Mutant of glutamate dehydrogenase from saccharomyces cerevisiae and application of mutant in synthesis of L-glufosinate-ammonium

Abstract

The invention discloses a mutant of glutamate dehydrogenase from saccharomyces cerevisiae and application of the mutant in synthesis of L-glufosinate-ammonium. According to the invention, single-site or multi-site mutation is carried out on wild-type glutamate dehydrogenase from saccharomyces cerevisiae as shown in SEQ ID NO.1 through a mutation transformation method for carrying out rational design analysis on amino acid residues in a binding pocket of glutamate dehydrogenase and a substrate and a geometrical shape in a molecular tunnel, and compared with the wild-type glutamate dehydrogenase, the single-site or multi-site mutation is carried out on the single-site or multi-site mutation of the wild-type glutamate dehydrogenase from saccharomyces cerevisiae as shown in SEQ ID NO.1; the enzyme activity of the obtained single-site or multi-site mutant is remarkably improved, and the L-glufosinate-ammonium acid prepared by applying the glutamate dehydrogenase mutant to biological catalysis can remarkably improve the feeding concentration and conversion rate of a substrate on the basis of high stereoselectivity, high conversion rate and high yield; the problems that existing glutamate dehydrogenase shows no activity or low activity on 2-carbonyl-4-(hydroxymethylphosphonyl) butyric acid in biological catalysis and the like are effectively solved.

Description

Technical Field

[0001] The present invention relates to an enzyme mutant and its application, in particular to an enzyme mutant derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and its application in L-phosphinothricin synthesis, belonging to the field of glutamate dehydrogenase mutants and their applications. Background Art

[0002] Glutamate dehydrogenase (EC 1.4.1.2–1.4.1.4) is abundant and diverse in source. It offers significant advantages in the preparation of chiral L-amino acids, including strict stereoselectivity, high yield, and ease of separation, and holds great promise for future applications. Therefore, utilizing glutamate dehydrogenase to biocatalyze the asymmetric amination of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to prepare L-phosphinothricin is of great significance in the development of a process for the preparation of L-glufosinate.

[0003] Chinese patent CN107630052A will be derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae Whole cells of an engineered bacterium co-expressing the glutamate dehydrogenase and formate dehydrogenase genes of the invention catalyze the conversion of 30 g / L of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to L-glufosinate in the presence of apple extract powder and spirulina powder as additives, achieving a maximum conversion rate of 95.8% and a conversion time of 16.8 hours. In the absence of additives, the conversion rate of 30 g / L of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to L-glufosinate was 82.3% and a conversion time of 26.1 hours. From the perspective of industrial application, the catalytic activity of the glutamate dehydrogenase in this patent is relatively low, which can lead to a series of problems in production, such as high enzyme catalyst preparation costs and heavy post-processing burdens for the conversion solution. Furthermore, the addition of apple extract powder and spirulina powder not only increases production costs but also makes subsequent product purification more difficult.

[0004] Therefore, the key to reducing the production cost of L-phosphinothricin is to mutate glutamate dehydrogenase through enzyme engineering technology and genetic engineering technology to improve the catalytic activity of glutamate dehydrogenase towards 2-carbonyl-4-(hydroxymethylphosphono)butyric acid. Summary of the Invention

