D-amino acid dehydrogenase mutant and application thereof in chiral D-amino acid synthesis

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a D-amino acid dehydrogenase mutant and application thereof in chiral D-amino acid synthesis. In order to obtain the D-amino acid dehydrogenase mutant with better catalytic performance, a two-round rapid evolution strategy based on Rosetta software is adopted to modify mesogen-diaminopimelate dehydrogenase from Symbiobacterium thermophilum, and the D-amino acid dehydrogenase mutant capable of efficiently catalyzing reductive amination of various alpha-keto acid substrates to generate corresponding D-amino acid is obtained. The obtained D-amino acid dehydrogenase mutant has 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, the highest substrate concentration reaches 0.5 M, and the substrate conversion rate is gt; the ee value is gt; therefore, the method has a relatively high practical application value.

Description

Technical Field

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

[0002] The basic chiral unit of protein is L-amino acid. Because it is ubiquitous in living organisms and participates in protein synthesis, L-amino acids have been studied in depth. However, as the enantiomer of L-amino acids, D-amino acids have been studied relatively little. It was not until recent decades that scientists began to gradually realize the important role of D-amino acids in biology. Unlike L-amino acids, D-amino acids exhibit unique characteristics in biochemical properties, molecular stability, and interaction with receptors, which makes their application in the biomedical field more and more extensive. In fact, D-amino acids are chiral synthetic building blocks for a variety of marketed drugs. For example, D-phenylglycine and D-p-hydroxyphenylglycine are key chiral intermediates for the synthesis of more than a dozen β-lactam antibiotics such as cephalexin, cefaclor, and amoxicillin; D-phenylalanine is an important precursor of the hypoglycemic drug nateglinide; D-tyrosine is the core component of the premature prevention drug atosiban; D-tert-leucine can be used to synthesize a variety of antiviral, anti-tumor and anti-inflammatory drugs. In addition to its application in the medical field, the application value of D-amino acids in food, cosmetics and agriculture has also been gradually recognized. 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 NADP-dependent +DAPDH is a coenzyme that can reversibly and selectively catalyze the D-type central oxidative deamination of the natural substrate meso-diaminopimelate to generate the corresponding L-2-amino-6-oxopimelic acid. In addition to its natural substrate, DAPDH can also catalyze the reductive amination of some α-keto acids to generate the corresponding D-amino acids, so it can also be called D-amino acid dehydrogenase. The reductive amination reaction catalyzed by DAPDH has the characteristics of strict stereoselectivity, high atom economy, and easy separation and purification of products. It is one of the ideal enzymatic synthesis methods of D-amino acids. According to genetic evolutionary relationships and functional differences, DAPDH can be divided into two subclasses, class I DAPDH and class II DAPDH. Class I DAPDH shows a more strict substrate specificity for its natural substrate meso-diaminopimelate, while class II DAPDH has a relatively wider substrate spectrum. Although class II DAPDH can catalyze more substrates, it still has high catalytic activity only for a few α-keto acids with simple structures and short side chains. In order to broaden its application range, there are many studies focusing on the substrate specific transformation of DAPDH. These works usually use multiple rounds of iterative fixed-site 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 DehydrogenaseforEfficientSynthesisofBulkyd-AminoAcids,ACS Catal.2024,14,11447-11456;Structure-guidedengineeringofmeso-diaminopimelatedehydrogenaseforenantioselectivereductiveaminationofstericallybulky2-ketoacids,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 D-phenylglycine precursor substrate benzoylformic acid, the mutant catalytic activity developed is still relatively low, resulting in its practical application being restricted.

[0004] Therefore, it is urgent to use 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] In order to overcome the above-mentioned shortcomings of the prior art, the present invention adopts a two-round rapid evolution strategy based on Rosetta to transform the meso-diaminopimelate dehydrogenase (StDAPDH) from Symbiobacterium thermophilum, obtains D-amino acid dehydrogenase mutants with significantly improved reductive amination activity for a series of large sterically hindered α-ketoacid substrates, and applies these mutants 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 at positions 121, 146, 154, 171, 181 and 227 of the StDPADH amino acid sequence shown in SEQ ID NO.13.

[0008] The present invention first uses D-phenylglycine, the corresponding product of benzoylformic acid (S1), as an input ligand, and uses the enzyme design program of Rosetta software to design the active pocket of StDAPDH, and obtains a total of 8 D-amino acid dehydrogenase mutants with significantly improved catalytic activity to benzoylformic acid. On this basis, the second round of enzyme design is carried out with the dominant conformation of D-phenylglycine, and the mutation of the mutant is counted and a simplified mutation library is designed. The optimized mutant (M2) obtained in the first round is used as the parent to construct the simplified mutation library, and then a high-throughput screening method is used for screening, and a D-amino acid dehydrogenase mutant with further improved catalytic activity to benzoylformic acid is successfully obtained.

