Formic acid dehydrogenase mutant and application thereof

Abstract

This invention relates to a formate dehydrogenase mutant and its applications, belonging to the field of enzyme engineering. The formate dehydrogenase mutant is derived by mutating one or more of the following positions in the amino acid sequence shown in SEQ ID NO.1: tryptophan at position 128 is mutated to lysine, alanine at position 225 is mutated to valine, tryptophan at position 595 is mutated to cysteine, asparagine at position 750 is mutated to cysteine, aspartic acid at position 759 is mutated to valine, and serine at position 888 is mutated to glutamic acid. The formate dehydrogenase mutant provided by this invention has significant advantages in substrate affinity, catalytic activity, and enzyme stability, thereby increasing its value in industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme technology, specifically relating to a formate dehydrogenase mutant and its applications. Background Technology

[0002] NAD-dependent formate dehydrogenase (FDH, EC 1.2.1.2) can metabolize nicotinamide adenine dinucleotide (NAD) into formaldehyde. + ( ) acts as a coenzyme, catalyzing the oxidation of formate to carbon dioxide, while simultaneously converting NAD+ into carbon dioxide. + It is reduced to NADH. This enzymatic reaction is considered a highly promising tool in coenzyme regeneration systems due to its inexpensive substrates, mild reaction conditions, easy removal of the byproduct carbon dioxide, and its environmental friendliness. It is widely used in the synthesis of chiral chemicals, preparation of pharmaceutical intermediates, and other fields.

[0003] However, naturally derived NAD-dependent formate dehydrogenases (such as FDH from Candida botrytis cinerea) suffer from inherent defects in industrial applications, including generally low catalytic efficiency, insufficient stability, and poor tolerance to organic solvents, severely limiting their economic viability and practicality. Although protein engineering strategies have been used to improve the performance of FDH, for example, patent (CN202310432052.5) ​​significantly improved enzyme activity and catalytic efficiency by constructing an FDH mutant containing a combination of mutations at five sites (positions 18, 57, 70, 235, and 316), thereby converting 150 g / L of fructose into more than 140 g / L of D-mannitol within 15 hours. This example illustrates that obtaining high-performance mutants through deep molecular modification of FDH is key to overcoming yield bottlenecks. Alternatively, patent (CN202310432052.5) ​​develops a carrier-free co-immobilization technique for phenylalanine dehydrogenase and formate dehydrogenase. This technique, by forming cross-linked enzyme aggregates, improves the temperature stability, pH tolerance, and storage stability of the immobilized FDH, making it more suitable for large-scale continuous production and reducing purification difficulty. This example demonstrates that advanced immobilization technology is an effective way to overcome the stability limitations of FDH and improve its process economy.

[0004] In summary, although existing technologies have optimized FDH through molecular modification and immobilization, there remains an urgent need in the field to develop a novel or comprehensively improved NAD-dependent formate dehydrogenase. An ideal enzyme formulation should synergistically achieve high catalytic efficiency, excellent stability (thermal and operational stability), and potentially broader coenzyme adaptability to meet diverse industrial biocatalytic needs. Summary of the Invention

[0005] One of the objectives of this invention is to provide a formate dehydrogenase mutant.

[0006] This invention modifies wild-type formate dehydrogenase (AaFDH, CP021524.1, gene sequence shown in SEQ ID NO.2) from Acetobacter ascendens using techniques such as random mutation and semi-rational design to obtain a formate dehydrogenase mutant with enhanced catalytic activity and stability. Further optimization is then used to improve its catalytic activity and stability.

[0007] The second objective of this invention is to provide a gene encoding the aforementioned formate dehydrogenase mutant, as well as a recombinant plasmid and expression vector containing the gene.

[0008] A third objective of this invention is to provide recombinant cells transformed with the aforementioned recombinant plasmid.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned formate dehydrogenase mutants or recombinant cells in the preparation of non-natural amino acids.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows:

[0011] A formate dehydrogenase mutant is provided, wherein at least one of the following positions in the amino acid sequence of the wild-type formate dehydrogenase is mutated: tryptophan at position 128 is mutated to lysine (Trp128Lys), alanine at position 225 is mutated to valine (Ala225Val), tryptophan at position 595 is mutated to cysteine ​​(Trp595Cys), asparagine at position 750 is mutated to cysteine ​​(Asn750Cys), aspartic acid at position 759 is mutated to valine (Asp759Val), and serine at position 888 is mutated to glutamic acid (Ser888Glu). The amino acid sequence of the wild-type formate dehydrogenase is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant plasmid containing the gene of the formate dehydrogenase mutant described above, the plasmid containing a vector for expressing the gene of the formate dehydrogenase mutant described above, preferably a PET series vector, such as PET28a, but not limited thereto.

