D-amino acid dehydrogenase mutants and their use in the synthesis of chiral d-amino acids

Abstract

The application belongs to the technical field of bio-chemical industry, and particularly relates to D-amino acid dehydrogenase mutants and application thereof in synthesis of chiral D-amino acids. In order to obtain D-amino acid dehydrogenase mutants with better catalytic performance, meso-diaminopimelic acid dehydrogenase from Symbiobacterium thermophilum is reformed by using a two-round fast evolution strategy based on Rosetta software, and D-amino acid dehydrogenase mutants capable of efficiently catalyzing reductive amination of various alpha-keto acid substrates to generate corresponding D-amino acids are obtained. The D-amino acid dehydrogenase mutants obtained by the application have high catalytic activity, wide substrate spectrum and strict stereoselectivity, and can be used for biological catalytic synthesis of various high-value chiral D-amino acids, with a highest substrate concentration of 0.5M, a substrate conversion rate of >99%, and an ee value of >99%, and have high practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and particularly relates to a D-amino acid dehydrogenase mutant and an application thereof in the synthesis of chiral D-amino acids. Background Art

[0002] L-amino acids, the fundamental chiral units of proteins, have been extensively studied due to their ubiquitous presence in living organisms and their involvement in protein synthesis. However, D-amino acids, their enantiomers, have received relatively little research. Only in recent decades have scientists begun to recognize their crucial role in biology. Unlike L-amino acids, D-amino acids exhibit unique properties in biochemical properties, molecular stability, and receptor interactions, leading to their increasing application in biomedicine. D-amino acids serve as chiral building blocks for numerous marketed drugs. For example, D-phenylglycine and D-p-hydroxyphenylglycine are key chiral intermediates in the synthesis of over a dozen β-lactam antibiotics, including cephalexin, cefaclor, and amoxicillin; D-phenylalanine is an important precursor for the hypoglycemic drug nateglinide; D-tyrosine is a core component of the premature birth prevention drug atosiban; and D-tert-leucine can be used to synthesize a variety of antiviral, anti-tumor, and anti-inflammatory drugs. Beyond their pharmaceutical applications, the value of D-amino acids in food, cosmetics, and agriculture is also gaining recognition. With the deepening of research on D-amino acids, their application prospects in various industries are becoming more and more extensive, and the market potential is huge. The development of their synthesis process is of great significance.

[0003] Meso-diaminopimelate dehydrogenase (DAPDH; EC1.4.1.16) is a type of enzyme that uses NADP +DAPDH is an oxidoreductase that acts as a coenzyme and reversibly and selectively catalyzes the D-central oxidative deamination of the natural substrate meso-diaminopimelate to produce the corresponding L-2-amino-6-oxopimelic acid. In addition to its natural substrate, DAPDH also catalyzes the reductive amination of some α-keto acids to produce the corresponding D-amino acids, hence its designation as a D-amino acid dehydrogenase. The reductive amination reaction catalyzed by DAPDH offers strict stereoselectivity, high atom economy, and easy product isolation and purification, making it an ideal enzymatic method for the synthesis of D-amino acids. Based on their genetic evolutionary relationships and functional differences, DAPDH can be divided into two subclasses: class I DAPDH and class II DAPDH. Class I DAPDH exhibits a relatively strict substrate specificity for its natural substrate, meso-diaminopimelate, while class II DAPDH has a relatively broader substrate spectrum. Although class II DAPDH can catalyze a wider range of substrates, it still exhibits high catalytic activity only for a few α-keto acids with simple structures and short side chains. In order to broaden its scope of application, many studies have focused on the substrate specificity modification of DAPDH. These works usually use multiple rounds of iterative site-directed saturation / half-saturation mutation strategies to transform the active pocket of DAPDH, significantly improving the catalytic activity of DAPDH to multiple large sterically hindered substrates (Hierarchical Engineering ofmeso-Diaminopimelate Dehydrogenasefor Efficient Synthesis of Bulky d-Amino Acids,ACS Catal.2024,14,11447-11456;Structure-guided engineering of meso-diaminopimelate dehydrogenase forenantioselective reductive amination ofsterically bulky 2-keto acids,Catal.Sci.Technol.2018,8,4994). However, these transformation works all need to carry out multiple rounds of time-consuming and labor-intensive screening, and need to target specific substrates, such as benzoylformic acid, the precursor substrate of D-phenylglycine, and the mutant catalytic activity developed is still relatively low, resulting in its practical application being restricted.