[0005] One of the objects of the present invention is to provide Saccharomyces cerevisiaeA mutant of a wild-type glutamate dehydrogenase ScGluDH derived from a plant, wherein the enzyme activity of the mutant is significantly improved compared to the wild-type glutamate dehydrogenase ScGluDH. When the mutant is used as a catalyst to catalyze the biotransformation of 2-carbonyl-4-(hydroxymethylphosphono)butyrate as a substrate, the substrate conversion rate and L-phosphinothricin yield are significantly improved compared to the wild-type glutamate dehydrogenase ScGluDH. The second object of the present invention is to provide a gene encoding a mutant of the wild-type glutamate dehydrogenase ScGluDH; The third object of the present invention is to provide an expression cassette or chimeric gene containing the gene encoding the mutant of the wild-type glutamate dehydrogenase ScGluDH, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector; A fourth object of the present invention is to apply the mutant of the wild-type glutamate dehydrogenase ScGluDH, its encoding gene, an expression cassette or chimeric gene containing the encoding gene of the mutant, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector to the synthesis or biotransformation of L-phosphinothricin.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions: One aspect of the present invention is to provide Saccharomyces cerevisiae A mutant of wild-type glutamate dehydrogenase ScGluDH derived from Saccharomyces cerevisiae The amino acid sequence of the wild-type glutamate dehydrogenase ScGluDH from the source is shown in SEQ ID NO.1; the mutant is a single-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to any one of single-site mutations of A148G, V378P, V378G or V378A; or a multi-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to any one of multiple-site mutations of A148G / V378A, A148G / V272R / L375A / V378A or A148G / I151L / V272R / L375A / V378A / A379L, wherein the English abbreviations of amino acids have the following meanings: A represents alanine, G represents glycine, V represents valine, P represents proline, R represents arginine, L represents leucine, and I represents isoleucine.

[0007] The single-site mutant "A148G" in the present invention means that the amino acid sequence is as shown in SEQ ID NO. Saccharomyces cerevisiae The 148th amino acid of the wild-type glutamate dehydrogenase ScGluDH derived from the invention is mutated from alanine (Ala, A) to glycine (Gly, G); the description of the remaining single-site mutants of the present invention is similar.

[0008] The multi-site mutant "A148G / V378A" in the present invention refers to the amino acid sequence shown in SEQ ID NO. Saccharomyces cerevisiae The 148th amino acid of the wild-type glutamate dehydrogenase ScGluDH of the source is mutated from alanine (Ala, A) to glycine (Gly, G), and the 378th valine (Val, V) is mutated to alanine (Ala, A); the description of the remaining multi-site mutants of the present invention is similar.

[0009] In order to solve the problem of low catalytic activity of glutamate dehydrogenase to 2-carbonyl-4-(hydroxymethylphosphono)butyric acid, the present invention first targets Saccharomyces cerevisiae The three-dimensional structure of the wild-type glutamate dehydrogenase (ScGluDH, SEQID NO. 1) protein was analyzed. Molecular simulation calculations were performed with 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as the substrate to obtain a reasonable three-dimensional structure of the complex between the two. Based on the three-dimensional structure of the complex, key residues that form important interactions with the substrate and important residues that constitute the active pocket cavity structure were found in the enzyme catalytic center of the wild-type ScGluDH protein. Secondly, in addition to the modification of the active pocket, the inventors speculated that the channel for substrate entry and exit of the active pocket may also have a significant impact on the catalytic activity and substrate selectivity of the enzyme. Therefore, focusing on the channel for substrate entry and exit of the active pocket, the inventors applied molecular simulation methods to explore the morphology of the channel for substrate entry and exit of the active pocket in the wild-type enzyme structure. The morphological characteristics of the channel were quantitatively described using indicators such as average diameter, bottleneck diameter, and length, and the key sites of the channel were determined, thereby screening out mutants that can improve substrate permeability.

[0010] Another aspect of the present invention is to provide Saccharomyces cerevisiae The gene encoding the mutant of wild-type glutamate dehydrogenase ScGluDH is derived from Glutamate.

[0011] Another aspect of the present invention is to provide an expression cassette, a chimeric gene, a recombinant expression vector containing the gene encoding the mutant, or a recombinant host cell containing the recombinant expression vector; wherein the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0012] The present invention further provides a method for preparing any of the mutants, comprising: (1) operably connecting the coding gene of the mutant to an expression regulatory element to construct a recombinant expression vector; (2) The recombinant expression vector is transformed into host cells, the host cells are cultured, the recombinant protein is induced to express, and then purified.