[0013] The present invention also provides a recombinant cell carrying the gene of the formate dehydrogenase mutant or carrying the recombinant plasmid described above. This recombinant cell can serve as a host for expressing the formate dehydrogenase mutant.

[0014] Preferably, the host bacteria of the recombinant cells are selected from one of Corynebacterium glutamicum, Bacillus, yeast, and Escherichia coli. In one embodiment of the present invention, the recombinant cells use Escherichia coli BL21(DE3) as the host bacteria.

[0015] This invention also provides the application of the formate dehydrogenase mutant or recombinant cells, wherein the application is to utilize the formate dehydrogenase mutant or recombinant cells to participate in NAD. + —The reaction of NADH provides NADH to the main enzyme reaction system or is used solely for the production of NADH.

[0016] This invention also provides the application of the above-mentioned formate dehydrogenase mutants or recombinant cells in the preparation of non-natural amino acids; the application method is as follows: adding the above-mentioned mutants or recombinant cells to a solution containing substrate amino acids or amino salts, amino acid dehydrogenase, formate, and NAD. + The reaction is carried out in the reaction system to obtain the product.

[0017] In one embodiment of the present invention, the reaction substrate includes, but is not limited to, glufosinate, tyramine, etc.

[0018] As an alternative implementation, the recombinant cells described above can be added to the reaction system in the form of wet bacterial cells or cell fragments thereof.

[0019] The beneficial effects of this invention compared to the prior art are as follows:

[0020] This invention is based on wild-type formate dehydrogenase (AaFDH, CP021524.1) derived from *Acetobacter ascendens*. Through random mutation and semi-rational molecular modification, a formate dehydrogenase mutant with significantly enhanced catalytic activity and stability was obtained. Applying this mutant to the synthesis of non-natural amino acids or amino salts, using inexpensive amino acids or amino salts as substrates, enables stable and efficient reactions with low cost and high yield, demonstrating significant industrial application and development potential. Attached Figure Description

[0021] Figure 1 This is a comparison of the relative enzyme activities of wild-type and mutant methyl dehydrogenase;

[0022] Figure 2 Is NADH in A 340 Absorbance standard curve at [location]. Detailed Implementation

[0023] The present invention will be further described below with reference to specific implementation examples, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following implementation examples are conventional methods or implemented under the conditions recommended in the manufacturer's instructions.

[0024] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0026] The formate dehydrogenase mutant constructed in this invention is derived from the wild-type formate dehydrogenase of *Acetobacter ascendens*, and its amino acid sequence is shown in SEQ ID NO.1. It is a new protein formed by substituting amino acids in the sequence of SEQ ID NO.1. The encoding gene for the wild-type formate dehydrogenase is shown in SEQ ID NO.2 of the sequence listing.

[0027] To obtain a formate dehydrogenase with high catalytic activity and stable properties, this invention uses the gene sequence of wild-type formate dehydrogenase (SEQ ID NO.2) as a basis and performs base mutations using error-prone PCR to obtain a formate dehydrogenase mutant.

[0028] The specific experimental procedure is as follows.

[0029] In the following experiments, the complete gene synthesis and primer synthesis were performed by BGI Genomics. The molecular biology experiments in the implementation examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, gene transformation, and preparation of culture media, were conducted according to the reaction conditions provided by the supplier or the instructions of the kit. Simple adjustments were made as necessary.

[0030] The materials and instruments used in the experiment are as follows:

[0031] Vectors and strains: The expression vector used was pET-28a, the plasmid was purchased from Novagen, and the host cell used was Escherichia coli BL21(DE3), purchased from TransGen Biotech Ltd.

[0032] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjusted to pH 7.0 with 1M sodium hydroxide, sterilized at 121°C for 20 min. (LB solid medium is prepared with 15 g / L agar powder added).

[0033] TB medium: tryptone 12 g / L, yeast extract 24 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.31 g / L, glycerol 5 g / L, pH 7.0-7.5, sterilized at 121℃ for 20 min. (TB solid medium is prepared with 15 g / L agar powder added).

[0034] Implementation Example 1: Construction of Recombinant Escherichia coli with Wild-Type Formate Dehydrogenase Gene

[0035] 1. For the formate dehydrogenase AaFDH derived from Acetobacter ascendens, based on the formate dehydrogenase gene sequence (ARW11429.1) published in GenBank: CP021524.1 (SEQ ID NO.2), the whole genome was synthesized by BGI, and restriction endonuclease sites BamHI and XhoI were introduced at both ends of the gene. The restriction fragment of formate dehydrogenase AaFDH was ligated with the restriction fragment of vector pET28a to obtain the recombinant plasmid pET28a-AaFDH.