[0004] Therefore, there is an urgent need to adopt more efficient and precise modification methods to modify DAPDH in order to obtain D-amino acid dehydrogenase mutants with better catalytic performance, thereby effectively improving its application potential in the enzymatic synthesis of large steric hindrance and complex chiral D-amino acids. Summary of the Invention

[0005] To overcome the above-mentioned shortcomings of the prior art, the present invention uses a two-round rapid evolution strategy based on Rosetta to transform meso-diaminopimelate dehydrogenase (StDAPDH) from Symbiobacterium thermophilum, obtaining D-amino acid dehydrogenase mutants with significantly improved reductive amination activity for a series of sterically hindered α-ketoacid substrates. These mutants are then applied to the asymmetric synthesis of various chiral D-amino acids.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a D-amino acid dehydrogenase mutant, which is a mutant formed by mutation of all or part of the amino acid residues 121, 146, 154, 171, 181 and 227 of the StDPADH amino acid sequence shown in SEQ ID NO.13.

[0008] The present invention first used D-phenylglycine, the product of benzoylformic acid (S1), as the input ligand and the enzyme design program in Rosetta software to design the active pocket of StDAPDH. A total of eight D-amino acid dehydrogenase mutants with significantly improved catalytic activity towards benzoylformic acid were obtained. Based on this, a second round of enzyme design was conducted using the dominant conformation of D-phenylglycine. The mutational profile of the mutants was statistically analyzed and a streamlined mutant library was designed. The optimal mutant (M2) obtained in the first round was used as the parent to construct this streamlined mutant library. Subsequently, high-throughput screening was used to successfully obtain D-amino acid dehydrogenase mutants with further improved catalytic activity towards benzoylformic acid.

[0009] Preferably, the D-amino acid dehydrogenase mutant is selected from at least one of the following mutants:

[0010] (1) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with isoleucine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 15;

[0011] (2) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by isoleucine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by leucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 16;

[0012] (3) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with valine, the amino acid residue at position 146 is substituted with isoleucine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with valine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 20;

[0013] (4) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with methionine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with valine, the amino acid sequence of the mutant being shown in SEQ ID NO. 22;

[0014] (5) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by methionine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 23;

[0015] (6) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with isoleucine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with valine, the amino acid sequence of the mutant being shown in SEQ ID NO. 24;

[0016] (7) A combined mutant in which the amino acid residue at position 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by leucine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by valine, the amino acid sequence of the mutant being shown in SEQ ID NO.25.

[0017] The second aspect of the present invention provides a gene encoding the D-amino acid dehydrogenase mutant described in the first aspect, wherein the gene encoding the D-amino acid dehydrogenase mutant is selected from at least one of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12.

[0018] The third aspect of the present invention provides a biological material comprising the encoding gene described in the second aspect, wherein the biological material comprises an expression vector and a recombinant engineered bacterium.

[0019] More preferably, the expression vector is pET-28a(+) comprising the gene encoding the D-amino acid dehydrogenase mutant.

[0020] More preferably, the recombinant engineered bacteria is Escherichia coli BL21 (DE3) into which a D-amino acid dehydrogenase mutant expression vector is introduced.

[0021] The fourth aspect of the present invention provides the use of the D-amino acid dehydrogenase mutant described in the first aspect, or the encoding gene described in the second aspect, or the biomaterial described in the third aspect in synthesizing chiral D-amino acids.

[0022] A fifth aspect of the present invention provides a method for catalytically synthesizing chiral D-amino acids, specifically comprising: adding an enzyme solution of the D-amino acid dehydrogenase mutant described in the first aspect or recombinant engineered bacterial cells capable of expressing the D-amino acid dehydrogenase mutant described in the first aspect to a mixed system containing an α-ketoacid substrate, an amino group donor, and a coenzyme regeneration system; and obtaining a chiral D-amino acid after a reductive amination reaction.

[0023] Preferably, the enzyme solution is a crude enzyme solution of a genetically engineered bacterium capable of expressing the D-amino acid dehydrogenase mutant described in the first aspect or a purified pure enzyme solution.

[0024] Preferably, the coenzyme regeneration system is a reduced nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme regeneration system, comprising formate dehydrogenase as a coenzyme regeneration enzyme, ammonium formate as a coenzyme regeneration substrate, and NADP +Formate dehydrogenase coenzyme regeneration system; or glucose dehydrogenase as coenzyme regeneration enzyme, glucose as coenzyme regeneration substrate, including NADP + Glucose dehydrogenase coenzyme regeneration system; or alcohol dehydrogenase as coenzyme regeneration enzyme, isopropanol as coenzyme regeneration substrate, including NADP + Alcohol dehydrogenase coenzyme regeneration system.

[0025] More preferably, the coenzyme regeneration system uses glucose dehydrogenase shown in SEQ ID NO.26 as the coenzyme regeneration enzyme, glucose as the coenzyme regeneration substrate, and contains NADP + Glucose dehydrogenase coenzyme regeneration system. NADP in the reaction solution + The dosage is 0.1~1.0mM.

