standard 6×His tag method is used to purify the target protein; 100mM Tris-HCl, 2mM MgCl2, 20mM isopropanol, 1mM NAD + pH7.5 is used as the enzyme activity determination system, and the expressed enzymes are determined for enzyme activity under the condition of 30°C; under this condition, 1U of enzyme is required to generate 1µmol of NADH per minute; after detection, the specific activity of the purified wild type LbADH is 10.5U / mg, and the specific activity of the mutant LbADH is 3.3U / mg, which is about 3.2 times higher.

[0049] Example Two: Shake flask fermentation of wild type and mutant LbADH expression strains

[0050] The wild type and mutant LbADH wet cells are prepared according to the following method:

[0051] The expression strain screened in Example One is inoculated into LB medium containing 25µg / L kanamycin, and is placed in a 37°C, 220rpm shaking incubator for culture; when the OD 600 of the culture solution reaches 2.0, it is inoculated into TB medium at a 5% (v / v) inoculation amount, and is placed in a 37°C, 200rpm shaking incubator for culture; when the OD 600 of the culture solution reaches 3.0, IPTG with a final concentration of 0.1mM is added as an inducer, and is induced at 25°C for 15h; the culture solution is centrifuged, the supernatant is discarded, and the cells are collected, i.e. the wet cells, which are stored at -20°C;

[0052] The wild type and mutant LbADH expression strains were subjected to high-density fermentation by using the general method, and wet bacterial bodies were obtained. The enzyme activity of the wild type alcohol dehydrogenase was 120 U / g wet bacterial body, and the enzyme activity of the mutant alcohol dehydrogenase was 440 U / g wet bacterial body;

[0053] The obtained wild type and mutant LbADH wet bacterial bodies were treated, and buffer was added to the wet bacterial bodies to dilute to a concentration of 20 g / L of wet bacterial body. The bacterial liquid after crushing by ultrasonic wave was subjected to protein electrophoresis according to the whole bacteria, supernatant after centrifugation, and precipitate after centrifugation. The protein electrophoresis result of the mutant alcohol dehydrogenase is shown in Figure 2 .

[0054] Example Three: Preparation of wild type and mutant LbADH crude enzyme liquid

[0055] The wild type and mutant LbADH crude enzyme liquid was prepared according to the following method:

[0056] 100 g of the wet bacterial body stored at -20 DEG C in Example Two was dissolved in 400 ml of 20 mM PBS solution with pH of 7.5. The cell was crushed by using a high-pressure homogenizer. 10 g of 5% polyethyleneimine was added to the enzyme liquid after crushing. The mixture was stirred for 30 min, and then centrifuged at 1000 rpm for 45 min. The precipitate was discarded, and the supernatant was the required crude enzyme liquid.

[0057] Example Four: Preparation of wild type and mutant LbADH immobilized whole cell

[0058] The wild type and mutant LbADH immobilized whole cell was prepared according to the following method:

[0059] 100 g of the wet bacterial body stored at -20 DEG C in Example Two was dissolved in 900 ml of buffer with pH of 8.5. The mixture was stirred at room temperature until completely dissolved. 6 g of diatomite and 30 g of 5% polyethyleneimine were added in sequence. The pH was adjusted to 8.5 by using acid. The mixture was stirred for 1 hour. Then, 5 g of 50% glutaraldehyde was added. The pH was controlled to be greater than 7.0 during the stirring process. The stirring was continued for 1 hour. The mixture was suction filtered, and then washed with purified water for 3 times. The filter cake after suction filtration was the required immobilized whole cell.

[0060] Example Five: Synthesis of R-3-hydroxybutyrate catalyzed by wild type and mutant LbADH in isopropyl alcohol system

[0061] Reaction 1: A 200 ml beaker was magnetically stirred. Ethyl acetoacetate 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 15 ml, NAD + 10 mg, and 10 ml of wild type LbADH crude enzyme liquid prepared in Example Three were added in sequence. The reaction was started by timing, and the reaction temperature was 25 DEG C.