[0036] 2. The plasmid pET28a-AaFDH was transformed into Escherichia coli BL21(DE3) competent cells by heat shock transformation to obtain recombinant Escherichia coli expressing wild-type formate dehydrogenase AaFDH.

[0037] Example 2: Error-prone PCR construction of a random mutant library of wild-type formate dehydrogenase AaFDH

[0038] 1. Using the wild-type formate dehydrogenase recombinant plasmid pET28a-AaFDH as a template, a random mutant library was constructed using error-prone PCR technology. The error-prone PCR kit was purchased from Takara Bio Inc. (Clontech, PT33128-1); the single-point mutation kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Novozymes, C214).

[0039] 2. Using the Takara Bio Error-Prone PCR Kit (Clontech, PT33128-1), error-prone PCR amplification was performed with plasmid pET28a-AaFDH as template and primers AaFDH-F and AaFDH-R.

[0040] The primer sequences used are as follows:

[0041] AaFDH-F (SEQ ID NO.3): acagcaaatgggtcgcggatccTACCGCGACCCGGCGGCGCTTA, AaFDH-R (SEQ ID NO.4): gtggtggtggtggtggtgc tcgagCACTTTTTCCACGTT CACCAGA; where lowercase letters are homologous arm sequences.

[0042] The PCR reaction system consisted of 50 μL: 1 μL template DNA (final concentration approximately 1 ng / μL), 1 μL each of forward primer (10 nM) and reverse primer (10 nM), 1 μL 50× Diversify dNTP Mix, 1 μL dGTP (2 mM), 2 μL MnSO4 (8 mM), 5 μL 10× TITANIUM Taq Buffer, 1 μL TITANIUM Taq Polym, and 37 μL ddH2O.

[0043] The error-prone PCR program was as follows: pre-denaturation at 225℃ for 30 seconds, followed by 25 cycles of the following program: 225℃, 30 seconds, 68℃, 3 minutes. The reaction was terminated with a final extension at 68℃ for 1 minute. Result: Amplified band of approximately 3051 bp was successfully obtained.

[0044] The amplified band and the pET28a double-digested (BamHI and XhoI) fragment of the vector were recovered using a DNA gel recovery kit. Homologous recombination was performed using a Novizan single mutant kit. The reaction system was directly heat-shocked to transform E. coli BL21(DE3) competent cells to obtain a formate dehydrogenase mutant library.

[0045] Construction of recombinant Escherichia coli: Escherichia coli BL21(DE3) competent cells were thawed on ice, and then 20 μL of the above fusion reaction system was added. After gentle mixing, the cells were placed on ice for 30 min, heat-shocked in a 42℃ water bath for 30 sec, and immediately placed on ice for 2 min. 800 μL of liquid LB medium was added, and the cells were cultured at 37℃ and 180 rpm for 1 h. The culture was then spread on LB solid medium plates containing 50 mg / L kanamycin and incubated upside down at 37℃ for 14-16 h to obtain engineered bacteria containing formate dehydrogenase mutants with recombinant plasmids.

[0046] Example 3 High-throughput screening of mutant libraries

[0047] 1. Preparation of wet bacterial cells from crude enzyme solution of mutant strains

[0048] Single clones of wild-type formate dehydrogenase recombinant *E. coli* and mutant recombinant *E. coli* obtained in Examples 1-2 were picked and placed into sterile 128-well plates. 1 ml of LB broth containing 50 mg / L kanamycin was added to each well, and the plates were incubated at 37°C and 180 rpm for 8 hours. Then, 500 μL of the bacterial culture was transferred at a 1:1 ratio to another 128-well plate containing 500 μL of LB broth containing 50 mg / L kanamycin and a final concentration of 0.2 M IPTG in each well. The plates were incubated overnight at 20°C and 180 rpm for 16 hours. After incubation, the cells were collected in 128-well plates by centrifugation at 8000 rpm for 5 minutes at room temperature, yielding recombinant *E. coli* wet cells containing wild-type and a large number of mutant genes, i.e., wild-type and mutant wet cells.

[0049] The whole cells obtained from the wild-type formate dehydrogenase engineered bacteria and its mutants constructed and cultured by the above method were used as catalysts.