[0026] Preferably, the α-keto acid substrate is selected from any one of the following S1-S10:

[0027]

[0028] Preferably, the concentration of the α-keto acid substrate is 100-500 mM, and the temperature of the reduction reaction is 25-50°C.

[0029] Preferably, the amino donor includes but is not limited to ammonium chloride (NH4Cl).

[0030] The sixth aspect of the present invention provides a method for catalytically synthesizing D-phenylglycine, specifically comprising: dissolving benzoylformic acid (S1), glucose, and an amino donor in a glycerol solution, adjusting the pH to 8.0-10.0, and then adding the enzyme solution of the D-amino acid dehydrogenase mutant described in the first aspect, the enzyme solution of the glucose dehydrogenase shown in SEQ ID NO. 26, and NADP + , D-phenylglycine is obtained after the reaction;

[0031] Alternatively, benzoylformic acid (S1) and an amino donor are dissolved in water, and the pH is adjusted to 1.0-3.0 to prepare a substrate concentrate. Glucose and an amino donor are dissolved in water, and the pH is adjusted to 8.0-10.0 to prepare a reaction solution. The substrate concentrate, the enzyme solution of the D-amino acid dehydrogenase mutant described in the first aspect, the enzyme solution of the glucose dehydrogenase shown in SEQ ID NO. 26, and NADP are then added. + Add it into the reaction solution, and slowly add the substrate concentrate into the reaction system during the reaction process, and D-phenylglycine is obtained after the reaction.

[0032] Preferably, the amino donor includes but is not limited to ammonium chloride (NH4Cl), and the glycerol solution is a 4-6% glycerol solution.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This study utilizes a two-round Rosetta-based rapid evolution strategy to engineer meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum. The resulting D-amino acid dehydrogenase mutants exhibit high catalytic activity, a broad substrate spectrum, and strict stereoselectivity. The highest reported specific activity for the model substrate S1 reached 29.47 U / mg-protein. These D-amino acid dehydrogenase mutants can be applied to the biocatalytic synthesis of various high-value chiral D-amino acids, including D-phenylglycine, achieving substrate conversions exceeding 99% and ee values ​​exceeding 99% at substrate concentrations as high as 0.5 M, demonstrating high practical application value. This study addresses the limitation of natural meso-diaminopimelate dehydrogenase in its low catalytic activity toward sterically hindered substrates, enriches the enzyme toolkit for the preparation of chiral D-amino acids, and provides new, highly efficient enzyme catalysts for the biocatalytic synthesis of high-value D-amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the construction of D-amino acid dehydrogenase mutants;

[0036] Figure 2 Set for catalytic constraints;

[0037] Figure 3 Viability assays for mutants designed for the first round;

[0038] Figure 4 Mutation statistics of mutants designed for the second round;

[0039] Figure 5 To streamline the screening results of the mutation library;

[0040] Figure 6 The reaction equation for the synthesis of D-amino acids by D-amino acid dehydrogenase mutants;

[0041] Figure 7 The reaction process of M12 catalyzing 0.3M S1 to synthesize D-phenylglycine;

[0042] Figure 8 This is the reaction process of M12 catalyzing 0.5M S1 to synthesize D-phenylglycine. DETAILED DESCRIPTION

[0043] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0045] The experimental methods described in the examples are conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experimental Guide" edited by J. Sambrook et al.

[0046] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombination cloning kit, and plasmid extraction kit used in the examples were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; gene synthesis, primer synthesis, and gene sequencing were all performed by Qingke Zixi Biotechnology (Guangzhou) Co., Ltd., and the instructions for use of the above reagents were referred to the product instructions.

[0047] The expression vector involved in the embodiment is pET-28a(+), and the host used is Escherichia coli BL21(DE3), all of which are deposited in the Marine Natural Products Laboratory of the School of Marine Sciences, Sun Yat-sen University where the inventors are located.

[0048] The α-keto acid substrates and amino acid standards used in the examples were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; kanamycin, ammonium chloride, isopropyl-β-D-thiogalactopyranoside (IPTG), NADP + and NADPH were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; other commonly used reagents can be purchased from the market.

[0049] The three-letter or single-letter amino acid expressions used in the examples all follow the amino acid code specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0050] The standard procedure for bacterial culture and protein purification described in the examples is as follows: After streaking and activating the recombinant engineered strain of the present invention [Escherichia coli BL21 (DE3) containing the corresponding mutant gene] from a glycerol tube, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin (Kan) and cultured at 37°C, 200 rpm, and shaking for 6 hours. Then, a 2% inoculum was transferred to 250 mL of fresh TB liquid medium also containing 50 μg / mL Kan, and cultured at 37°C, 200 rpm, and shaking for 3 hours. IPTG was added to a final concentration of 0.5 mM, and induced at 25°C for 16 hours. After the culture was completed, the culture was centrifuged at 12,000 g and 4°C for 10 minutes, the bacteria were collected, the cells were ultrasonically disrupted, and the protein was purified by nickel affinity chromatography.