[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 amino acid sequence of meso-diaminopimelate dehydrogenase 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 isoleucine, the amino acid sequence of the mutant is shown in SEQ ID NO.15;

[0011] (2) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase 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 is shown in SEQ ID NO.16;

[0012] (3) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13, wherein the amino acid residue at position 121 is replaced by valine, the amino acid residue at position 146 is replaced by isoleucine, the amino acid residue at position 154 is replaced by leucine, the amino acid residue at position 171 is replaced by valine, 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 is 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 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 valine, the amino acid sequence of the mutant being shown in SEQ ID NO.22;

[0014] (5) 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 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 methionine, the amino acid residue at position 181 is replaced with isoleucine and the amino acid residue at position 227 is replaced with isoleucine, the amino acid sequence of the mutant is 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 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 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 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.

[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, SEQID 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] The fifth aspect of the present invention provides a method for catalytic synthesis of chiral D-amino acids, specifically: adding the enzyme solution of the D-amino acid dehydrogenase mutant described in the first aspect or the 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 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 + , after the reaction, D-phenylglycine is obtained;

[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. Then, 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 added. + Add it into the reaction solution, and slowly drop the substrate concentrate into the reaction system during the reaction, 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] The present invention adopts a two-round rapid evolution strategy based on Rosetta to transform the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum, and the obtained D-amino acid dehydrogenase mutant has high catalytic activity, a wide substrate spectrum, and strict stereoselectivity. The highest specific enzyme activity for the model substrate S1 can reach 29.47U / mg-protein, which is the highest level reported so far. These D-amino acid dehydrogenase mutants can be applied to the biocatalytic synthesis of various high-value chiral D-amino acids such as D-phenylglycine, with the highest substrate concentration reaching 0.5M, the substrate conversion rate>99%, and the ee value>99%, showing a high practical application value. The present invention solves the limitation of the low catalytic activity of natural meso-diaminopimelate dehydrogenase to large sterically hindered substrates, enriches the enzyme tool library for the preparation of chiral D-amino acids, and provides a new and efficient enzyme catalyst 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 specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can 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 Experiment 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 all purchased from Nanjing Novezan Biotechnology Co., Ltd.; gene synthesis, primer synthesis and gene sequencing were all completed by Qingke Zixi Biotechnology (Guangzhou) Co., Ltd., and the use of the above reagents was 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 preserved in the Marine Natural Products Laboratory of the School of Marine Sciences of Sun Yat-sen University where the inventor works.

[0048] The α-keto acid substrates and amino acid standards used in the examples were purchased from Shanghai Bidex 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 the cultivation of the bacteria and protein purification described in the embodiment is as follows: after streaking and activating the recombinant engineering strain [Escherichia coli BL21 (DE3) containing the corresponding mutant gene] constructed by the present invention from the glycerol tube, a single colony is inoculated into 5mL LB liquid medium containing 50μg / mL kanamycin (Kan), and cultured at 37°C, 200rpm for 6h. Then, the inoculum is transferred to 250mL of fresh TB liquid medium containing 50μg / mL Kan at a 2% inoculum amount, cultured at 37°C, 200rpm for 3h, IPTG is added to a final concentration of 0.5mM, and induced culture is performed at 25°C for 16h. After the cultivation is completed, the culture solution is centrifuged at 12000g, 4°C for 10min, the bacteria are collected, the cells are ultrasonically broken, and the protein is purified by nickel column affinity chromatography.

[0051] The standard detection system of D-amino acid dehydrogenase activity described in the embodiment is: 0.001-0.1 mg of pure enzyme, 10 mM α-keto acid substrate, 0.12 mM NADPH, the total system is 1 mL, and the reaction medium is 1M NH4Cl-NH3·H2O buffer with pH = 9.0. The reaction temperature is 30°C, the reaction is triggered by adding enzyme solution, and then the OD within 1 minute is detected by spectrophotometer. 340nm Changes in absorbance value.

[0052] The definition of the enzyme activity unit (U) in the examples is: 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 is 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 embodiment, the concentration and ee value of amino acids in the reaction solution were analyzed by chiral HPLC. Chromatographic conditions: chromatographic column: Chiral AAOA 5u 150*4.6mm; detection wavelength: 254nm; flow rate: 1.0mL / min; column temperature: 25°C. The mobile phase and retention time of each amino acid analysis are shown in Table 1:

