Beta-amino acid dehydrogenase mutants and their use in the synthesis of aromatic beta-amino acid compounds

Abstract

This invention provides a β-amino acid dehydrogenase mutant and its application in the synthesis of aromatic β-amino acid compounds. Specifically, the β-amino acid dehydrogenase is derived from *Marininema halotolerans*. L β-Erythro-3,5-diaminohexanoate dehydrogenase. The mutant protein of this β-amino acid dehydrogenase is a non-natural protein, and the mutant protein exhibits significantly enhanced activity in catalyzing the synthesis of β-amino acids from aromatic β-keto acids. Furthermore, the mutant protein contains mutations in two or more core amino acids related to the enzyme's catalytic activity in the wild-type β-amino acid dehydrogenase. The β-amino acid dehydrogenase mutant provided by this invention has a broadened substrate spectrum and can significantly improve the activity of β-amino acid dehydrogenase in catalyzing the synthesis of β-amino acids.

Description

Technical Field

[0001] This invention relates to the field of enzymes and enzyme engineering, and more specifically, to a mutant of β-amino acid dehydrogenase with enhanced catalytic activity and its application in the catalytic synthesis of chiral aromatic β-amino acid compounds. Background Technology

[0002] Chiral aromatic β-amino acids are important building blocks for the synthesis of many complex drug molecules and bioactive substances, and have significant applications in medicine, chemical engineering, and other fields. For example, (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid can be used to synthesize sitagliptin, an important drug for treating type II diabetes; (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-(4-hydroxyphenyl) can be used to synthesize some anti-inflammatory and antifungal drugs. Therefore, developing efficient and green methods for the preparation of chiral aromatic β-amino acids is of great research significance. Compared with chemical synthesis methods, enzymatic catalysis generally has the advantages of high selectivity, mild reaction conditions, and environmental friendliness.

[0003] Amino acid dehydrogenases (EC1.4.1.X) are a class of oxidoreductases dependent on the cofactors NAD(H) or NADP(H), catalyzing reversible oxidative deamination of amino acids and reductive amination of keto acids. They possess significant advantages and potential applications in the synthesis of amino acids and chiral amine compounds. β-amino acid dehydrogenases can asymmetrically reductively amination β-keto acids to synthesize the corresponding β-amino acids. However, the only β-amino acid dehydrogenase reported in the literature is L-erythro-3,5-diaminohexanoate dehydrogenase (3,5-DAHDH, EC.1.4.1.11) in the lysine degradation pathway. Furthermore, this type of enzyme exhibits significant catalytic activity only towards its natural substrate, L-erythro-3,5-diaminohexanoate, demonstrating strong substrate specificity and a very limited range of substrates it can convert. Previous research, through molecular modification and extensive screening of 3,5-DAHDH derived from Candidatus Cloacamonas acidaminovorans, obtained mutants with significantly enhanced activity against non-natural substrates. For the first time, a two-step method was used to convert β-ketonitriles or β-keto ester substrates by coupling β-amino acid dehydrogenase with nitrile hydrolase or lipase, thus realizing the synthesis of chiral β-amino acids (Patent Application No.: 202110133031.4).

[0004]

[0005] However, the existing β-amino acid dehydrogenase mutants still have very low catalytic efficiency for aromatic β-amino acids, which cannot meet the requirements of industrial production. Therefore, modifying β-amino acid dehydrogenases through directed evolution to further improve their catalytic activity is of great significance for the research and industrial application of chiral aromatic β-amino acids. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a β-amino acid dehydrogenase mutant modified through genetic engineering. Specifically, the modified β-amino acid dehydrogenase mutant exhibits significantly enhanced activity in synthesizing chiral aromatic β-amino acids.

[0007] The first aspect of the present invention provides a β-amino acid dehydrogenase mutant, wherein the β-amino acid dehydrogenase mutant protein has at least 90% identity with the amino acid sequence shown in SEQ ID NO.:2, and the mutant protein has the ability to synthesize chiral β-amino acids and has significantly improved catalytic activity.

[0008] The β-amino acid dehydrogenase mutant provided by this invention is obtained by first: mutating glutamic acid (E) at position 307 to serine (S) and glycine (G) at position 320 to serine (S) at positions 1 to 353 of amino acids corresponding to SEQ ID NO.:1, and the amino acid sequence is SEQ ID NO.:2.