[0051] The standard assay system for D-amino acid dehydrogenase activity described in the examples is: 0.001-0.1 mg of pure enzyme, 10 mM α-keto acid substrate, 0.12 mM NADPH, a total volume of 1 mL, and a reaction medium of 1 M NH4Cl-NH3·H2O buffer at pH 9.0. The reaction temperature is 30°C, and the reaction is initiated by adding the enzyme solution. The OD value is then measured spectrophotometrically within 1 minute. 340nm Changes in absorbance values.

[0052] The enzyme activity unit (U) described in the examples is defined as the amount of enzyme required to consume 1 μmol NADPH per minute under standard reaction conditions.

[0053] In the embodiment, the concentration of the reaction solution S1 was analyzed by non-chiral HPLC. Chromatographic conditions: the chromatographic column was Athena C18, 5μm, 4.6×250mm; mobile phase: 50mM pH 7.5 phosphate buffer: acetonitrile = 0.9:0.1; detection wavelength: 220nm; flow rate: 1mL / min; column temperature: 30℃.

[0054] In the examples, chiral HPLC was used to analyze the concentration and ee value of the amino acids in the reaction solution. Chromatographic conditions: Chiral AAOA 5u 150*4.6mm column; detection wavelength: 254nm; flow rate: 1.0mL / min; column temperature: 25°C. The mobile phase and retention time for each amino acid analysis are shown in Table 1:

[0055] Table 1 Mobile phases and retention times used in HPLC analysis of different amino acid products

[0056]

[0057]

[0058] Example 1: Construction, screening and activity verification of D-amino acid dehydrogenase mutants

[0059] like Figure 1 As shown, it specifically includes the following parts:

[0060] 1. The first round of design based on Rosetta

[0061] The online tool BCL:ConfServer (http: / / carbon.structbio.vanderbilt.edu / index.php / bclconf) was used to search for small molecule conformations of the input ligand, D-phenylglycine, the product corresponding to benzoylformic acid (S1). The meso-diaminopimelate dehydrogenase (StDAPDH, NCBI accession number: BAD40410.1, SEQ ID NO.13) from Symbiobacterium thermophilum was used as the design target. The interaction pattern between the enzyme protein and the small molecule in the crystal structure of StDAPDH (PDB ID: 3WBF) was analyzed, and the catalytic geometric constraints ( Figure 2 , Table 2), and select design sites (W121, F146, H154, T171, R181, H227). Subsequently, the enzymedesign program of Rosetta was used to design the design site of StDAPDH. The design mutants provided by the program were screened and sorted, and 8 design mutants were finally selected for subsequent wet experiment verification. These 8 D-amino acid dehydrogenase mutants are numbered M1-M8, and the amino acid sequences are shown in SEQ ID NO.14-21. The genes corresponding to these 8 mutants (as shown in SEQ ID NO.1-8) were handed over to Guangzhou Qingke Zixi Biotechnology Co., Ltd. for synthesis and connected to pET-28a (+) plasmid (N-terminal His-tag purification tag) to construct a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells to obtain a recombinant engineering bacterium comprising the D-amino acid dehydrogenase mutant gene.

[0062] The obtained recombinant engineered bacteria were fermented and the protein was purified, and the activity of the purified protein was determined using the D-amino acid dehydrogenase activity standard detection system. Figure 3 As shown, it was found that the eight designed D-amino acid dehydrogenase mutants all had a certain catalytic activity towards S1 (M1-M8, amino acid sequences are shown in SEQ ID NOs. 14-21). For example, the catalytic activities of M2, M3, and M7 reached 22.38 U / mg-protein, 12.94 U / mg-protein, and 12.88 U / mg-protein, respectively. Among them, the catalytic activity of M2 was significantly higher than the highest level reported by previous researchers (15.96 U / mg-protein reported in Chinese invention patent CN118360265A).

