D-threonine aldolase mutant and application thereof in preparation of D-configuration beta-hydroxyl-alpha, alpha-dialkyl-alpha-amino acid

Abstract

The invention discloses a D-threonine aldolase mutant and an application of the D-threonine aldolase mutant in preparation of D-configuration beta-hydroxyl-alpha, alpha-dialkyl-alpha-amino acid. The D-threonine aldolase mutant is obtained by mutating wild type D-threonine aldolase of which the accession number is SUY79794.1 in the NCBI (National Center of Biotechnology Information). According to the D-threonine aldolase mutant, pyridoxal phosphate and manganese ions are used as cofactors to catalyze condensation of D-alanine, D-serine, p-methylsulfonyl benzaldehyde, p-nitrobenzaldehyde, 2, 4-dihydroxy benzaldehyde and the like to form D-configuration beta-hydroxyl-alpha, alpha-dialkyl-alpha-amino acid. The D-configuration beta-hydroxyl-alpha, alpha-dialkyl-alpha-amino acid synthesized by the invention has the following advantages: (1) the production process is simple, and the reaction conditions are mild; (2) the D-threonine aldolase is high in selectivity, and the optical purity of the product is high; (3) splitting is not needed, and the atom utilization rate can reach 100%; (4) the downstream separation and purification process is simple and conforms to the green chemistry concept; and (5) the amino substrate spectrum is broadened, the aldehyde group substrate spectrum is wide, and various products can be produced.

Description

D-threonine aldolase mutant and its application in the preparation of D-configuration β-hydroxy-α,α-dialkyl-α-amino acids Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to D-threonine aldolase mutants and their application in the preparation of D-configuration β-hydroxy-α,α-dialkyl-α-amino acids. Background Technology

[0002] D-β-hydroxy-α,α-dialkyl-α-amino acids are important pharmaceutical components, serving as conformational modifiers for enzyme inhibitors and physiologically active peptides. Due to their unique chemical properties and biological activity, D-β-hydroxy-α,α-dialkyl-α-amino acids have broad application and market prospects in the pharmaceutical field. D-β-hydroxy-α,α-dialkyl-α-amino acids have two chiral centers and two isomers: D-syn-β-hydroxy-α,α-dialkyl-α-amino acids and D-anti-β-hydroxy-α,α-dialkyl-α-amino acids.

[0003] D-β-hydroxy-α,α-dialkyl-α-amino acids are not naturally occurring substances. Their synthesis methods mainly include chemical synthesis and biocatalysis. The chemical synthesis of D-β-hydroxy-α,α-dialkyl-α-amino acids is complicated, requires harsh conditions, and is difficult, resulting in low optical purity and low yield of the product. There are currently three main biocatalytic pathways for obtaining D-β-hydroxy-α,α-dialkyl-α-amino acids: (1) Alanine racemic enzyme as a catalyst to catalyze the reaction of 3-nitrobenzaldehyde with D-alanine to obtain α-methyl-3-nitro-β-phenylserine. (2) α-methylserine hydroxymethyltransferase as a catalyst to catalyze the reaction of formaldehyde with D-alanine to obtain α-methylserine. (3) D-threonine aldolase as a catalyst to catalyze the reaction of amino acids and their analogues with various aldehydes to obtain D-β-hydroxy-α,α-dialkyl-α-amino acids. The first two pathways have low yields, typically less than 20%, and the selectivity during synthesis is low, making it difficult to obtain single-configuration mutants. However, current methods for synthesizing D-β-hydroxy-α, α-dialkyl-α-amino acids using D-threonine aldolases still face some challenges. Currently reported D-threonine aldolases have limited amino substrate recognition, almost exclusively recognizing glycine to catalyze the production of D-β-hydroxy-α-amino acids. Only two D-threonine aldolases can catalyze other amino acids, but their enzyme activity and selectivity are low, resulting in low production efficiency. Therefore, further exploration of D-threonine aldolases with broad amino substrate recognition, high selectivity, and high enzyme activity, and optimization of the D-β-hydroxy-α, α-dialkyl-α-amino acid production system, is of great significance. Summary of the Invention

[0004] This invention addresses the shortcomings of existing synthetic processes for D-β-hydroxy-α,α-dialkyl-α-amino acids by providing a D-threonine aldolase mutant and its application in the preparation of D-configuration β-hydroxy-α,α-dialkyl-α-amino acids.