[0013] In a preferred embodiment, the expression control element is selected from one or more of a promoter, a terminator, an enhancer, a transposon, a leader sequence or a marker gene.

[0014] Another aspect of the present invention is to Saccharomyces cerevisiae A mutant of a wild-type glutamate dehydrogenase ScGluDH from a source is used for the biosynthesis of L-phosphinothricin, comprising: using the single-site mutant or the multi-site mutant as a catalytic enzyme and 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate to carry out an enzymatic reaction to obtain L-phosphinothricin.

[0015] In a preferred embodiment of the present invention, the reaction temperature of the enzymatic reaction is controlled at 28-38°C; preferably, the reaction temperature of the enzymatic reaction is controlled at 35°C; and the pH value of the enzymatic reaction process is controlled at 6.5-7.5.

[0016] In a preferred embodiment of the present invention, in order to achieve a better catalytic effect, formate dehydrogenase can be added to the enzymatic reaction, and the formate dehydrogenase and the mutant of glutamate dehydrogenase ScGluDH can be used as catalysts for the catalytic reaction.

[0017] Compared with the prior art, the main beneficial effects of the present invention include: 1. The present invention is based on the rational design of the active pocket and channel of glutamate dehydrogenase, the rational design strategy of enzyme based on protein and substrate structure, and the molecular simulation calculation method of the enzyme derived from Saccharomyces cerevisiae The active pocket and channel wall of glutamate dehydrogenase were mutated to obtain various mutants with significantly improved enzyme activity. These mutants were used as catalysts to significantly improve the substrate conversion rate and the production concentration of L-phosphinothricin ammonium using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as the substrate, effectively solving the problem of glutamate dehydrogenase showing inactivity or low activity in biocatalysis using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as the substrate.

[0018] 2. Compared with the wild-type glutamate dehydrogenase, the catalytic activity of the mutant of the glutamate dehydrogenase obtained in the present invention for the substrate 2-carbonyl-4-(hydroxymethylphosphono)butyric acid is increased by up to 256 times. When the substrate feed concentration is 180 g / L, the substrate conversion rate can reach up to 100% after 10 hours of conversion, and the maximum concentration of L-glufosinate generated can reach 177.3 g / L, with an ee value of >99.9%, indicating that the mutant of the glutamate dehydrogenase provided by the present invention has good industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1It is the active pocket binding site residue of glutamate dehydrogenase shown in SEQ ID NO.1. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications or replacements fall within the scope of protection of the present invention.

[0021] Definitions of terms used in this invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0022] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphamidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions ( Mol Cell. Probes 8:91-98 (1994)).

[0023] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide equally applies to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0024] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally understood by those skilled in the art.

[0025] The term "recombinant host cell strain" or "host cell" refers to a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0026] The term "operably linked" refers to a functional connection between two or more elements. Operably linked elements may be contiguous or non-contiguous.