[0063] Among them, the StDPADH-M2 mutant is a combination mutant formed by replacing the amino acid residue 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 with isoleucine, the amino acid residue 146 with valine, the amino acid residue 154 with leucine, the amino acid residue 171 with isoleucine, the amino acid residue 181 with isoleucine, and the amino acid residue 227 with isoleucine. The amino acid sequence of the mutant is shown in SEQ ID NO.15. The StDPADH-M3 mutant is a combination mutant formed by replacing the amino acid residue 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 with isoleucine, the amino acid residue 146 with valine, the amino acid residue 154 with leucine, the amino acid residue 171 with isoleucine, the amino acid residue 181 with isoleucine, and the amino acid residue 227 with leucine. The amino acid sequence of the mutant is shown in SEQ ID NO.16. The StDPADH-M7 mutant is a combination mutant formed by replacing the 121st amino acid residue of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 with valine, the 146th amino acid residue with isoleucine, the 154th amino acid residue with leucine, the 171st amino acid residue with valine, the 181st amino acid residue with isoleucine, and the 227th amino acid residue with isoleucine. The amino acid sequence of the mutant is shown in SEQ ID NO.20.

[0064] Table 2 Distance and angle constraint setting parameters

[0065]

[0066] 2. The second round of evolution based on Rosetta

[0067] (1) Second round of design and mutation library design

[0068] The D-phenylglycine conformation corresponding to the M2 mutant was selected as the dominant conformation for a new round of enzyme design, and other parameters remained the same as the previous round of design process. The mutation types contained in each site of the designed mutants obtained by screening and sorting were statistically analyzed ( Figure 4 Based on the statistical results, a streamlined combinatorial mutation library was designed, specifically: isoleucine was introduced at position 121, leucine was introduced at position 154, isoleucine was introduced at position 181, leucine, methionine, and valine were randomly introduced at position 146, isoleucine, valine, leucine, and methionine were randomly introduced at position 171, and isoleucine, alanine, and valine were randomly introduced at position 227.

[0069] (2) Construction of a streamlined combinatorial mutation library

[0070] Specific primers were synthesized (Table 3). Using the pET-28a(+)-StDAPDH-M2 recombinant plasmid containing the M2 mutant gene obtained in part 1 as a template, PCR was performed with primer pair 146_VTG-F and 171-R to obtain linearized fragment I. PCR was performed with primer pair mixed I (171_VTG-F:171_ATT-F = 3:1) and 227-R to obtain linearized vector fragment II. PCR was then performed with primer mixed II (227_RTT-F:227_GCA-F = 2:1) and 146-R to obtain linearized vector fragment III. The specific PCR system is shown in Table 4. The specific PCR amplification conditions are: 1) pre-denaturation at 95°C for 3 min; 2) denaturation at 95°C for 15 s; annealing at 56°C for 15 s; extension at 72°C for 30-150 s; 30-35 cycles; 3) post-extension at 72°C for 5 min; and storage at 4°C.

[0071] Following the instructions for the ClonExpress MultiS One-Step Cloning Kit, the three fragments were digested with Dpn I and recovered on gel. The three fragments were then recombined in a single reaction and transformed into competent E. coli BL21 (DE3) cells. These recombinant E. coli cells containing the genes for the different D-amino acid dehydrogenase mutants constituted the constructed streamlined combinatorial mutation library.

[0072] Table 3 Primers required for streamlining the combinatorial mutation library

[0073]

[0074] Table 4 PCR amplification system

[0075] Components volume DNA polymerase (2×PhantaMaxMasterMix) 25 μL Upstream primer (10 pmol / μL) 2μL Downstream primer (10 pmol / μL) 2μL Plasmid template 1 μL <![CDATA[ddH2O]]> 20 μL

[0076] (3) High-throughput screening of mutation libraries

[0077] Add 200 μL of LB culture medium (containing 50 μg / mL of kanamycin) to a sterilized 96-deep-well plate, and use a sterilized pipette tip to pick a single colony into the 96-deep-well plate. Then, culture the deep-well plate at 37°C and 200 rpm for 6 hours, which is called the primary plate. Add 400 μL of LB culture medium (containing 50 μg / mL of kanamycin) to another sterilized 96-deep-well plate as the secondary plate, and draw 50 μL of bacterial solution from the primary plate to the secondary plate. Add 20% glycerol to the primary plate and place it in a -80°C refrigerator for long-term storage. Place the secondary plate at 37°C for shaking culture for 3 hours, then add IPTG with a final concentration of 0.5 mM for induction, and then culture the secondary plate at 25°C and 200 rpm for 16 hours.

[0078] After centrifugation at 4000 rpm and 4°C for 20 minutes, collect the cells and then freeze at -80°C for at least 24 hours. Remove the secondary plate from -80°C and thaw at room temperature for 0.5 hours. Then, add 400 μL of lysis solution (50 mM pH 7.5 phosphate buffer, 2 mg / mL lysozyme, 10 mg / L DNase I) to each well, shake to suspend the cells, and incubate on a shaker at 37°C and 200 rpm for 1.5 hours. After incubation, centrifuge at 4000 rpm and 4°C for 20 minutes, and remove the supernatant for enzyme activity determination.