[0009] The mutants subsequently obtained were β-amino acid dehydrogenase mutants obtained by mutating one or more sites, including sites 119, 155, 175, 176, 321, and 325, of amino acids 1 to 353 in the amino acid sequence SEQ ID NO.:2 corresponding to the β-amino acid dehydrogenase mutant.

[0010] In another preferred embodiment, the catalytic substrate of the β-amino acid dehydrogenase includes β-amino acid or β-keto acid compounds.

[0011] In another preferred embodiment, the β-amino acids and β-keto acids are selected from the following group:

[0012] In another preferred embodiment, the β-amino acid dehydrogenase mutant has significantly enhanced activity in catalyzing the reaction of β-keto acid compounds or in the kinetic resolution of racemic β-amino acid compounds to generate chiral β-amino acid compounds.

[0013] A second aspect of this invention provides the application of a β-amino acid dehydrogenase mutant or a genetically engineered bacterium encoding it in the preparation of aromatic chiral β-amino acid compounds, comprising the steps of:

[0014] (i) The β-amino acid dehydrogenase mutant described in the first aspect of the present invention is contacted with a reaction substrate to carry out a catalytic reaction, thereby obtaining the aromatic β-amino acid compound;

[0015] (ii) Optionally, the aromatic β-amino acid compounds are isolated and purified.

[0016] Specifically, the catalytic reaction uses wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing β-amino acid dehydrogenase mutants as catalysts, β-keto acid compounds obtained by hydrolyzing β-keto esters / nitrile compounds with lipohydrolases as substrates, or β-amino acid racemic compounds as substrates, and a buffer solution with a pH of 6.0-11.0 as the reaction medium, and is carried out at 25℃-50℃.

[0017] In another preferred embodiment, the reaction has one or more characteristics selected from the group consisting of:

[0018] (i) The reaction system contains 10-100 g / L of bacterial cells, more preferably 30-60 g / L;

[0019] (ii) The pH of the reaction system is 6.0-11.0, preferably 7-10, and more preferably 8.5;

[0020] (iii) The reaction system temperature is 25℃-50℃, preferably 25℃-35℃, and more preferably 30℃.

[0021] The co-solvent for the reaction system may be unco-solvent, or may be acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, 1,4-dioxane, preferably unco-solvent, methanol, ethanol, dimethyl sulfoxide, acetone, and more preferably dimethyl sulfoxide.

[0022] In another preferred embodiment, the β-amino acid dehydrogenase mutant has one or more features selected from the group consisting of:

[0023] (a) Compared with wild-type β-amino acid dehydrogenase, the substrate concentration catalyzed is 1-200 g / L, more preferably 10-100 g / L;

[0024] (b) The conversion rate of β-amino acid compounds obtained by catalysis is ≥50%, preferably ≥90%, and more preferably ≥95%, compared with wild-type β-amino acid dehydrogenases;

[0025] (c) The ee value of the β-amino acid compounds obtained by catalysis is ≥20%, preferably ≥90%, and more preferably ≥99%, compared with wild-type β-amino acid dehydrogenases; Attached Figure Description

[0026] Figure 1 Electrophoretic analysis results of purified β-amino acid dehydrogenase (3,5-DAHDH) mutant protein;

[0027] Where M stands for Marker, and 1-8 are some mutant proteins. Detailed Implementation

[0028] the term

[0029] As used in this article, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "V159A" indicates that valine V at position 159 is mutated to alanine A, "V159X" indicates that valine V at position 159 is mutated to any amino acid, i.e., the saturated mutant library at position 159, and so on.

[0030] In a preferred embodiment of the present invention, the method for preparing the β-amino acid dehydrogenase mutant of the present invention is as follows: Escherichia coli is used as the expression host.

[0031] Specifically, the preparation method includes the following steps: (1) The gene of the corresponding mutation site of β-amino acid dehydrogenase is constructed into the pET-21b(+) expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) The recombinant plasmid is transformed into host bacterial cells (preferably Escherichia coli BL21(DE3)) to obtain the corresponding engineered strain. (3) The engineered strain is inoculated into LB medium, cultured at 37°C for 6 hours, and 0.5 mM isopropyl thiogalactoside (IPTG) is added and cultured at 25°C for 6-12 hours. (4) The bacterial cells are collected by centrifugation.