[0027] Example 1 Construction and screening of glutamate dehydrogenase mutants In the present invention, the molecular simulation calculation method is used based on the substrate binding site characteristics and channel characteristics to Saccharomyces cerevisiae The binding pocket and molecular tunnel of the glutamate dehydrogenase ScGluDH from the source were analyzed and scanned. Figure 1 ) showed that the binding pocket of ScGluDH has certain spatial restrictions when binding to the substrate, which may lead to reduced substrate binding efficiency; tunnel scanning results showed that some channels may be narrow or blocked, which may limit the effective entry of the substrate and the smooth release of the product. To this end, a series of mutants were designed to Saccharomyces cerevisiae A series of mutants were obtained by rationally designing and analyzing the amino acid residues in the substrate binding pocket and the geometric shape of the molecular tunnel of the wild-type glutamate dehydrogenase ScGluDH, among which, Saccharomyces cerevisiae The amino acid sequence of the wild-type glutamate dehydrogenase ScGluDH from the source is shown in SEQ ID NO. 1: 1 MSEPEFQQAY EEVVSSLEDS TLFEQHPEYR KVLPIVSVPE RIIQFRVTWE NDKGEQEVAQ 61 GYRVQYNSAK GPYKGGLRFH PSVNLSILKF LGFEQIFKNS LTGLDMGGGK GGLCVDLKGR 121 SNNEIRRICY AFMRELSRHI GQDTDVPAGD IGVGGREIGY LFGAYRSYKN SWEGVLTGKG 181 LNWGGSLIRP EATGYGLVYY TQAMIDYATN GKESFEGKRV TISGSGNVAQ YAALKVIELG 241 GTVVSLSDSKGCIISETGIT SEQVADISSA KVNFKSLEQI VNEYSTFSEN KVQYIAGARP 301 WTHVQKVDIA LPCATQNEVS GEEAKALVAQ GVKFIAEGSN MGSTPEAIAV FETARSTATG 361 PSEAVWYGPP KAANLGGVAV SGLEMAQNSQ RITWTSERVD QELKRIMINC FNECIDYAKK 421 YTKDGKVLPS LVKGANIASF IKVSDAMFDQ GDVF (SEQ ID NO. 1).

[0028] A series of mutants obtained by subjecting the wild-type glutamate dehydrogenase represented by SEQ ID NO.1 to single-site or multi-site mutations are shown in Table 1, wherein the English abbreviations of the amino acids have the following meanings: A represents alanine, G represents glycine, V represents valine, P represents proline, R represents arginine, L represents leucine, I represents isoleucine, Q represents glutamine, N represents asparagine, D represents aspartic acid, E represents glutamic acid, K represents lysine, R represents arginine, H represents histidine, F represents phenylalanine, W represents tryptophan, and S represents serine.

[0029] Table 1 Mutation sites of glutamate dehydrogenase ScGluDH

[0030] Note: The various mutants in Table 1 are obtained by mutating the wild-type glutamate dehydrogenase ScGluDH with the amino acid sequence shown in SEQ ID NO.1.

[0031] Test Example 1 Glutamate dehydrogenase ScGluDH mutant enzyme activity detection test The coding genes of the wild-type glutamate dehydrogenase and its mutants derived from SEQ ID NO. 1 in Example 1 were sent to a gene synthesis company for full gene synthesis and cloned into the pET28a vector. The recombinant vectors containing the target genes were transformed into Escherichia coli BL21 (DE3). After sequencing verification, positive colonies were picked, cultured, and induced to prepare wet bacteria expressing the wild-type glutamate dehydrogenase and its mutants shown in SEQ ID NO. 1.

[0032] In addition, the method of bioconversion of L-glufosinate in the publication number CN 107630052A (invention name: Bioconversion method of L-glufosinate ammonium) Saccharomyces cerevisiae Glufosinate dehydrogenase (Note: This is derived from Saccharomyces cerevisiae The nucleotide sequence of the glufosinate-ammonium dehydrogenase gene is shown as SEQ ID NO. 1 in the specification of CN 107630052A) was cloned into the pET28a vector and transformed into Escherichia coli BL21 (DE3). After verification by sequencing, positive colonies were selected, cultured, and induced to prepare wet cells expressing the glutamate dehydrogenase shown in SEQ ID NO. 2.