[0050] 2. Preliminary screening

[0051] Detection of formate dehydrogenase activity:

[0052] Formate dehydrogenase (NADH) can be measured by observing the change in the absorption peak of NADH at 340 nm. The specific steps are as follows: Take phosphate buffer (10 mmol / L pH 8.0, containing 0.1 mmol / L β-mercaptoethanol), 2 mmol NAD... + 1 ml of a 2 mmol sodium formate reaction solution was mixed with 1 ml of whole-cell bacterial culture and reacted at 30 °C for 5 min. The change in the absorption peak of the product NADH at 340 nm was detected using a spectrophotometer. Following the above enzyme activity detection method, the absorbance change of NADH for each monoclonal strain in the formate dehydrogenase mutant library was measured, and mutant recombinant strains with absorbance changes of NADH greater than those of wild-type formate dehydrogenase AaFDH were screened.

[0053] 3. Secondary screening

[0054] The wet cells of the strains obtained in the initial screening were used as catalysts for secondary screening. The secondary screening method was the same as the initial screening method, except for the increase in the reaction volume. The change in absorbance of NADH produced by the wild-type formate dehydrogenase AaFDH recombinant strain was defined as 100% relative enzyme activity.

[0055] Through the above steps, approximately 2000 mutant strains were screened, and 11 dominant mutant strains with significantly enhanced catalytic activity were obtained, named AaFDHm-1, AaFDHm-2, AaFDHm-3, AaFDHm-4, AaFDHm-5, AaFDHm-6, AaFDHm-7, AaFDHm-8, AaFDHm-9, AaFDHm-10, and AaFDHm-11, respectively. The results are as follows... Figure 1 As shown.

[0056] 4. Nucleotide sequence determination of formate dehydrogenase mutant

[0057] The formate dehydrogenase mutants AaFDHm-1, AaFDHm-2, AaFDHm-3, AaFDHm-4, AaFDHm-5, AaFDHm-6, AaFDHm-7, AaFDHm-8, AaFDHm-9, AaFDHm-10, and AaFDHm-11 obtained from the above screening were inoculated into 3 ml of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 180 rpm for 14-16 h in a shaker. The bacterial cells were collected by centrifugation at 12000 rpm for 1 min at room temperature. Plasmid extraction was performed according to the instructions of the plasmid extraction kit. This experiment used the plasmid purification kit (AG21001) from Aikerui Biotechnology, but it is not limited to this company's plasmid purification kit; any commercial plasmid extraction kit can be used. The extracted plasmids were sent to a sequencing company for sequencing.

[0058] Sequencing results showed that the amino acid sequences of the formate dehydrogenase mutants AaFDHm-1, AaFDHm-2, AaFDHm-3, AaFDHm-4, AaFDHm-5, AaFDHm-6, AaFDHm-7, AaFDHm-8, AaFDHm-9, AaFDHm-10, and AaFDHm-11 in this embodiment were mutated at different positions. The mutation sites of AaFDHm-1 and AaFDHm-6 were identical, with tryptophan at position 128 being mutated to lysine. The mutation sites of AaFDHm-3 and AaFDHm-10 are the same, with alanine at position 225 being mutated to valine. The mutation site of AaFDHm-2 is tryptophan at position 595 being cysteine. The mutation sites of AaFDHm-4, AaFDHm-8, and AaFDHm-11 are the same, with asparagine at position 750 being mutated to cysteine. The mutation site of AaFDHm-7 is aspartic acid at position 759 being mutated to valine. The mutation sites of AaFDHm-5 and AaFDHm-9 are the same, with serine at position 888 being mutated to glutamic acid. That is, BL21(DE3) / PET28a-AaFDHmW128K, BL21(DE3) / PET28a-AaFDHmA225V, BL21(DE3) / PET28a-AaFDHmW595C, BL21(DE3) / PET28a-AaFDHmN750C, BL21(DE3) / PET28a-AaFDHmD759V, and BL21(DE3) / PET28a-AaFDHmS888E are obtained.

[0059] Implementation Example 4: Fusion of Formate Dehydrogenase Multisite Mutants and Construction of Engineered Bacteria

[0060] Because formate dehydrogenase mutants AaFDHm-5, AaFDHm-9, and AaFDHmS888E catalyze NAD... + The highest activity of synthesized NADH was found. Therefore, multi-site mutations were further constructed based on AaFDHm-5, AaFDHm-9, and AaFDHmS888E.

[0061] Formate dehydrogenase multisite site-directed mutagenesis was performed using a Novizan single-point mutagenesis kit (Novozymes, C214), and the primer design is shown in Table 2.