[0005] The specific technical solution is as follows: In a first aspect, the present invention provides a D-threonine aldolase mutant, wherein the mutant has three or fewer substitutions relative to SEQ ID NO.2 and has less than 100% sequence identity with SEQ ID NO.2, wherein the substitutions are selected from: S255A, S255C, S255G, S255I, S255K, S255L, S255M, S255N, S255P, S255Q, S255R, S255D, S255E, S255F, S255H, S255T, S255V, S255W, S255Y, D327A, Y190A, Y190C, Y190D, Y190E, Y190F, Y190G, Y190H, Y190I, Y190K, Y190L, Y190M, Y190N, Y190P, Y190Q, Y190R, Y190S, Y190T, Y190V, Y190W, H358A, H358C, H358D, H358E, H358F, H358G, H358I, H358K, H358L, H358M, H358N, H358P, H358Q, H358R, H358S, H358T, H358V, H358W, and H358Y, or combinations thereof.

[0006] Furthermore, the substitute is selected from: S255A, S255T, D327A, Y190F and H358V, or a combination thereof.

[0007] In a second aspect, the present invention provides a gene encoding the D-threonine aldolase mutant.

[0008] In a third aspect, the present invention provides a recombinant vector comprising a gene encoding a D-threonine aldolase mutant.

[0009] Furthermore, the recombinant vector is a pET28a plasmid containing a gene encoding a D-threonine aldolase mutant.

[0010] In a fourth aspect, the present invention provides a recombinant microorganism comprising the aforementioned gene or recombinant vector.

[0011] In a fifth aspect, the present invention provides a catalyst comprising the aforementioned D-threonine aldolase mutant or recombinant microorganism; the catalyst being an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.

[0012] In a sixth aspect, the present invention provides the use of the D-threonine aldolase mutant, the recombinant microorganism, or the catalyst in the preparation of D-β-hydroxy-α,α-dialkyl-α-amino acids; wherein the D-β-hydroxy-α,α-dialkyl-α-amino acids are D-syn-β-hydroxy-α,α-dialkyl-α-amino acids or D-anti-β-hydroxy-α,α-dialkyl-α-amino acids.

[0013] The present invention provides a method for preparing D-β-hydroxy-α,α-dialkyl-α-amino acids in a seventh aspect, using D-alanine or D-serine as an amino acid substrate, a benzaldehyde derivative as an aldehyde substrate, pyridoxal phosphate and manganese ions as cofactors, and employing the catalyst to carry out a condensation reaction to generate D-β-hydroxy-α,α-dialkyl-α-amino acids; wherein the benzaldehyde derivative is one of p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, and 2,4-dihydroxybenzaldehyde; and the D-β-hydroxy-α,α-dialkyl-α-amino acid is D-syn-β-hydroxy-α,α-dialkyl-α-amino acid or D-anti-β-hydroxy-α,α-dialkyl-α-amino acid.

[0014] Furthermore, the reaction is carried out in an organic solvent-water mixture or an aqueous solution.

[0015] More preferably, the organic solvent is DMSO.

[0016] More preferably, the amount of DMSO added does not exceed 20% of the total volume.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The production process is simple and the reaction conditions are mild; (2) The selectivity of D-threonine aldolase is high. The product has high optical purity; (3) No separation is required and the atom utilization rate can reach 100%; (4) The downstream separation and purification process is simple and conforms to the concept of green chemistry; (5) The aldehyde substrate has a broad spectrum and can produce a variety of products. Attached Figure Description

[0018] Figure 1 shows the reaction equations for the formation of D-β-hydroxy-α,α-dialkyl-α-amino acids by D-threonine aldolase catalyzing the reaction of D-alanine, D-serine, and various aldehydes.

[0019] Figure 2 shows the high-performance liquid chromatography (HPLC) spectra of the substrate and product in the reaction solution after the preparation of products 1 and 2 by D-threonine aldolase.

[0020] Figure 3 shows the high-performance liquid chromatography (HPLC) spectra of the substrate and product in the reaction solution after the preparation of products 7 and 8 by D-threonine aldolase.

[0021] Figure 4 shows the mass spectra of the substrate and product in the reaction solution after the preparation of products 1 and 2 by D-threonine aldolase.

[0022] Figure 5 shows the mass spectra of the substrate and product in the reaction solution after the preparation of products 7 and 8 by D-threonine aldolase. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0026] The reagents used in the catalytic process, including D-serine, D-alanine, p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, 2,4-dihydroxybenzaldehyde, pyridoxal phosphate, and manganese chloride, were all commercially available analytical grade.