[0062] Reaction 2: 200 mL beaker, magnetic stirring, acetoacetic acid ethyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 15 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 10 ml of crude enzyme solution of mutant LbADH of Example III at time zero;

[0063] Reaction 3: 200 mL beaker, magnetic stirring, acetoacetic acid methyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 15 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 10 ml of crude enzyme solution of wild type LbADH of Example III at time zero;

[0064] Reaction 4: 200 mL beaker, magnetic stirring, acetoacetic acid methyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 15 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 10 ml of crude enzyme solution of mutant LbADH of Example III at time zero;

[0065] Reaction 5: 200 mL beaker, magnetic stirring, acetoacetic acid ethyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 23 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 2.0 g of immobilized whole cells of wild type LbADH of Example IV at time zero;

[0066] Reaction 6: 200 mL beaker, magnetic stirring, acetoacetic acid ethyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 23 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 2.0 g of immobilized whole cells of mutant LbADH of Example IV at time zero;

[0067] Reaction 7: 200 mL beaker, magnetic stirring, acetoacetic acid ethyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 10 ml, purified water 13 ml, NAD + 10 mg, reaction temperature 25 °C, start reaction by adding 2.0 g of immobilized whole cells of mutant LbADH of Example IV at time zero;

[0068] Reaction 8: 200 mL beaker, magnetic stirring, acetoacetic acid ethyl ester 35 ml, isopropyl alcohol 40 ml, pH 7.5 0.1 M PBS 5 ml, purified water 18 ml, NAD +10mg, reaction temperature is 25°C, add 2.0g of the mutant LbADH immobilized whole cell of Example 4 to start the reaction;

[0069] Reaction 9: in a 200ml beaker, magnetic stirring, add ethyl acetoacetate 35ml, isopropyl alcohol 40ml, purified water 23ml, NAD + 10mg, reaction temperature is 25°C, add 2.0g of the mutant LbADH immobilized whole cell of Example 4 to start the reaction;

[0070] Reaction 10: in a 200ml beaker, magnetic stirring, add ethyl acetoacetate 35ml, isopropyl alcohol 40ml, pH7.5 0.1M PBS 5ml, purified water 18ml, NAD + 5mg, reaction temperature is 25°C, add 2.0g of the mutant LbADH immobilized whole cell of Example 4 to start the reaction;

[0071] Reaction 11: in a 200ml beaker, magnetic stirring, add ethyl acetoacetate 35ml, isopropyl alcohol 40ml, pH7.5 0.1M PBS 5ml, purified water 18ml, NAD + 2mg, reaction temperature is 25°C, add 2.0g of the mutant LbADH immobilized whole cell of Example 4 to start the reaction;

[0072] Reaction 12: in a 200ml beaker, magnetic stirring, add ethyl acetoacetate 35ml, isopropyl alcohol 40ml, pH7.5 0.1M PBS 5ml, purified water 18ml, reaction temperature is 25°C, add 2.0g of the mutant LbADH immobilized whole cell of Example 4 to start the reaction.

[0073] Sample at different time points of reactions 1-12, calculate the conversion rate, the results are shown in Table 1;

[0074] Table 1 reaction results of different systems

[0075]

[0076] From the data in Table 1, under the same conditions, with ethyl acetoacetate as the substrate, the conversion rate of wild-type LbADH is lower than that of mutant LbADH, and the chirality of the product of the mutant enzyme is obviously higher than that of the wild-type enzyme, which may be due to the change of the catalytic pocket of the mutant enzyme compared with the wild-type, or the relative proportion of other enzymes acting on ethyl acetoacetate is reduced due to the increase of specific activity of the mutant enzyme; the reaction results can prove that the mutant enzyme is obviously superior to the wild-type enzyme in terms of acetoacetate conversion rate and product ee value.

[0077] Using methyl acetoacetate as a substrate, the ee values ​​of the products are all no greater than 98%, which has little application value.

[0078] When immobilized whole cells are used as the enzyme source, the effect of phosphate buffer concentration as low as 0 mM on catalysis is not particularly significant. However, considering the possible influence of residual phosphate in the immobilized cells, 5 mM phosphate buffer should be used when immobilized cells are reused.

[0079] No coenzyme NAD + At that time, the product conversion rate decreased significantly; coenzyme NAD + When the dosage is as low as 2 mg, it has no significant effect on substrate conversion.