[0062] Table 2. Primers for site-directed mutagenesis of formate dehydrogenase

[0063] ;

[0064] Using the vector PET28a-AaFDHmS888E as a template and W128K-F / W128K-R in the table as primers, site-directed mutagenesis was performed to mutate tryptophan at position 128 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E to lysine, thus obtaining PET28a-AaFDHmS888E-W128K.

[0065] Using the vector PET28a-AaFDHmS888E as a template and A225V-F / A225V-R in the table as primers, site-directed mutagenesis was performed to mutate alanine at position 225 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E to valine, thus obtaining PET28a-AaFDHmS888E-A225V.

[0066] Using the vector PET28a-AaFDHmS888E as a template and W595C-F / W595C-R in the table as primers, site-directed mutagenesis was performed to mutate tryptophan at position 595 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E to cysteine, thus obtaining PET28a-AaFDHmS888E-W595C.

[0067] Using the vector PET28a-AaFDHmS888E as a template and N750C-F / N750C-R in the table as primers, site-directed mutagenesis was performed to mutate asparagine at position 750 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E to cysteine, thus obtaining PET28a-AaFDHmS888E-N750C.

[0068] Using the vector PET28a-AaFDHmS888E as a template and D759V-F / D759V-R in the table as primers, site-directed mutagenesis was performed to mutate the aspartic acid at position 759 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E to valine, thus obtaining PET28a-AaFDHmS888E-D759V.

[0069] Using the vector PET28a-AaFDHmS888E-W128K as a template and A225V-F / A225V-R in the table as primers, site-directed mutagenesis was performed to mutate alanine at position 225 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K to valine, thus obtaining PET28a-AaFDHmS888E-W128K-A225V.

[0070] Using the vector PET28a-AaFDHmS888E-W128K as a template and W595C-F / W595C-R in the table as primers, site-directed mutagenesis was performed to mutate tryptophan at position 595 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K to cysteine, thus obtaining PET28a-AaFDHmS888E-W128K-W595C.

[0071] Using the vector PET28a-AaFDHmS888E-W128K as a template and N750C-F / N750C-R in the table as primers, site-directed mutagenesis was performed to mutate asparagine at position 750 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K to cysteine, thus obtaining PET28a-AaFDHmS888E-W128K-N750C.

[0072] Using the vector PET28a-AaFDHmS888E-W128K as a template and D759V-F / D759V-R in the table as primers, site-directed mutagenesis was performed to mutate the aspartic acid at position 759 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K to valine, thus obtaining PET28a-AaFDHmS888E-W128K-D759V.

[0073] Using the vector PET28a-AaFDHmS888E-W128K-A225V as a template and W595C-F / W595C-R in the table as primers, site-directed mutagenesis was performed to mutate tryptophan at position 595 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V to cysteine, resulting in PET28a-AaFDHmS888E-W128K-A225V-W595C.

[0074] Using the vector PET28a-AaFDHmS888E-W128K-A225V as a template and N750C-F / N750C-R in the table as primers, site-directed mutagenesis was performed to mutate the asparagine at position 750 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V to cysteine, thus obtaining PET28a-AaFDHmS888E-W128K-A225V-N750C.

[0075] Using the vector PET28a-AaFDHmS888E-W128K-A225V as a template and D759V-F / D759V-R in the table as primers, site-directed mutagenesis was performed to mutate the aspartic acid at position 759 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V to valine, thus obtaining PET28a-AaFDHmS888E-W128K-A225V-D759V.

[0076] Using the vector PET28a-AaFDHmS888E-W128K-A225V-W595C as a template and N750C-F / N750C-R in the table as primers, site-directed mutagenesis was performed to mutate the asparagine at position 750 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V-W595C to cysteine, thus obtaining PET28a-AaFDHmS888E-W128K-A225V-W595C-N750C.

[0077] Using the vector PET28a-AaFDHmS888E-W128K-A225V-W595C as a template and D759V-F / D759V-R in the table as primers, site-directed mutagenesis was performed to mutate the aspartic acid at position 759 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V-W595C to valine, resulting in PET28a-AaFDHmS888E-W128K-A225V-W595C-N759C.

[0078] Using the vector PET28a-AaFDHmS888E-W128K-A225V-W595C-N750C as a template and D759V-F / D759V-R in the table as primers, site-directed mutagenesis was performed to mutate the aspartic acid at position 759 of the amino acid sequence of the formate dehydrogenase mutant AaFDHmS888E-W128K-A225V-W595C-N750C to valine, resulting in PET28a-AaFDHmS888E-W128K-A225V-W595C-N750C-N759C.