[0079] 80 μL of activity mixture (0.5 M pH 9.0 NH4CL-NH 3· HO buffer, 0.5mM NADPH, 5mM S1 substrate), then aspirate 20μL of supernatant enzyme solution and add it to the reaction plate to start the reaction. After incubation at 25°C for 30 minutes, measure the absorbance at 340nm using a microplate reader. Lower absorbance values ​​indicate higher catalytic activity. Mutants with lower absorbance values ​​than the control (M2 mutant) were selected for subsequent activity verification.

[0080] (4) Verification of S1 catalytic activity

[0081] After screening a total of 192 single clones, the positive mutants obtained were sequenced and their specific mutations were analyzed. Subsequently, the mutants were fermented and purified, and the activity of the purified protein was determined using a standard activity detection system. Figure 5 As shown, through verification, a total of four D-amino acid dehydrogenase mutants M9-M12 (amino acid sequences are shown in SEQ ID NO.22-25, and gene sequences are shown in SEQ ID NO.9-12) with higher catalytic activity towards S1 than M2 were obtained, among which M12 had the highest catalytic activity, reaching 29.47 U / mg-protein, which is the highest level reported so far.

[0082] Among them, the StDPADH-M9 mutant is a combination mutant formed by replacing the amino acid residue 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 with isoleucine, the amino acid residue 146 with valine, the amino acid residue 154 with leucine, the amino acid residue 171 with methionine, the amino acid residue 181 with isoleucine, and the amino acid residue 227 with valine. The amino acid sequence of the mutant is shown in SEQ ID NO.22. The StDPADH-M10 mutant is a combination mutant formed by replacing the amino acid residue 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 with isoleucine, the amino acid residue 146 with valine, the amino acid residue 154 with leucine, the amino acid residue 171 with methionine, the amino acid residue 181 with isoleucine, and the amino acid residue 227 with isoleucine. The amino acid sequence of the mutant is shown in SEQ ID NO.23. The StDPADH-M11 mutant is a combination mutant in which the amino acid residue at position 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13 is replaced with isoleucine, the amino acid residue at position 146 is replaced with valine, the amino acid residue at position 154 is replaced with leucine, the amino acid residue at position 171 is replaced with isoleucine, the amino acid residue at position 181 is replaced with isoleucine, and the amino acid residue at position 227 is replaced with valine. The amino acid sequence of the mutant is shown in SEQ ID NO. 24. The StDPADH-M12 mutant is a combination mutant in which the amino acid residue at position 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13 is replaced with isoleucine, the amino acid residue at position 146 is replaced with valine, the amino acid residue at position 154 is replaced with leucine, the amino acid residue at position 171 is replaced with leucine, the amino acid residue at position 181 is replaced with isoleucine, and the amino acid residue at position 227 is replaced with valine. The amino acid sequence of the mutant is shown in SEQ ID NO. 25.

[0083] Example 2: Determination of the catalytic activity of D-amino acid dehydrogenase mutants on different ketoacid substrates

[0084] Taking the D-amino acid dehydrogenase mutants M2 and M12 as examples, the mutants M2 and M12 were fermented and protein purified. The catalytic activity of the D-amino acid dehydrogenase mutants towards 10 wild-type difficult-to-catalyze ketoacid substrates was determined using pure enzymes. To better evaluate the modification effect, the StDAPDH mutant (W121L / H227I) reported in the literature was used as a control (Structure-guided engineering of meso-diaminopimelate dehydrogenase for enantioselective reductive amination of sterically bulky 2-keto acids, Catal. Sci. Technol. 2018, 8, 4994). The activity assay system was: 1-10 mM α-ketoacid substrate (S1 to S10), 0.12 mM NADPH, a total system of 1 mL, and a reaction medium of 1 M NH4CL-NH3·H2O buffer at pH = 9.0. The reaction temperature was 30°C, and the reaction was triggered by adding enzyme solution, and then the OD within 1 minute was detected using a spectrophotometer. 340nm Changes in absorbance values.

[0085] Table 5D- Catalytic activity of amino acid dehydrogenase mutants towards different ketoacid substrates

[0086]

[0087]

[0088] a The four data separated by slashes ( / ) represent the amount of enzyme added in the enzyme activity assay system of wild type, W121L / H227I, M2, and M12 respectively;

[0089] b ND: no detectable catalytic activity.