[0027] In this invention, the reactions in which D-serine, D-alanine, and p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, and 2,4-dihydroxybenzaldehyde are converted to D-configuration β-hydroxy-α,α-dialkyl-α-amino acids under the catalysis of D-threonine aldolase are shown in the following formula:

[0028]

[0029] This invention uses high-performance liquid chromatography (HPLC) to analyze the concentrations of substrates and products in the reaction solution and monitor the progress of the reaction.

[0030] The HPLC analysis method was as follows: The chromatographic column was CHIRALPAK ZWIX (-) (DAICELCO., LTD, Japan); the mobile phase was 1 L methanol, 1.9 mL formic acid, and 2.6 mL ethylenediamine; the flow rate was 0.5 mL / min; the column temperature was 40℃; and the detection wavelength was 225 nm. The peak characteristics are shown in Figure 3-5: aldehyde 2.9 min, D-syn-β-hydroxy-α, α-dialkyl-α-amino acid approximately 5.2 min, and D-anti-β-hydroxy-α, α-dialkyl-α-amino acid approximately 6.4 min.

[0031] This invention characterizes the selectivity of D-threonine aldolase and its mutants using the de value, as shown in the following formula:

[0032] Example 1: Construction of Wild-Type D-Threonine Aldolase Engineered Bacteria. The D-threonine aldolase gene was retrieved from the NCBI database, and the D-threonine aldolase (XD10) with accession number SUY79794.1 was selected, with its base sequence shown in SEQ ID NO.1. The five amino acid sequences were converted into nucleotide sequences through codon optimization, as shown in SEQ ID NO.2. The XD10 nucleotide sequence was chemically synthesized (Qingke Biotechnology) and integrated between the BamHI and HindIII restriction sites of the expression vector pET-28a(+). Finally, the constructed plasmid was introduced into *E. coli* BL21(DE3) host cells to construct an engineered bacterium with high amino substrate recognition of D-threonine aldolase.

[0033]

[0034] SEQ ID NO.2: MKHIPEHLQTLVGQAVKRIDTPALVIDLDAMDRNIERMARFAQQHGVLWRPHAKLHKSAHIAHLLEHAGACGHCVQKVSEAEALAQGGITNIFIS NEVIAQTKLARVAALAKALNAQGGRLALAVDCEEGITRLARALQDAQAGDAAMDVLVEINVGQNRCGAEPGEAALALAQAIAAQPVLRFAGLQAYHGGAQ HLRGAEERKTAIEQVLKLVNRTRHEFDRAGLAIPLITGAGTGTMVNEAASGIYGEIQPGSFLFMDADYATNQRDAAQPLFEHALYVKTQVMSLSSDYAVCDAGHKAHAIDSGLPRLHALAPENALRFANGGDEHGVLHPDADAGNHSGWLPALGETLWLIPHCDPTVNLHDHLIGVRGGRLKGVVERIFTVDGRGALT.

[0035] Example 2 Construction of D-Threonine Aldolase Mutant 1. Activation and Plasmid Extraction of Engineered Bacteria The engineered bacteria (E. coli BL21(DE3), obtained in Example 1) were activated and cultured using LB medium with the following formula: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The mixture was then sterilized at 121°C for 20 min. The solid culture medium was LB medium with 1-2% agar added.

[0036] The preserved engineered bacterial glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured overnight at 37°C and 220 rpm. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored long-term at -20°C.

[0037] 2. Site-directed gene mutation: Gene mutation was obtained by whole plasmid PCR.

[0038] Table 1 PCR amplification system

[0039] PCR amplification program: (1) Pre-denaturation: 98℃ for 5 min; (2) Denaturation: 98℃ for 30 s; Annealing: 60℃ for 30 s; Extension: 72℃ for 90 s; 30 cycles in total; (3) Post-extension: 72℃ for 10 min; (4) Store at 4℃.

[0040] After PCR amplification, the amplification product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplification product was a single band, approximately 6500 bp in size. The amplification product was purified and recovered using a DNA purification kit. The specific steps were followed according to the kit's instructions. The primers are shown in Table 2.

[0041] Table 2. Mutation primers for different mutants

[0042] 3. Construction of the mutant engineered bacteria: The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into E. coli BL21(DE3) competent cells, plated, and single colonies were picked and cultured in LB broth. Positive transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, sterile glycerol was added to a final concentration of 15%, and the cells were labeled and placed in... Store at 80℃ for later use.

[0043] Example 3. Cultivation of bacterial cells and preparation of crude enzyme solution. 1. Composition of bacterial cell culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, then sterilized at 121°C for 20 min.