[0055] Table 1 Mobile phases and retention times used for 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 mode 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 the design site (W121, F146, H154, T171, R181, H227). Subsequently, the design site of StDAPDH was designed using the enzymedesign program of Rosetta. The design mutants given 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 plus 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 containing 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 pure protein was determined using the D-amino acid dehydrogenase activity standard detection system. Figure 3 As shown, it was found that the 8 designed D-amino acid dehydrogenase mutants all had a certain catalytic activity against S1 (M1-M8, the 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 which 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 combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with isoleucine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with isoleucine, and the amino acid sequence of the mutant is shown in SEQ ID NO.15. The StDPADH-M3 mutant is a combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with isoleucine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with leucine, and the amino acid sequence of the mutant is shown in SEQ ID NO.16. The StDPADH-M7 mutant is a combined 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, and 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 corresponding D-phenylglycine conformation in the M2 mutant was selected as the dominant conformation for a new round of enzyme design, and other parameters were kept consistent with 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 simplified combinatorial mutation library was designed, specifically: isoleucine was introduced into position 121, leucine was introduced into position 154, isoleucine was introduced into position 181, leucine, methionine, and valine were randomly introduced into position 146, isoleucine, valine, leucine, and methionine were randomly introduced into position 171, and isoleucine, alanine, and valine were randomly introduced into position 227.

[0069] (2) Construction of streamlined combinatorial mutation libraries

[0070] Specific primers were synthesized (Table 3), and the pET-28a(+)-StDAPDH-M2 recombinant plasmid containing the M2 mutant gene obtained in part 1 was used as a template. PCR was performed using 146_VTG-F and 171-R as a primer pair to obtain linearized fragment I. PCR was performed using mixed primer I (171_VTG-F: 171_ATT-F = 3:1) and 227-R as a primer pair to obtain linearized vector fragment II. PCR was then performed using mixed primer II (227_RTT-F: 227_GCA-F = 2:1) and 146-R as a primer pair 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; 4) storage at 4°C.

[0071] Referring to the instruction manual of the ClonExpress MultiS One Step Cloning Kit, the three fragments were digested with DpnⅠ and recovered by gel, and then the three fragments were recombined in one reaction system and then transformed into E. coli BL21 (DE3) competent cells. These recombinant E. coli containing different D-amino acid dehydrogenase mutant genes are the simplified combinatorial mutation library constructed.

[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) 25μL Upstream primer (10pmol / μL) 2μL Downstream primer (10pmol / μL) 2μL Plasmid template 1μL <![CDATA[ddH2O]]> 20μL

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

[0077] Add 200 μL of LB medium (containing 50 μg / mL of kanamycin) to a sterilized 96-deep-well plate, and use a sterilized pipette tip to pick up a single colony into the 96-deep-well plate. Then place the deep-well plate at 37°C and 200 rpm for 6 hours, which is called the primary plate. Add 400 μL of LB 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 place the secondary plate at 25°C and 200 rpm for 16 hours.

[0078] After centrifugation at 4000rpm and 4℃ for 20min, collect the cells and then freeze them at -80℃ for more than 24h. Take the secondary plate out from -80℃ and thaw it at room temperature for 0.5h. Then add 400μL of lysate (50mM pH7.5 phosphate buffer, 2mg / mL lysozyme, 10mg / L DNaseⅠ) to each well, shake to suspend the cells, and incubate them on a shaker at 37℃ and 200rpm for 1.5h. After incubation, centrifuge at 4000rpm and 4℃ for 20min, and take the supernatant for enzyme activity determination.

[0079] Add 80 μL of the activity test mixture (0.5 M pH 9.0 NH4CL-NH 3· H2O buffer, 0.5mM NADPH, 5mM S1 substrate), and then aspirate 20μL of supernatant enzyme solution and add it to the reaction plate to start the reaction. After reacting at 25℃ for 30min, use a microplate reader to measure the absorbance at 340nm. The lower the absorbance value, the higher the catalytic activity. Select mutants with lower absorbance values ​​than the control (M2 mutant) for subsequent activity verification

[0080] (4) Verification of the catalytic activity of S1

[0081] After screening a total of 192 single clones, the positive mutants obtained by screening were sequenced and their specific mutations were analyzed. Subsequently, the obtained mutants were fermented and purified, and the activity of the purified pure protein was determined using an activity standard detection system. Figure 5 As shown, through verification, a total of 4 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 combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with methionine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with valine, and the amino acid sequence of the mutant is shown in SEQ ID NO.22. The StDPADH-M10 mutant is a combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with methionine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with isoleucine, and the amino acid sequence of the mutant is shown in SEQ ID NO.23. The StDPADH-M11 mutant is a combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with isoleucine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with valine, and the amino acid sequence of the mutant is shown in SEQ ID NO.24. The StDPADH-M12 mutant is a combined mutant formed by replacing the amino acid residue at position 121 of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.13 with isoleucine, the amino acid residue at position 146 with valine, the amino acid residue at position 154 with leucine, the amino acid residue at position 171 with leucine, the amino acid residue at position 181 with isoleucine, and the amino acid residue at position 227 with valine, and 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 D-amino acid dehydrogenase mutants M2 and M12 as examples, mutants M2 and M12 were fermented and purified. The catalytic activity of D-amino acid dehydrogenase mutants on 10 wild-type difficult-to-catalyze keto acid substrates was determined using pure enzymes. In order 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 enantioselectivereductive amination ofsterically bulky 2-keto acids, Catal. Sci. Technol. 2018, 8, 4994). The activity detection system is: 1-10mM α-keto acid substrate (S1 to S10), 0.12mM NADPH, the total system is 1mL, and the reaction medium is 1M NH4CL-NH3·H2O buffer at pH=9.0. The reaction temperature was 30°C. The reaction was triggered by adding enzyme solution, and then the OD within 1 min was detected by spectrophotometer. 340nm Changes in absorbance value.