[0090] Example 3: Synthesis of chiral D-amino acids catalyzed by D-amino acid dehydrogenase mutants

[0091] The reaction equation for the synthesis of chiral D-amino acids catalyzed by D-amino acid dehydrogenase mutants is as follows Figure 6 As shown, glucose dehydrogenase (SEQ ID NO. 26) is used to achieve NADPH regeneration. The specific synthesis method and system are as follows:

[0092] The D-amino acid dehydrogenase mutant and glucose dehydrogenase were fermented and purified according to standard methods to obtain pure enzymes. The reaction system was: 0.1-2 mg pure D-amino acid dehydrogenase mutant enzyme, 1 mg pure glucose dehydrogenase enzyme, 100 mM α-keto acid substrate (S1 to S10), 120 mM glucose, 2.5 M NH4Cl, 0.5 mM NADP. + The total system volume was 1 mL, and the reaction medium was a 0.2 M sodium carbonate-sodium bicarbonate buffer solution at pH 9.0. After 24 hours of reaction at 35°C and 800 rpm, a 20 μL sample was taken and mixed with 980 μL of chiral HPLC mobile phase (2 mM copper sulfate aqueous solution: isopropanol = 95:5). The mixture was passed through a membrane and analyzed by chiral HPLC for the concentration and ee value of the amino acid product. The results are shown in Table 6. Compared with the wild type, the D-amino acid dehydrogenase mutant provided by the present invention exhibited significantly higher catalytic efficiency, with a D-amino acid product yield of >99% and an ee value greater than 99%.

[0093] Table 6 Synthesis of chiral D-amino acids catalyzed by D-amino acid dehydrogenase mutants

[0094]

[0095]

[0096] a Wild-type StDAPDH was used as a control to evaluate the effects of the mutants;

[0097] b ND: No amino acid product was detected in the reaction solution.

[0098] Example 4: Synthesis of D-phenylglycine by M12 catalyzing 0.3M S1

[0099] D-amino acid dehydrogenase mutant M12 (SEQ ID NO. 25) and glucose dehydrogenase (SEQ ID NO. 26) were fermented and purified according to standard methods to obtain pure enzymes.

[0100] The synthetic reaction system is 20 mL, and its preparation method is as follows: weigh 300mM S1, 360mM glucose, and 1M NH4Cl, dissolve them in 5% glycerol solution, add ammonia water to adjust the pH to 9.0, then add 5mg pure D-amino acid dehydrogenase mutant enzyme, 20mg pure glucose dehydrogenase enzyme, 0.25mM NADP + Start the reaction. The reaction apparatus uses a magnetic stirrer, and the reaction temperature is controlled at 40°C in a water bath. During the reaction, the pH is maintained at 9.0 by dropwise addition of aqueous ammonia. The concentration of S1 in the system is measured by achiral HPLC to monitor the reaction progress. The concentration of D-phenylglycine and the ee value in the reaction solution are also measured by chiral HPLC.

[0101] The reaction process of M12 catalyzing 0.3M S1 to synthesize D-phenylglycine is as follows Figure 7 The results showed that the substrate conversion rate of 0.3M S1 reached more than 99.0% within 10 h, the concentration of D-phenylglycine produced was 44.54 g / L, and the space-time yield reached 108 g·L -1 ·d -1 , ee value>99.0%, indicating that the D-phenylglycine prepared by M12 has very high optical purity.

[0102] Example 5: Synthesis of D-phenylglycine by M12 catalyzing 0.5M S1

[0103] D-amino acid dehydrogenase mutant M12 (SEQ ID NO. 25) and glucose dehydrogenase (SEQ ID NO. 26) were fermented and purified according to standard methods to obtain pure enzymes.

[0104] The 0.5M S1 catalytic reaction was carried out by substrate flow addition and feeding. The total reaction system was 20mL. The preparation method was as follows: 1.5g S1 and 0.53g NH4Cl were weighed, dissolved in 7mL deionized water, and ammonia was added dropwise to adjust the pH to 1.0 and then the volume was adjusted to 10mL with deionized water to prepare a substrate concentrate. 2.16g glucose and 0.53g NH4Cl were weighed, dissolved in 7mL deionized water, ammonia was added dropwise to adjust the pH to 9.0 and then the volume was adjusted to 10mL with deionized water to prepare a reaction solution. 2mL substrate concentrate, 20mg pure enzyme of D-amino acid dehydrogenase mutant, 20mg pure enzyme of glucose dehydrogenase and 0.5mM NADP were added. + Add to the reaction solution to initiate the reaction. Add the remaining 8 mL of substrate concentrate to the reaction system at a flow rate of 0.02 mL / min. The reaction apparatus is a magnetic stirrer, and the reaction temperature is controlled at 40°C in a water bath. During the reaction, the pH is controlled at 9.0 by dropwise addition of aqueous ammonia. The concentration of S1 in the system is measured by achiral HPLC to monitor the reaction progress. The concentration and ee value of D-phenylglycine in the reaction solution are also measured by chiral HPLC.