[0080] Example 6: Reusable synthesis of ethyl R-3-hydroxybutyrate from whole cells immobilized with mutant LbADH

[0081] Reaction system: In a 1000mL beaker, with magnetic stirring, 180mL ethyl acetoacetate, 200mL isopropanol, 25mL 0.1M PBS (pH 7.5), 95mL purified water, and NAD+. + 10 mg of the mutant LbADH immobilized whole cells from Example 4 were added at room temperature (25°C), and the reaction was started. After 24 hours of reaction, the mixture was filtered through a double-layered filter paper using a Buchner funnel until no liquid dripped. The filter cake, which is the immobilized whole cell, was then added to the reaction solution for the next reaction. The results of the number of times the mutant LbADH immobilized whole cells were used and the conversion rate are shown in Table 2.

[0082] Table 2. Transformation results of immobilized whole-cell mutant LbADH for reuse.

[0083]

[0084] As shown in Table 2, the substrate conversion rate of the mutant LbADH-immobilized whole cells remained unchanged after five repeated uses over 24 hours.

[0085] Example 7: Synthesis of ethyl R-3-hydroxybutyrate in a 3L whole-cell system immobilized with mutant LbADH

[0086] Reaction system 1: In a 5L three-necked flask, with mechanical stirring, 1080ml ethyl acetoacetate, 1200ml isopropanol, 150ml 0.1M PBS (pH 7.5), 570ml purified water, and NAD+. +60mg, water bath temperature set to 30°C, add mutant LbADH immobilized whole cells of Example Four 60.0g, start reaction by timing; 6 hours into the reaction, substrate conversion rate 58.4%, 24 hours, substrate conversion rate 89.3%, significantly lower than the conversion rate in Example Six, 38 hours conversion rate 91.3%, it is speculated that the acetone produced by the dehydrogenation of isopropyl alcohol cannot be volatilized in a large system, resulting in a higher concentration of acetone in the reaction system, which produces inhibition; install a vacuum water pump to extract vacuum and a reflux device on the reactor, 2 hours after vacuum extraction, conversion rate 95.4%, 4 hours after vacuum extraction, conversion rate 97.4%, 8 hours after vacuum extraction, conversion rate 98.9%;

[0087] Reaction system 2: 5L three-necked bottle, mechanical stirring, ethyl acetoacetate 1080ml, isopropyl alcohol 1200ml, pH7.5 0.1M PBS 150ml, purified water 570ml, NAD + 60mg, water bath temperature set to 30°C, install a vacuum water pump to extract vacuum and a reflux device, add mutant LbADH immobilized whole cells of Example Four 60.0g, start reaction by timing; 6 hours into the reaction, conversion rate 75.8%, 12 hours into the reaction, conversion rate 94.0%, 16 hours into the reaction, conversion rate 96.5%; the conversion rate increases slowly from 12h to 16h, it is speculated that the vacuum extraction force is too large and the reflux water temperature is too high, causing a large amount of isopropyl alcohol to be extracted, after adding 50ml of isopropyl alcohol, continue to extract vacuum for 4 hours, conversion rate 98.8%;

[0088] Reaction system 3: 5L three-necked bottle, mechanical stirring, ethyl acetoacetate 1080ml, isopropyl alcohol 1200ml, pH7.5 0.1M PBS 150ml, purified water 570ml, NAD + 60mg, water bath temperature set to 30°C, install a bottom aeration (1.5L / min) and reflux device (control cooling water temperature 5-10°C), add mutant LbADH immobilized whole cells of Example Four 60.0g, start reaction by timing; 6 hours into the reaction, conversion rate 77.4%, 12 hours into the reaction, conversion rate 94.0%, 16 hours into the reaction, conversion rate 98.3%;

[0089] From the above experimental results, it can be seen that when the reaction system is scaled up, the acetone produced by the reaction cannot be removed in time, which will seriously inhibit the progress of the reaction, which can be solved by vacuum extraction (isopropyl alcohol needs to be added in batches during the reaction) or bottom aeration combined with a reflux device, but the cooling water temperature in the reflux device needs to be strictly controlled at 5-10°C to achieve the effect of removing acetone without removing isopropyl alcohol and substrate.