[0079] The site-directed mutagenesis PCR system (50 μL) consisted of: 25 μL 2×Max buffer, 1 μL dNTP, template plasmid with a final concentration of 1 ng / μL, 2 μL each of forward and reverse primers (10 mM each), 1 μL Phanta Max Super-Fidelity DNA polymerase, and finally ddH2O to bring the total volume to 50 μL.

[0080] PCR reaction program: 95℃ pre-denaturation for 30 sec; 30 cycles, 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 6 min; final extension at 72℃ for 5 min.

[0081] Add 1 μL of the amplified fragment directly. Dpn I. After digestion at 37℃ for 1 hour, agarose gel electrophoresis was performed. The fragments were recovered and subjected to recombination. The reaction mixture consisted of 50-400 ng of the above-mentioned... Dpn Digestion product I, 4 μL 5×CE II Buffer, 2 μL LExnase II, and ddH2O to a final volume of 20 μL. Mix well and incubate at 37°C for 30 min, then immediately cool on ice for 10 min. Transform E. coli BL21(DE3) competent cells using the heat shock method. Spread on LB agar plates containing 50 mg / L kanamycin and incubate upside down at 37°C for 14–16 h. Recombinant bacteria containing multi-site mutations are obtained.

[0082] The recombinant bacteria that were verified to be correct by sequencing were preserved and used to further test their activity in catalyzing the synthesis of NADH.

[0083] Example 5 Determination of enzyme activity and kinetic parameters of wild-type formate dehydrogenase and its mutants

[0084] 1. Purification of the target protein

[0085] The dominant formate dehydrogenase mutant and wild-type formate dehydrogenase AaFDH obtained in Example 4 were used to obtain wet bacterial cells according to step 1 of Example 3. The cells were washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mM pH 8.0 phosphate buffer containing 0.3 M NaCl and 30 mM imidazole. The cells were sonicated for 10 min, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The protein was purified using a His-tagged purification column (Roche, COHISC-RO, 6781535001).

[0086] Formate dehydrogenase mutants PET28a-AaFDHmS888E, PET28a-AaFDHmS888E-W128K, PET28a-AaFDHmS888E-A225V, PET28a-AaFDHmS888E-W595C, PET28a-AaFDHmS888E-N750C, and PET28a-AaFDHmS888E-D759V were obtained. PET28a-AaFDHmS888E-W128K-A225V, PET28a-AaFDHmS888E-W128K-W595C, PET28a-AaFDHmS8 88E-W128K-N750C, PET28a-AaFDHmS888E-W128K-D759V, PET28a-AaFDHmS888E-W128K-A225V -W595C, PET28a-AaFDHmS888E-W128K-A225V-N750C, PET28a-AaFDHmS888E-W128K-A225V-D7 59V, PET28a-AaFDHmS888E-W128K-A225V-W595C-N750C, PET28a-AaFDHmS888E-W128K-A225V The concentrations of purified enzyme solutions of formate dehydrogenase pET28a-AaFDH (W595C-D759V, PET28a-AaFDHmS888E-W128K-A225V-W595C-N750C-D759V, and wild-type formate dehydrogenase pET28a-AaFDH) were determined using the modified Bradford protein concentration assay kit from Sangon Biotech (Sangon Biotech, C503041). Specific methods can be found on the Sangon Biotech website. All proteins were diluted to the same concentration using 50 mM pH 8.0 phosphate buffer.

[0087] 2. Enzyme activity assay:

[0088] The catalytic system consisted of: 10 mM pH 8.0 phosphate buffer (containing 0.1 mmol / L β-mercaptoethanol), 2 mM NAD+, and 2 mM NAD+. + 2mM sodium formate was added, and the reaction was carried out at 30°C for 5 min. The product NADH was detected by spectrophotometer. 340 The absorbance was used to calculate the NADH yield based on the standard curve.

[0089] Standard curve: Prepare NADH standard solutions with different concentration gradients, and determine the concentrations of each solution at different concentrations within the range of A. 340 The absorbance was used to construct a standard curve, and the resulting standard curve is shown below. Figure 2 As shown.

[0090] Enzyme activity (U) definition: The amount of enzyme required to generate 1 uM NADH per minute is defined as one unit of enzyme activity.

[0091] Enzyme activity: The number of enzyme activity units per milligram of enzyme protein. The results are shown in Table 3.

[0092] Table 3. Specific enzyme activities of formate dehydrogenase and its mutants

[0093] .

[0094] 3. Analyze the kinetic parameters of formate dehydrogenase and its dominant mutants.