[0032] Example 1: Preparation of recombinant expression plasmid and recombinant expression transformant of β-amino acid dehydrogenase mutant E307S / G320S

[0033] The nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO.:1 was fully synthesized and cloned into the restriction endonuclease sites NdeI and HindIII of the pET-21b(+) vector to obtain the recombinant plasmid pET-21b(+)-3,5-DAHDH. This plasmid was further transformed into the expression host E. coli BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET-21b(+)-3,5-DAHDH.

[0034] Construction of the β-amino acid dehydrogenase mutant E310S: Using pET-21b(+)-3,5-DAHDH as a template, primers E307S-F:CC were designed. AGC GGCCTGGGTAAAGATG, E307S-R:GGCC GCTThe mutant E307S was constructed using the whole plasmid PCR method (GGCGCCCAGTGCTG), and PCR was performed using the high-fidelity polymerase KOD-plus. The PCR reaction conditions were as follows: In a 50 μL PCR reaction system, 5 μL of 10×KOD buffer, 5 μL of dNTP (2 mM), 2 μL of MgSO4 (25 mM), 20–100 ng of template, 1 μL of each pair of mutant primers (10 μM), 1 μL of KOD polymerase, and sterile distilled water were added to a final volume of 50 μL. The PCR reaction program was as follows: (1) denaturation at 94℃ for 3 min, (2) denaturation at 94℃ for 30 sec, (3) annealing at 55℃ for 30 sec, (4) extension at 68℃ for 7 min. Steps (2) to (4) were performed for a total of 20–30 cycles. The PCR products were stored at 4℃. After the PCR products were verified by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37℃ for 2 h. The digestion products were transformed into E. coli BL21(DE3) competent cells and plated on plates containing ampicillin. The plates were then incubated at 37°C for approximately 12 hours. Positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET-21b(+)-3,5-DAHDH-E307S.

[0035] Construction of the β-amino acid dehydrogenase mutant E307S / G320S: Using pET-21b(+)-3,5-DAHDH-E307S plasmid as a template, the combined mutant E307S / G320S was constructed by the whole plasmid PCR method described above, and finally the recombinant expression transformant E.coli BL21(DE3) / pET-21b(+)-3,5-DAHDH-E307S / G320S was obtained.

[0036] Example 2: Construction and screening of a saturated mutant library of β-amino acid dehydrogenases

[0037] A saturated mutant library of β-amino acid dehydrogenases was constructed: L119X / E307S / G320S, P155X / E307S / G320S, C175X / E307S / G320S, G176X / E307S / G320S, E307S / G320S / F321X, and E307S / G320S / H325X. Using pET-21b(+)-3,5-DAHDH-E307S / G320S as a template, site-directed saturation mutagenesis was performed at sites 119, 155, 175, 176, 321, and 325. The construction methods for the recombinant expression plasmids and recombinant expression transformants are as described in Example 1. Mutants whose mutations were confirmed by sequencing were picked and cultured in 24-well plates, and the activity of the expressed protein was detected. The catalytic activity of β-amino acid dehydrogenase in the oxidative deamination direction was calculated by detecting the change in NADPH absorbance at 340 nm. The substrates tested were (R)-β-phenylalanine (A), (S)-β-homophenylalanine (B), and (S)-β-homotyrosine (C). The results are shown in Table 1. The mutation sites that improved the catalytic activity of β-amino acid dehydrogenase were 119Q, 155L / I / W, 175W / F / Y / H / S, 176F / Y, 321S / T, and 325I / F / R / N / Q / M / T / S / C / A / P / K. The mutants with the best activity were 1, 2, 3, and 4, and their protein sequences are shown in SEQ ID NO.: 3-6.

[0038] Table 1. Results of mutation screening using E307S / G320S as a template.

[0039]

[0040]

[0041] Example 3: Construction of a β-amino acid dehydrogenase combinatorial mutant library

[0042] Construction of combinatorial mutants of β-amino acid dehydrogenases: Based on the results of previous saturation mutagenesis and screening, the mutants with the best activity enhancement for different β-amino acid substrates in Example 2 were selected as templates to construct combinatorial mutant libraries.