[0105] The reaction process of M12 catalyzing 0.5M S1 to synthesize D-phenylglycine is as follows Figure 8 The results showed that the substrate conversion rate of 0.5M S1 reached more than 99.0% in 12h, the concentration of D-phenylglycine produced was 74.76g / L, and the space-time yield was 149.52g·L -1 ·d -1 , ee value>99.0%, indicating that the D-phenylglycine prepared by M12 has very high optical purity.

[0106] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A D-amino acid dehydrogenase mutant, characterized in that The D-amino acid dehydrogenase mutant is selected from the following mutants: (1) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with isoleucine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 15; (2) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by isoleucine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by leucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 16; (3) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with valine, the amino acid residue at position 146 is substituted with isoleucine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with valine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 20; (4) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with methionine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with valine, the amino acid sequence of the mutant being shown in SEQ ID NO. 22; (5) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by methionine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO. 23; (6) a combined mutant of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO. 13, wherein the amino acid residue at position 121 is substituted with isoleucine, the amino acid residue at position 146 is substituted with valine, the amino acid residue at position 154 is substituted with leucine, the amino acid residue at position 171 is substituted with isoleucine, the amino acid residue at position 181 is substituted with isoleucine, and the amino acid residue at position 227 is substituted with valine, the amino acid sequence of the mutant being shown in SEQ ID NO. 24; (7) A combined mutant in which the amino acid residue at position 121 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.13 is replaced by isoleucine, the amino acid residue at position 146 is replaced by valine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by leucine, the amino acid residue at position 181 is replaced by isoleucine, and the amino acid residue at position 227 is replaced by valine, the amino acid sequence of the mutant being shown in SEQ ID NO.

25.

2. The gene encoding the D-amino acid dehydrogenase mutant according to claim 1, characterized in that The coding gene is selected from one of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.

12.

3. The biological material comprising the gene encoding method according to claim 2, characterized in that: The biological materials include expression vectors and recombinant engineering bacteria.

4. Use of the D-amino acid dehydrogenase mutant according to claim 1, or the encoding gene according to claim 2, or the biomaterial according to claim 3 in synthesizing chiral D-amino acids using α-keto acids as substrates, characterized in that: The α-keto acid is selected from any one of the following S1-S10:

5. A method for catalytic synthesis of chiral D-amino acids, characterized in that: adding an enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1 or a recombinant engineered bacterial cell capable of expressing the D-amino acid dehydrogenase mutant according to claim 1 to a mixed system containing an α-keto acid substrate, an amino donor, and a coenzyme regeneration system, and performing a reductive amination reaction to obtain a chiral D-amino acid; The α-keto acid substrate is selected from any one of the following S1-S10:

6. The method for catalytic synthesis of chiral D-amino acids according to claim 5, characterized in that: The enzyme solution is a crude enzyme solution of a genetically engineered bacterium capable of expressing the D-amino acid dehydrogenase mutant according to claim 1 or a purified pure enzyme solution; the coenzyme regeneration system is a reduced nicotinamide adenine dinucleotide phosphate coenzyme regeneration system, including formate dehydrogenase as a coenzyme regeneration enzyme, ammonium formate as a coenzyme regeneration substrate, and NADP + Formate dehydrogenase coenzyme regeneration system; or glucose dehydrogenase as coenzyme regeneration enzyme, glucose as coenzyme regeneration substrate, including NADP + Glucose dehydrogenase coenzyme regeneration system; or alcohol dehydrogenase as coenzyme regeneration enzyme, isopropanol as coenzyme regeneration substrate, including NADP + Alcohol dehydrogenase coenzyme regeneration system.

7. The method for catalytic synthesis of chiral D-amino acids according to claim 5, characterized in that: The concentration of the α-keto acid substrate is 100-500 mM, and the temperature of the reduction reaction is 25-50°C.

8. A method for catalytic synthesis of D-phenylglycine, characterized in that: Benzoylformic acid, glucose, and an amino donor are dissolved in a glycerol solution, and after adjusting the pH to 8.0-10.0, the enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1, the enzyme solution of the glucose dehydrogenase shown in SEQ ID NO. 26, and NADP are added. + , D-phenylglycine is obtained after the reaction; Alternatively, benzoylformic acid and an amino donor are dissolved in water, and the pH is adjusted to 1.0-3.0 to prepare a substrate concentrate. Glucose and an amino donor are dissolved in water, and the pH is adjusted to 8.0-10.0 to prepare a reaction solution. The substrate concentrate, the enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1, the enzyme solution of the glucose dehydrogenase shown in SEQ ID NO. 26, and NADP are then added. + Add it into the reaction solution, and slowly add the substrate concentrate into the reaction system during the reaction process, and D-phenylglycine is obtained after the reaction.

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