[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 the 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 is 1mL, and the reaction medium is 0.2M sodium carbonate-sodium bicarbonate buffer with pH=9.0. After reacting at 35℃ and 800rpm for 24h, 20μL of sample is taken, mixed in 980μL chiral HPLC mobile phase (2mM copper sulfate aqueous solution: isopropanol=95:5), passed through the membrane, and the concentration and ee value of the amino acid product are analyzed by chiral HPLC. The results are shown in Table 6. Compared with the wild type, the D-amino acid dehydrogenase mutant provided by the present invention shows significantly higher catalytic efficiency, the yield of D-amino acid product is>99%, and the ee value is greater than 99%.

[0093] Table 6 D-amino acid dehydrogenase mutants catalyze the synthesis of chiral D-amino acids

[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] The 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, and then add 5mg pure enzyme of D-amino acid dehydrogenase mutant, 20mg pure enzyme of glucose dehydrogenase, and 0.25mM NADP + The reaction was started. The reaction device was a magnetic stirrer, and the reaction temperature was controlled at 40°C by a water bath. During the reaction, the pH was controlled at 9.0 by dropping ammonia water. The concentration of S1 in the system was determined by non-chiral HPLC to monitor the progress of the reaction, and the concentration and ee value of D-phenylglycine in the reaction solution were determined 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 was 44.54 g / L, and the space-time yield was 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] The 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 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, 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 start the reaction. The remaining 8 mL of substrate concentrate was added to the reaction system at a flow rate of 0.02 mL / min. The reaction device was a magnetic stirrer, and the reaction temperature was controlled at 40°C by a water bath. During the reaction, the pH was controlled at 9.0 by adding ammonia water. The concentration of S1 in the system was determined by non-chiral HPLC to monitor the reaction progress, and the concentration and ee value of D-phenylglycine in the reaction solution were determined 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 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. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A D-amino acid dehydrogenase mutant, characterized in that The D-amino acid dehydrogenase mutant is a mutant formed by mutation of all or part of the amino acid residues at positions 121, 146, 154, 171, 181 and 227 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.

13.

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

3. The gene encoding the D-amino acid dehydrogenase mutant according to claim 2, characterized in that: The coding gene 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.

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

5. Use of the D-amino acid dehydrogenase mutant according to claim 1 or 2, or the encoding gene according to claim 3, or the biomaterial according to claim 4 in synthesizing chiral D-amino acids.

6. A method for catalytic synthesis of chiral D-amino acids, characterized in that: The enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1 or 2 or the recombinant engineered bacterial cells capable of expressing the D-amino acid dehydrogenase mutant according to claim 1 or 2 are added to a mixed system containing an α-keto acid substrate, an amino donor and a coenzyme regeneration system, and a chiral D-amino acid is obtained after a reductive amination reaction.

7. A method for catalytic synthesis of chiral D-amino acids according to claim 6, 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 2, 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.

8. The method for catalytic synthesis of chiral D-amino acids according to claim 6, characterized in that: The α-keto acid substrate is selected from any one of the following S1-S10:

9. The method for catalytic synthesis of chiral D-amino acids according to claim 6, 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.

10. 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, an enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1 or 2, an enzyme solution of glucose dehydrogenase shown in SEQ ID NO. 26, and NADP are added. + , after the reaction, D-phenylglycine is obtained; Alternatively, benzoylformic acid and an amino donor are dissolved in water, the pH is adjusted to 1.0-3.0, and then a substrate concentrate is prepared; glucose and an amino donor are dissolved in water, the pH is adjusted to 8.0-10.0, and then a reaction solution is prepared; then the substrate concentrate, the enzyme solution of the D-amino acid dehydrogenase mutant according to claim 1 or 2, the enzyme solution of the glucose dehydrogenase shown in SEQ ID NO. 26, and NADP + Add it into the reaction solution, and slowly drop the substrate concentrate into the reaction system during the reaction, and D-phenylglycine is obtained after the reaction.

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