[0043] First, mutations were performed using E307S / G320S / H325I as a template. The construction methods for the recombinant expression plasmid and recombinant expression transformant were as described in Example 1, and the activity assay methods were as described in Example 2. The activities of the three β-amino acid substrates in Example 2 were measured, and the results are shown in Table 2. The mutation sites that increased the catalytic activity of β-amino acid dehydrogenases were 119Q, 155L / I / W, 175W / Y / H / S, G176Y, and F321T. The mutants with the highest activity, 5 and 6, were selected, and their protein sequences are shown in SEQ ID NO.: 7-8.

[0044] Table 2 shows the mutation results using E307S / G320S / H325I as a template.

[0045]

[0046] Using E307S / G320S / H325R as a template, mutations were performed. The construction methods for the recombinant expression plasmid and recombinant expression transformant were as described in Example 1, and the activity assay methods were as described in Example 2. The activities of the three β-amino acid substrates in Example 2 were measured, and the results are shown in Table 3. The mutation sites that increased the catalytic activity of β-amino acid dehydrogenases were 155L / I / W, 179H / S, and F321T. The mutant with the highest activity, denoted as 7, was selected, and its protein sequence is shown in SEQ ID NO.:9.

[0047] Table 3 shows the mutation results using E307S / G320S / H325R as a template.

[0048]

[0049] Mutations were performed using E307S / G320S / H325F as a template. The construction methods for the recombinant expression plasmid and recombinant expression transformant were as described in Example 1, and the activity assay methods were as described in Example 2. The activities of the three β-amino acid substrates in Example 2 were measured, and the results are shown in Table 4. The mutants with enhanced β-amino acid dehydrogenase catalytic activity were obtained as 159L / I, 179S, and F321T, which are mutants 8, and their protein sequences are shown in SEQ ID NO.:10.

[0050] Table 4 shows the mutation results using E307S / G320S / H325F as a template.

[0051]

[0052] Example 4: Induction, expression, and purification of β-amino acid dehydrogenase mutants

[0053] Single colonies of the genetically engineered bacteria of mutants 1-8 were inoculated into 4 mL of LB broth containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured overnight at 37°C and 200 rpm in a shaker to obtain the seed culture. The overnight seed culture was then transferred at a 1% inoculation rate to 50 mL of LB broth containing ampicillin and cultured at 37°C and 200 rpm until OD (dose elapsed). 600The pH was approximately 0.6-1.0. 0.5 mM IPTG was added, and the mixture was incubated at 25°C and 200 rpm for 8-12 hours. The bacterial cells were collected by centrifugation at 4°C and 6000 rpm. The cells were resuspended in sodium phosphate buffer (50 mM, pH 8.0, containing 5% glycerol, 200 mM NaCl, and 30 mM imidazole), homogenized using a high-pressure homogenizer, and centrifuged at 4°C and 12000 rpm. The supernatant was then collected and purified using metal affinity chromatography (nickel column). The imidazole concentration in the sodium phosphate buffer used to elute the target protein was 120 mM. After ultrafiltration to remove salt and concentration, the target protein yielded a pure enzyme solution of the β-amino acid dehydrogenase mutant. SDS-PAGE electrophoresis showed that the purified protein had a single band, as shown in the image. Figure 1 As shown, M represents the marker, 1 is the purified protein of mutant C175H / E307S / G320S, 2 is the purified protein of mutant E307S / G320S / H325F, 3 is the purified protein of mutant E307S / G320S / H325I, 4 is the purified protein of mutant E307S / G320S / H325R, 5 is the purified protein of mutant C175H / E307S / G320S / H325I, 6 is the purified protein of mutant C175S / E307S / G323S / H325I, 7 is the purified protein of mutant C175H / E307S / G323S / H325R, and 8 is the purified protein of mutant C175S / E307S / G320S / H325F. The results show that the method of this embodiment can obtain relatively pure mutant proteins with a single subunit protein molecular weight of 39kD and a purity of >95%.

[0054] Example 5: Method for catalyzing β-keto acid compounds by recombinant bacteria with β-amino acid dehydrogenase mutants

[0055] Ethyl 3-oxo-4-phenyl-butyrate substrate (2M, dissolved in DMSO) was added to sodium carbonate buffer (pH 8.5, 100mM) to a final concentration of 0.5M. 5 mg / mL of lipohydrolase Novozyme 435 was added, and the mixture was hydrolyzed at 30°C for 3 hours to obtain the β-keto acid substrate. During the hydrolysis reaction, the pH was adjusted to approximately 8.5 using solid sodium carbonate.