[0033] The publication number is CN 107630052A (invention name: Biotransformation method of L-glufosinate ammonium) Saccharomyces cerevisiae The amino acid sequence of the glufosinate dehydrogenase is shown in SEQ ID NO.2: 1 MSEPEFQQAY EEVVSSLEDS TLFEQHPEYR KVLPIVSVPE RIIQFRVTWE NDKGEQEVAQ 61 GYRVQYNSAK GPYKGGLRFH PSVNLSILKF LGFEQIFKNS LTGLDMGGGK GGLCVDLKGR 121 SNNEIRRPCY PFMRELSRHI GQDTDVPAGD IGVGGREIGY LFGAYRSYKN SWEGVLTGKG 181 LNWGGSLIRP EATGYGLVYY TQAMIDYATN GKESFEGKRV TISGSGNVAQ YAALKVIELG 241 GTVVSLSDSKGCVISETGIT SEQVADISSA KVNFKSLEQI VNEYSTFSEN KVQYIAGARP 301 WTHVQKVDIA LPCATQNEVS GEEAKALVAQ GVKFIAEGSN MGSTPEAIAV FETARSTATG 361 PSEAVWYGPP KAANLGGVAV SGLEMAQNSQ RITWTSERVD QELKRIMINC FNECIDYAKK 421 YTKDGKVLPS LVKGANIASF IKVSDAMFDQ GDVF (SEQ ID NO. 2).

[0034] Various wet bacterial cells used in the present invention can be prepared according to the following general preparation method: a recombinant strain E. coli BL21 (DE3) glycerol bacteria containing a target enzyme (glutamate dehydrogenase or its various mutants, formate dehydrogenase) encoding gene is inoculated into an LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 15 hours, inoculated into a fresh LB liquid culture medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, cultured at 37°C and 180 rpm for 3 hours, and then IPTG with a final concentration of 0.1 mM is added to the culture solution for induction. After culture at 25°C and 180 rpm for 16 hours, the culture is centrifuged at 4000 rpm for 15 minutes, and the collected precipitate is the wet bacterial cell containing the target enzyme.

[0035] 40 g / L of the collected wet cells containing glutamate dehydrogenase and 10 g / L of the wet cells containing formate dehydrogenase were resuspended and mixed, cooled, and lysed. The crude enzyme solution prepared was used as a catalyst, 30 g / L 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid was used as a substrate, 0.3 g / L NADP was used as a coenzyme, 15 g / L ammonium formate was used as an amino donor, the reaction medium was 50 mM sodium phosphate buffer at pH 7.5, the reaction was carried out at 35°C and 180 rpm for 10 min, and after terminating the reaction, the concentration and chirality of the product L-glufosinate were analyzed and detected by HPLC.

[0036] According to the above-mentioned specific reaction conditions, the catalytic activities of the wild-type glutamate dehydrogenase with the amino acid sequence shown in SEQ ID NO.1 and its various glutamate dehydrogenase mutants, and the glufosinate dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 towards 2-carbonyl-4-(hydroxymethylphosphono)butyrate were respectively measured.

[0037] The enzyme activity test results are shown in Table 2.

[0038] Table 2 Glutamate dehydrogenase activity test results

[0039] Note: “-” means no test was performed.

[0040] The enzyme activity test results in Table 2 show that the modification and mutation of the glutamate dehydrogenase ScGluDH shown in SEQ ID NO. 1 can significantly improve its catalytic activity for 2-carbonyl-4-(hydroxymethylphosphono)butyrate by molecular simulation calculation methods based on rational design of substrate binding site characteristics and channel characteristics. The most significant improvement in enzyme activity is the combined mutant A148G / V272R / L375A / V378A of the binding pocket site and the channel site, whose enzyme activity is 256 times that of the wild-type enzyme and 10 times that of the glufosinate dehydrogenase from Saccharomyces cerevisiae shown in SEQ ID NO. 2 (which is the glufosinate dehydrogenase described in CN107630052A).

[0041] Experimental Example 2: Preparation of L-phosphinothricin using wet bacteria containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The reaction system, containing 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate as the substrate, 0.3 g / L NADP, and 82 g / L ammonium formate, was used in a 100 mL reaction. The wet cell concentration of the wild-type glutamate dehydrogenase ScGluDH (SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-phosphinothricin formation concentration and ee value were determined using a chiral derivatization reagent.