[0044] After activating the engineered bacteria from Examples 1 and 2 by streak plating, single colonies were inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking. Then, 2% of the inoculum was transferred to 50 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD (outlet capacity) was reached. 600 When the concentration reaches approximately 0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce culture at 18°C ​​for 18-24 h. After culture, centrifuge the culture at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and wash the cells twice with 100 mM pH 7.5 phosphate buffer. Store at -80°C for later use.

[0045] 2. Preparation of crude enzyme solution: After the culture was completed, the bacterial cells collected were suspended in 1M pH 8.0 phosphate buffer at a 4:1 ratio. The cells were then sonicated at 400W for 30 cycles, with each sonication lasting 3 seconds and a 7-second interval. The lysate was centrifuged at 12000 rpm at 4℃ for 10 minutes to remove the precipitate. The supernatant obtained was the crude enzyme solution containing D-threonine aldolase.

[0046] Example 4: Preparation of Product 1 from D-Threonine Aldolase Mutant in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutants, synthesizing D-syn-β-hydroxy-α,α-dialkyl-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M D-alanine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 8h, with samples taken at intervals to determine the concentrations and de values ​​of the substrate and product. The data at the end of the reaction are shown in Table 3.

[0047] Example 5: Preparation of Product 1 from D-Threonine Aldolase Mutant in Water-DMSO Solution. Following the method in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-syn-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M D-alanine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and product were measured. The data at the end of the reaction are shown in Table 3.

[0048] Table 3. de values ​​and final conversion rates of product 1 prepared by different mutants in different systems.

[0049] Example 6: Preparation of Product 2 from D-Threonine Aldolase Mutant in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-anti-β-hydroxy-α,α-dialkyl-α-amino acid threonine aldolase and its mutants were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M D-alanine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 8h, with samples taken at intervals to detect the concentrations and de values ​​of the substrate and product. The data at the end of the reaction are shown in Table 4.

[0050] Example 7: Preparation of Product 2 from D-Threonine Aldolase Mutant in Water-DMSO. Following the method described in Example 3, engineered bacteria expressing D-anti-β-hydroxy-α-amino acid threonine aldolase and its mutants were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M D-alanine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and product were measured. The data at the end of the reaction are shown in Table 4.

[0051] Table 4. de values ​​and final conversion rates of product 2 prepared by different mutants in different systems.

[0052] Example 8: Preparation of Product 7 from D-Threonine Aldolase Mutant in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutants, synthesizing D-syn-β-hydroxy-α,α-dialkyl-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M D-serine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 8h, with samples taken at intervals to determine the concentrations and de values ​​of the substrate and product. Data at the end of the reaction are shown in Table 5.

[0053] Example 9: Preparation of Product 7 from D-Threonine Aldolase Mutant in Water-DMSO. Following the method described in Example 3, engineered bacteria expressing D-syn-β-hydroxy-α-amino acid threonine aldolase and its mutants were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M D-serine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and product were measured. The data at the end of the reaction are shown in Table 5.

[0054] Table 5. de values ​​and final conversion rates of product 7 prepared by different mutants in different systems.

[0055] Example 10: Preparation of Product 8 from D-Threonine Aldolase Mutant in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-anti-β-hydroxy-α,α-dialkyl-α-amino acid threonine aldolase and its mutants were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M D-serine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 8h, with samples taken at intervals to determine the concentrations and de values ​​of the substrate and product. Data at the end of the reaction are shown in Table 6.

[0056] Example 11 Preparation of Product 8 from D-Threonine Aldolase Mutant in Water-DMSO Following the method in Example 3, engineered bacteria expressing D-anti-β-hydroxy-α-amino acid threonine aldolase and its mutants were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M D-serine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and product were measured. Data at the end of the reaction are shown in Table 6.

[0057] Table 6. de values ​​and final conversion rates of product 8 prepared by different mutants in different systems.

[0058] Example 12: Preparation of Products 3 and 4 from D-Threonine Aldolase Mutants in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-syn / anti-β-hydroxy-α,α-dialkyl-α-amino acid aldolase and its mutants were cultured and crude enzyme solutions were obtained. The reaction system (1L) contained 1M D-alanine, 50mM p-nitrobenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and products were measured. The data at the end of the reaction are shown in Table 7.