[0056] (1) The β-amino acid dehydrogenase mutant 4 of the present invention (protein sequence as shown in SEQ ID NO.: 6) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 24 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. +Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration approximately 20 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 96%, the separation yield was 86%, and the ee value was ≥99% (S).

[0057] (2) The β-amino acid dehydrogenase mutant 5 of the present invention (protein sequence as shown in SEQ ID NO.:7) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 24 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. + Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration approximately 20 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 95%, the separation yield was 89%, and the ee value was ≥99% (S).

[0058] (3) The β-amino acid dehydrogenase mutant 6 of the present invention (protein sequence as shown in SEQ ID NO.:8) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 24 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. + Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration approximately 20 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 95%, the separation yield was 85%, and the ee value was ≥99% (S).

[0059] Example 6: Method for synthesizing chiral β-amino acid compounds catalyzed by recombinant bacteria with β-amino acid dehydrogenase mutants

[0060] Ethyl 3-oxo-4-(2,4,5-trifluorophenyl)-butyrate (2M, dissolved in DMSO) was added to sodium carbonate buffer (pH 8.5, 100mM) to a final concentration of 0.5M. Then, 5 mg / mL of lipohydrolase Novozyme 435 was added, and the mixture was hydrolyzed at 30°C for 3 hours to obtain the β-keto acid substrate. During the hydrolysis reaction, the pH was adjusted to approximately 8.5 using solid sodium carbonate.

[0061] (1) The β-amino acid dehydrogenase mutant 4 of the present invention (protein sequence as shown in SEQ ID NO.: 6) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 24 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. + Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration 25 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 97%, the separation yield was 85%, and the ee value was ≥99% (S).

[0062] (2) The β-amino acid dehydrogenase mutant 5 of the present invention (protein sequence as shown in SEQ ID NO.:7) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 24 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. + Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration 25 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 95%, the separation yield was 81%, and the ee value was ≥99% (S).

[0063] (3) The β-amino acid dehydrogenase mutant 7 of the present invention (protein sequence as shown in SEQ ID NO.: 9) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 1.8 g of bacterial cells were resuspended in 27 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 100 mM, containing 200 mM NH4Cl), and 0.5 g / L NADP was added. +Three equivalents of glucose and 60 mg of glucose dehydrogenase lyophilized enzyme powder were added to 6 mL of the above-mentioned 0.5 M β-keto acid substrate (final concentration 25 g / L). The mixture was reacted on a shaker at 30 °C and 200 rpm for 4 h, after which the reaction was stopped. After the reaction was completed, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the yield was 95%, the separation yield was 83%, and the ee value was ≥99% (S).

[0064] Example 7: Method for catalyzing β-amino acid racemic compounds by recombinant bacteria with β-amino acid dehydrogenase mutants

[0065] (1) The β-amino acid dehydrogenase mutant 4 of the present invention (protein sequence as shown in SEQ ID NO.: 6) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and substrate 3-amino-3-phenylpropionic acid was added to a final concentration of 17 g / L. 0.5 g / L NADP was added. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 6 h, after which the reaction was stopped. After the reaction, HPLC was used for detection, and the cells were separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 40%, and the ee value was ≥99% (S).

[0066] (2) The β-amino acid dehydrogenase mutant 5 of the present invention (protein sequence as shown in SEQ ID NO.:7) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and substrate 3-amino-3-(4-fluorophenyl)propionic acid was added to a final concentration of 18 g / L, along with 0.5 g / L NADP. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 4 h before stopping. After the reaction, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 42%, and the ee value was ≥99% (S).

[0067] (3) The β-amino acid dehydrogenase mutant 7 of the present invention (protein sequence as shown in SEQ ID NO.: 9) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and the substrate 3-amino-4-(2,4,5-trifluorophenyl)butyric acid was added to a final concentration of 23 g / L, along with 0.5 g / L NADP. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 4 h, after which the reaction was stopped. After the reaction, HPLC was used for detection, and the cells were separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 43%, and the ee value was ≥99%(R).