[0095] Using the purified wild-type formate dehydrogenase and its mutants described in section 1 above, kinetic analysis was performed at a fixed concentration of sodium formate (2 mM) at 30°C and pH 8.0. Measurements were taken with different substrates (NAD... + Enzyme activity was measured at concentrations of 2-20 mM. Based on the obtained data, a curve was plotted showing the relationship between the maximum reaction rate (Vmax) and substrate concentration, and the Km value was obtained using the Michaelis-Menten equation. The Kcat value was calculated using Vmax and enzyme concentration. The results are shown in Table 4 below.

[0096] Table 4 Comparison of kinetic parameters of formate dehydrogenase and its mutants

[0097] ;

[0098] Comparison of the Km value of the mutant with that of the wild type reveals that the amino acid substitutions in the formate dehydrogenase mutant significantly increase the enzyme's interaction with the substrate NAD. + Its affinity for NAD+, compared to wild-type formate dehydrogenase, catalyzes NAD+. + Their abilities have been greatly improved.

[0099] Example 6: Determination of the thermal stability of enzymes

[0100] Determination of optimal reaction temperature: The purified mutant enzyme solutions AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, and AaFDHmS888E-W128K were placed in the same assay system and reacted at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃) to measure their enzyme activity. The highest enzyme activity was taken as 100%. The results are shown in Table 4.

[0101] Table 4. Optimal catalytic temperature for mutants

[0102] ;

[0103] As shown in Table 4, the optimal reaction temperature for the formate dehydrogenase mutant is 40℃.

[0104] Thermal stability assay: Purified wild-type formate dehydrogenase AaFDH and formate dehydrogenase mutants AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, and AaFDHmS888E-W128K were incubated at 40℃, 50℃, 60℃, and 70℃ for 6 h, respectively, and then reacted at 40℃ for 30 min to determine their relative enzyme activity. The untreated enzyme activity was taken as 100%. The results are shown in Table 5.

[0105] Table 5. Relative enzyme activities of formate dehydrogenase and its mutants after treatment at different temperatures.

[0106] ;

[0107] As shown in Table 5, the relative enzyme activity of the formate dehydrogenase mutants gradually decreased with increasing incubation temperature. However, the relative enzyme activities of mutants AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, and AaFDHmS888E-W128K were all higher than those of wild-type formate dehydrogenase AaFDH under the same treatment conditions. This demonstrates that the thermostability of the formate dehydrogenase mutants is higher than that of the wild-type formate dehydrogenase, with mutant AaFDHmS888E-W128K-N750C exhibiting the best thermostability.

[0108] Example 7 Fermentation and Application of Formate Dehydrogenase Mutant

[0109] 1. Select strains containing recombinant plasmids AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, AaFDHmS888E-W128K, AaFDHmS888E-W128K-A225V, and AaFDHmS888E-W128K-A225V-W595C-N750C-D759V, and inoculate them into 3 mL of LB liquid medium containing 50 mg / L kanamycin. Incubate overnight at 37°C and 180 rpm. Then, inoculate 1% (V / V) of the mixture into 150 mL of TB medium and incubate at 30°C and 180 rpm until the OD600 reaches 0.4-0.6. Add 0.2 mM kanamycin. IPTG was used to induce culture for another 6 hours, followed by centrifugation at 10,000 rpm for 10 minutes to collect the cells, which were then stored at -80°C.

[0110] 2. Following the method in step 1 of Example 7, the wild-type formate dehydrogenase AaFDH strain was subjected to shake-flask fermentation, the bacterial cells were collected, and stored in a -80°C refrigerator.

[0111] 3. Add the wet bacterial cells obtained in steps 1 and 2 above to a final concentration of 10 g / L in a 50 mL catalytic reaction system. Substrate: 10 mM pH 8.0 phosphate buffer (containing 0.1 mmol / L β-mercaptoethanol), 2 mM NAD. + , 2mM sodium formate.

[0112] Specifically, the reaction catalyst and substrate were added to a 500ml shake flask, making the reaction volume 50ml. The reaction was carried out at 200rpm / min, with the temperature controlled at 40℃, for 30min. Samples were taken periodically, and the supernatant was collected by centrifugation at 10000rpm. The concentration of NADH in A was then detected using a spectrophotometer. 340 The absorbance was used to calculate the NADH yield based on the standard curve. The results are shown in Table 6.

[0113] Table 6. Synthesis of NADH by formate dehydrogenase and its mutants

[0114] ;

[0115] As shown in Table 6, under the same reaction conditions, the mutant AaFDHmS888E-W128K-N750C produced the highest NADH yield, with a substrate molar conversion rate of 98.5%, which is approximately 11 times that of the wild-type formate dehydrogenase AaFDH. The above examples demonstrate that the mutant AaFDHmS888E-W128K-N750C has advantages in application due to its high catalytic activity and good enzyme activity stability.