[0042] After 24 h of conversion, 178.2 g / L of substrate remained, with a conversion rate of 1.0%. The generated concentration of L-glufosinate was 1.1 g / L, and the ee value was >99.9%.

[0043] The amino acid sequence of formate dehydrogenase is shown in SEQ ID NO.3 below: 1 MATVLCVLYP DPVDGYPPHY VRDTIPVITR YADGQTAPTP AGPPGFRPGE LVGSVSGALG 61 LRGYLEAHGH TLIVTSDKDG PDSEFERRLP DADVVISQPF WPAYLTAERI ARAPKLRLAL 121 TAGIGSDHVD LDAAARAHIT VAEVTGSNSI SVAEHVVMTT LALVRNYLPS HAIAQQGGWN 181 IADCVSRSYD VEGMHFGTVG AGRIGLAVLR RLKPFGLHLH YTQRHRLDAA IEQELGLTYH 241 ADPASLAAAV DIVNLQIPLY PSTEHLFDAA MIARMKRGAY LINTARAKLV DRDAVVRAVT 301 SGHLAGYGGD VWFPQPAPAD HPWRAMPFNG MTPHISGTSL SAQARYAAGT LEILQCWFDG 361 RPIRNEYLIV DGGTLAGTGA QSYRLT (SEQ ID NO. 3).

[0044] Experimental Example 3: Preparation of L-phosphinothricin using wet bacteria containing phosphinothricin dehydrogenase shown in SEQ ID NO. 2 as catalyst The 100ml reaction system contained 180g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3g / L NADP, and 82g / L ammonium formate. The wet cell concentration of glufosinate dehydrogenase (CN 107630052A) shown in SEQ ID NO. 2 was 40g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-glufosinate concentration and ee value were determined using a chiral derivatization reagent.

[0045] After 24 h of conversion, 155.3 g / L of substrate remained, with a conversion rate of 13.7%. The generated concentration of L-glufosinate was 24.1 g / L, and the ee value was >99.9%.

[0046] Experimental Example 4: Preparation of L-phosphinothricin using the wet cell of mutant A148G containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the mutant A148G glutamate dehydrogenase ScGluDH (SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-glufosinate formation concentration and ee value were determined using a chiral derivatization reagent.

[0047] After 21 h of conversion, 1.6 g / L of substrate remained, with a conversion rate of 99.1%. The generated concentration of L-glufosinate was 175.7 g / L, and the ee value was >99.9%.

[0048] Experimental Example 5: Preparation of L-phosphinothricin using the mutant V378P wet cell containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the glutamate dehydrogenase mutant V378P (ScGluDH) shown in SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-phosphinothricin formation concentration and ee value were determined using a chiral derivatization reagent.

[0049] After 24 h of conversion, 143.8 g / L of substrate remained, with a conversion rate of 20.1%. The generated concentration of L-glufosinate was 35.7 g / L, and the ee value was >99.9%.

[0050] Experimental Example 6: Preparation of L-phosphinothricin using the mutant V378G wet cell containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the glutamate dehydrogenase mutant V378G (ScGluDH) shown in SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-phosphinothricin formation concentration and ee value were determined using a chiral derivatization reagent.

[0051] After 24 h of conversion, the remaining substrate was 93.6 g / L, the conversion rate was 48.0%, the generated concentration of L-glufosinate was 85.1 g / L, and the ee value was >99.9%.

[0052] Experimental Example 7: Preparation of L-phosphinothricin using the wet bacterial cell of mutant V378A containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the mutant V378A of the glutamate dehydrogenase ScGluDH (SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-glufosinate formation concentration and ee value were determined using a chiral derivatization reagent.

[0053] After 24 h of conversion, 85.7 g / L of substrate remained, the conversion rate was 52.4%, the concentration of L-glufosinate produced was 92.9 g / L, and the ee value was >99.9%.