[0090] Example Eight: Mutant LbADH and glucose dehydrogenase cooperate to convert and synthesize R-3-hydroxybutyrate ethyl ester

[0091] In this embodiment, the glucose dehydrogenase used is derived from Bacillus megaterium IWG3, reference Crystal Structure of Glucose Dehydrogenase from Bacillus megaterium IWG3, constructed and prepared by the company, which will not be described in detail in the present application, and glucose dehydrogenases from other sources can also be used;

[0092] Reaction system: 5L three-necked flask, mechanical stirring, ethyl acetoacetate 450ml, purified water 1800ml, anhydrous glucose 720g, NAD + 60mg, the water bath temperature was set to 30℃, 300ml of the crude enzyme solution of the mutant LbADH of Example Three was added, 300ml of the crude enzyme solution of glucose dehydrogenase was added, and the reaction was started; the reaction was carried out for 2 hours, the conversion rate was 69.8%, the reaction was carried out for 4 hours, the conversion rate was 89.6%, the reaction was carried out for 6 hours, and the conversion rate was 99.3%.

[0093] It can be seen from the above embodiments of the present application that the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant has a conversion rate of >98% under the condition of a substrate concentration of 350g / L for 24 hours; 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 Yikelaye, but the ee value of the product generated by the enzyme provided in the present application is greater than 99.95%, which is significantly better than the enzymes provided by the above two applications, and the enzyme of the present application can be used for coenzyme NADH regeneration in cooperation with glucose dehydrogenase under the condition of equivalent substrate concentration and conversion rate, co-production of D-gluconic acid-1,5-lactone without reducing the yield and ee value of R-3-hydroxybutyric acid ethyl ester, further reducing the cost.

[0094] Although the present application has been described in detail by referring to preferred embodiments, the present application is not limited to this. Various equivalent modifications and replacements can be made to the embodiments of the present application by those skilled in the art without departing from the spirit and essence of the present application, and these modifications and replacements should be included in the scope of the present application. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The sequence shown in SEQ ID NO.1 was subjected to multiple mutations at S143T, E145L, K160R, and Y190F sites. The amino acid sequence of the resulting 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.

2. A mutant alcohol dehydrogenase expression strain, characterized in that, The preparation process is as follows: the nucleic acid sequence of the S143T / E145L / K160R / Y190F alcohol dehydrogenase mutant as described in claim 1 is ligated into the pET vector through the NdeI / XhoI restriction site to obtain the recombinant vector; the recombinant vector is transformed into the expression system by heat shock to obtain the mutant LbADH expression strain; The expression system includes an Escherichia coli expression system.

3. The application of an alcohol dehydrogenase mutant in the production of ethyl R-3-hydroxybutyrate, characterized in that, Using wet cells, crude enzyme solution, or immobilized whole cells prepared by high-density fermentation of the mutant LbADH expression strain described in claim 2 as the enzyme catalytic system, and ethyl acetoacetate as the substrate, the conversion rate is >98% after 24 hours of reaction.

4. The application as described in claim 3, characterized in that, Coenzyme NAD needs to be added + Alternatively, NADH; using isopropanol or glucose as a coenzyme regeneration substrate.

5. The application as described in claim 4, characterized in that, Using isopropanol as a coenzyme regeneration substrate, no additional enzymes are needed, and the generated acetone is removed by vacuuming or aeration.

6. The application as described in claim 4, characterized in that, Glucose is used as a coenzyme regeneration substrate, with the addition of glucose dehydrogenase.

7. The application as described in claim 4, characterized in that, The preparation method of mutant alcohol dehydrogenase immobilized whole cells is as follows: Take 100g of wet cells frozen after high-density fermentation, dissolve them in 900mL of buffer solution with pH 8.0-9.0, stir at room temperature until completely dissolved, add 6g of diatomaceous earth, add 30g of 5% polyethyleneimine, adjust the pH to 8.0-9.0 with acid, stir for 1 hour, then add 5g of 50% glutaraldehyde, control the pH to >7.0, continue stirring for 1 hour, filter, and then wash 2-3 times with purified water. After filtration, the immobilized whole cells are obtained.

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