[0068] (4) The β-amino acid dehydrogenase mutant 5 of the present invention (protein sequence as shown in SEQ ID NO.:7) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and substrate 3-amino-4-phenylbutyric acid was added to a final concentration of 18 g / L, along with 0.5 g / L NADP. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 4 h, after which the reaction was stopped. After the reaction, HPLC was used for detection, and the cells were separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 40%, and the ee value was ≥99%(R).

[0069] (5) The β-amino acid dehydrogenase mutant 6 of the present invention (protein sequence as shown in SEQ ID NO.:8) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and substrate 3-amino-3-(2-methylphenyl)propionic acid was added to a final concentration of 18 g / L, along with 0.5 g / L NADP. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 4 h, after which the reaction was stopped. After the reaction, HPLC was used for detection, and the mixture was separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 41%, and the ee value was ≥99% (S).

[0070] (6) The β-amino acid dehydrogenase mutant 7 of the present invention (protein sequence as shown in SEQ ID NO.: 9) was induced to express according to the method of Example 4. The bacterial cells were collected by centrifugation and used as a biocatalyst. 2.5 g of bacterial cells were resuspended in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 9.5, 100 mM), and substrate 3-amino-3-(3,4-dimethoxyphenyl)propionic acid was added to a final concentration of 30 g / L, along with 0.5 g / L NADP. + 0.18 g / L riboflavin and 0.5 g of riboflavin reductase (derived from E. coli) recombinant expression cells were added, and the reaction was carried out at 37 °C and 200 rpm on a shaker for 4 h, after which the reaction was stopped. After the reaction, HPLC was used for detection, and the cells were separated and purified using a strongly acidic cation exchange resin. The results showed that the conversion rate was approximately 50%, the separation yield was 45%, and the ee value was ≥99% (S). sequence list <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> β-Amino Acid Dehydrogenase Mutants and Their Application in the Catalytic Synthesis of Chiral Aromatic β-Amino Acid Compounds <130> amino acid sequences <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 1 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Glu Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Gly 305 310 315 320 Phe Ala Pro Gly His Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Ser <210> 2 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 2 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly His Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Ser <210> 3 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 3 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val His Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly His Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum <210> 4 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 4 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Phe Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Ser <210> 5 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 5 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Ile Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum <210> 6 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 6 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum <210> 7 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 7 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val His Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Ile Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum <210> 8 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 8 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Ser Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Ile Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum <210> 9 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 9 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val His Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Ser <210> 10 <211> 353 <212> PRT <213> Candidatus Cloacamonas acidaminovorans <400> 10 Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val 1 5 10 15 Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala 20 25 30 Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu 35 40 45 Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln 50 55 60 Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg 65 70 75 80 Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly 85 90 95 Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val 100 105 110 Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser 115 120 125 Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val 130 135 140 Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro 145 150 155 160 Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Ser Gly 165 170 175 Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val 180 185 190 Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala 195 200 205 Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser 210 215 220 Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val 225 230 235 240 Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu 245 250 255 Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn 260 265 270 Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly 275 280 285 Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu 290 295 300 Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser 305 310 315 320 Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe 325 330 335 Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr 340 345 350 Serum

Claims

1. A mutant β-amino acid dehydrogenase protein, characterized in that, The amino acid sequence of the mutant protein is shown in any one of SEQ ID NO:4, 5 or 7.

2. A method for preparing aromatic β-amino acid compounds, characterized in that, Including the following steps: The β-amino acid dehydrogenase mutant protein of claim 1 is contacted with a reaction substrate to carry out a catalytic reaction, thereby obtaining the β-amino acid, wherein the β-amino acid compound is an aromatic chiral compound.

3. The method for preparing aromatic β-amino acid compounds as described in claim 2, characterized in that, It also includes the step of separating and purifying the β-amino acid.

4. The method for preparing aromatic β-amino acid compounds as described in claim 2, characterized in that, Using keto acids obtained from hydrolysis by lipase or nitrile hydrolase as substrates, chiral β-amino acids are catalyzed to obtain chiral β-amino acids in the presence of a coenzyme regeneration system.

5. The method for preparing aromatic β-amino acid compounds as described in claim 2, characterized in that, Using racemic β-amino acids as substrates, chiral resolution of β-amino acids is catalyzed in the presence of a coenzyme regeneration system.

Images