[0054] Experimental Example 8: Preparation of L-phosphinothricin using the mutant A148G / V378A wet cell containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the glutamate dehydrogenase ScGluDH mutant A148G / V378A (SEQ ID NO. 1) and formate dehydrogenase (SEQ ID NO. 3) was 40 g / L, and the wet cell concentration was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-glufosinate formation concentration and ee value were determined using a chiral derivatization reagent.

[0055] After 19.5 h of conversion, 2.3 g / L of substrate remained, with a conversion rate of 98.7%. The generated concentration of L-glufosinate was 175.0 g / L, and the ee value was >99.9%.

[0056] Experimental Example 9: Preparation of L-phosphinothricin using the mutant A148G / V272R / L375A / V378A wet cell containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the glutamate dehydrogenase ScGluDH mutant A148G / V272R / L375A / V378A (SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-phosphinothricin formation concentration and ee value were determined using a chiral derivatization reagent.

[0057] After 10 h of conversion, the substrate remained at 0.0 g / L, the conversion rate was 100%, the generated concentration of L-glufosinate was 177.3 g / L, and the ee value was >99.9%.

[0058] Experimental Example 10: Preparation of L-phosphinothricin using wet bacterial cells containing the wild-type glutamate dehydrogenase ScGluDH mutant A148G / I151L / V272R / L375A / V378A / A379L shown in SEQ ID NO.1 as a catalyst The 100-ml reaction system contained 180 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyrate substrate, 0.3 g / L NADP, and 82 g / L ammonium formate. The wet cell concentration of the glutamate dehydrogenase ScGluDH mutant (A148G / I151L / V272R / L375A / V378A / A379L, represented by SEQ ID NO. 1) was 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO. 3) was 10 g / L. The reaction temperature was maintained at 35°C, and the pH was controlled at 7.4. Substrate residue was monitored by HPLC, and the L-phosphinothricin formation concentration and ee value were determined using a chiral derivatization reagent.

[0059] After 24 h of conversion, 134.5 g / L of substrate remained, with a conversion rate of 25.3%. The generated concentration of L-glufosinate was 44.8 g / L, and the ee value was >99.9%.

Claims

1. A mutant of glutamate dehydrogenase, characterized in that: The mutant is a mutant obtained by subjecting the amino acid sequence of glutamate dehydrogenase shown in SEQ ID NO.1 to any one of single-site or multi-site mutations of A148G, V378P, V378G, V378A, A148G / V378A, A148G / V272R / L375A / V378A or A148G / I151L / V272R / L375A / V378A / A379L, wherein the English abbreviations of the amino acids have the following meanings: A represents alanine, G represents glycine, V represents valine, P represents proline, R represents arginine, L represents leucine, and I represents isoleucine.

2. A gene encoding the mutant according to claim 1.

3. A chimeric gene comprising the coding gene according to claim 2.

4. A recombinant expression vector containing the coding gene according to claim 2.

5. A recombinant host cell containing the recombinant expression vector according to claim 4.

6. A method for preparing the mutant according to claim 1, characterized in that: include: (1) The coding gene of the mutant according to claim 1 is operably connected to an expression regulatory element to construct a recombinant expression vector; (2) The recombinant expression vector is transformed into host cells, the host cells are cultured, the recombinant protein is induced to express, and then purified.

7. Use of the mutant according to claim 1, the encoding gene according to claim 2, the chimeric gene according to claim 3, the recombinant expression vector according to claim 4 or the recombinant host cell according to claim 5 in the synthesis of L-phosphinothricin.

8. A method for biosynthesis of L-phosphinothricin ammonium, characterized in that: include: The mutant according to claim 1 is used as a catalytic enzyme and 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid is used as a substrate to carry out an enzymatic reaction to obtain L-phosphinothricin ammonium.

9. The biosynthesis method according to claim 8, characterized in that The reaction temperature of the enzymatic reaction is controlled at 28-38° C.; the pH value of the reaction process of the enzymatic reaction is controlled at 6.5-7.5.

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