[0059] Example 13: Preparation of Products 9 and 10 from D-threonine Aldolase Mutants in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-syn / anti-β-hydroxy-α,α-dialkyl-α-amino acid aldolase and its mutants were cultured, and crude enzyme solutions were obtained. The reaction system (1L) contained 1M D-serine, 50mM p-nitrobenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and products were measured. The data at the end of the reaction are shown in Table 7.

[0060] Table 7. de values ​​and conversion rates of D-threonine aldolase mutants (product 3 / 4, product 9 / 10)

[0061] Example 12: Preparation of Products 5 and 6 from D-threonine Aldolase Mutants in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-syn / anti-β-hydroxy-α,α-dialkyl-α-amino acid aldolase and its mutants were cultured, and crude enzyme solutions were obtained. The reaction system (1 L) contained 1 M D-alanine, 50 mM 2,4-dihydroxybenzaldehyde, 50 μM pyridoxal phosphate, and 0.1 mM manganese chloride, with 250 mL of crude enzyme solution. The reaction was carried out at 30°C for 1 h, after which the concentrations and de values ​​of the substrate and products were measured. The data at the end of the reaction are shown in Table 8.

[0062] Example 13: Preparation of Products 11 and 12 from D-Threonine Aldolase Mutants in Aqueous Solution. Following the method described in Example 3, engineered bacteria expressing D-syn / anti-β-hydroxy-α,α-dialkyl-α-amino acid aldolase and its mutants were cultured, and crude enzyme solutions were obtained. The reaction system (1L) contained 1M D-serine, 50mM 2,4-dihydroxybenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations and de values ​​of the substrate and products were measured. The data at the end of the reaction are shown in Table 8.

[0063] Table 8. de values ​​and conversion rates of D-threonine aldolase mutants (product 5 / 6, product 11 / 12)

Claims

1. A D-threonine aldolase mutant, characterized in that, The mutant has three or fewer substitutions relative to SEQ ID NO.2 and has less than 100% sequence identity with SEQ ID NO.2, wherein the substitutions are selected from: S255A, S255C, S255G, S255I, S255K, S255L, S255M, S255N, S255P, S255Q, S255R, S255T, S255D, S255E, S255F, S255H, S255V, S255W, S255Y, D327A, Y190A, Y190C, Y190D, Y190E, Y190F, Y190G, Y190H, Y190I, Y190K, Y190L, Y190M, Y190N, Y190P, Y190Q, Y190R, Y190S, Y190T, Y190V, Y190W, H358A, H358C, H358D, H358E, H358F, H358G, H358I, H358K, H358L, H358M, H358N, H358P, H358Q, H358R, H358S, H358T, H358V, H358W, and H358Y, or combinations thereof.

2. The D-threonine aldolase mutant as described in claim 1, characterized in that, The replacement is selected from: S255A, S255T, D327A, Y190F and H358V, or a combination thereof.

3. A gene encoding a D-threonine aldolase mutant as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 3.

5. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the gene of claim 3 or the recombinant vector of claim 4.

6. A catalyst, characterized in that, The catalyst comprises the D-threonine aldolase mutant of claim 1 or 2 or the recombinant microorganism of claim 5; the catalyst is an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.

7. The use of a D-threonine aldolase mutant as described in claim 1 or 2, or a recombinant microorganism as described in claim 5, or a catalyst as described in claim 6 in the preparation of D-β-hydroxy-α,α-dialkyl-α-amino acids; wherein, The D-β-hydroxy-α, α-dialkyl-α-amino acid is either D-syn-β-hydroxy-α, α-dialkyl-α-amino acid or D-anti-β-hydroxy-α, α-dialkyl-α-amino acid.

8. A method for preparing D-β-hydroxy-α,α-dialkyl-α-amino acids, characterized in that, Using D-alanine or D-serine as the amino acid substrate, benzaldehyde derivative as the aldehyde substrate, pyridoxal phosphate and manganese ions as cofactors, and employing the catalyst described in claim 6, a condensation reaction is carried out to generate D-β-hydroxy-α,α-dialkyl-α-amino acid; wherein, the benzaldehyde derivative is one of p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, and 2,4-dihydroxybenzaldehyde; and the D-β-hydroxy-α,α-dialkyl-α-amino acid is D-syn-β-hydroxy-α,α-dialkyl-α-amino acid or D-anti-β-hydroxy-α,α-dialkyl-α-amino acid.

9. The method as described in claim 8, characterized in that, The reaction is carried out in an organic solvent-water mixture or an aqueous solution.

10. The method as described in claim 8, characterized in that, The organic solvent is DMSO, and the amount of organic solvent added does not exceed 20% of the total volume.

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