[0116] Example 8: Application of formate dehydrogenase mutants in the preparation of non-natural amino acids or amino salts.

[0117] 1. Wild-type formate dehydrogenase strain AaFDH stored at -80℃, strains containing recombinant plasmids AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, and AaFDHmS888E-W128K were selected and combined with glutamate dehydrogenase to catalyze the reaction using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) as a substrate.

[0118] Specifically, the collected bacterial cells were added to 50 mL of the catalytic reaction system at a final concentration of 10 g / L. The substrates were: 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) 250 mM, ammonium formate 0.5 M, and NAD. + 0.15mM, glutamate dehydrogenase 10g / L.

[0119] The catalyst and substrate were added to a 500 ml shake flask, making a total reaction volume of 50 ml. The reaction was carried out at 200 rpm / min, with formic acid used to maintain the pH at 8.0-8.2 and the temperature at 30-40℃. The reaction was allowed to proceed for 10-30 min, with samples taken periodically. The supernatant was collected by centrifugation at 6000 rpm. After processing the supernatant using OPA derivatization, the L-glufosinate content was determined by HPLC. The results are shown in Table 7.

[0120] Table 7. Synthesis of L-glufosinate by formate dehydrogenase and its mutants

[0121] ;

[0122] "—" indicates that no product was detected.

[0123] 2. Wild-type formate dehydrogenase strain AaFDH, stored at -80℃, and strains containing recombinant plasmids AaFDHmS888E-W128K-N750C, AaFDHmS888E-N750C, and AaFDHmS888E-W128K, were combined with amine oxidase to catalyze the reaction using tyramine as a substrate.

[0124] Specifically, the collected bacterial cells were added to 50 mL of the catalytic reaction system at a final concentration of 10 g / L. The substrates were: tyramine 90 mM, ammonium formate 225 mM, and NAD+. + 0.15mM, catalase 0.5ml / L, amine oxidase 10g / L.

[0125] The reaction catalyst and substrate were added to a 500 ml shake flask, the reaction volume was 50 ml, the rotation speed was 200 rpm / min, the pH was controlled at 6.9-7.1 with formic acid, the temperature was controlled at 30-40℃, the reaction was carried out for 10-30 min, samples were taken at regular intervals, the supernatant was collected by centrifugation at 6000 rpm, and the content of tyrosol (p-hydroxyphenylethanol) was detected by HPLC.

[0126] Specifically, the HPLC detection method is as follows: mobile phase: 30% methanol + 70% Wahaha purified water + 0.1% glacial acetic acid; 210 nm, 1 ml / min.

[0127] The results are shown in Table 8.

[0128] Table 8. Results of catalytic reactions using tyramine as a substrate in wild-type strains and mutants.

[0129] ;

[0130] "—" indicates that no product was detected.

[0131] A novel formate dehydrogenase mutant was obtained through mutation screening at different amino acid sites in wild-type formate dehydrogenase, which exhibits improved resistance to the substrate NAD. + The affinity for formate dehydrogenase is significantly enhanced, its catalytic activity is greatly improved, and its thermal stability is significantly enhanced. This modification expands the application range of formate dehydrogenase in the synthesis or detection of amino acids and other related products, and increases its industrial application value.

[0132] Wild-type formate dehydrogenase amino acid sequence (SEQ ID NO.1):

[0133]

[0134] Wild-type formate dehydrogenase nucleotide sequence (SEQ ID NO.2):

[0135]

Claims

1. A mutant formate dehydrogenase enzyme, characterized in that, The mutant is formed by mutations at the following positions in the amino acid sequence of the wild-type formate dehydrogenase: tryptophan at position 128 is mutated to lysine, asparagine at position 750 is mutated to cysteine, and serine at position 888 is mutated to glutamic acid. The amino acid sequence of the wild-type formate dehydrogenase is shown in SEQ ID NO.1, and the mutant is S888E-W128K-N750C.

2. The formate dehydrogenase mutant of claim 1, wherein, The nucleotide sequence of the wild-type formate dehydrogenase gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid comprising a gene encoding the formate dehydrogenase mutant of claim 1.

4. A recombinant cell, characterized in that, The recombinant cell carries the recombinant plasmid as described in claim 3.

5. The recombinant cell of claim 4, wherein The recombinant cells use Corynebacterium glutamicum, Bacillus, Escherichia coli or Pichia pastoris as host cells.

6. Use of the formate dehydrogenase mutant according to claim 1 for the preparation of NADH, characterized in that, The substrate in the application is a phosphate buffer containing sodium formate, NAD + The product is NADH.

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