A mutant of glutamate dehydrogenase derived from Saccharomyces cerevisiae and its application in the synthesis of L-glufosinate

Abstract

The present invention discloses a mutant of glutamate dehydrogenase derived from Saccharomyces cerevisiae and its application in the synthesis of L-glufosinate. The present invention performs single-site or multi-site mutagenesis on the wild-type glutamate dehydrogenase derived from Saccharomyces cerevisiae shown in SEQ ID NO.1 by a mutagenesis and transformation method of rationally designing and analyzing the amino acid residues in the substrate-binding pocket of glutamate dehydrogenase and the geometry in the molecular tunnel. Compared with the wild-type glutamate dehydrogenase, the enzyme activity of the obtained single-site or multi-site mutant is significantly improved. Biocatalytic preparation of L-glufosinate acid using the glutamate dehydrogenase mutant can significantly increase the feeding concentration and conversion rate of the substrate on the basis of high stereoselectivity, high conversion rate, and high yield, effectively solving the problems that the existing glutamate dehydrogenase shows inactivity or low activity in the biocatalysis of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.

Description

Technical Field

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

[0002] Glutamate dehydrogenase (Glutamate dehydrogenase, EC 1.4.1.2–1.4.1.4) is rich in sources and diverse, and has significant advantages such as strict stereoselectivity, high yield, and easy separation in the field of preparing chiral L-amino acids, with a very broad application prospect. Therefore, it is of great significance to utilize glutamate dehydrogenase for biocatalyzing 2-oxo-4-(hydroxymethylphosphinyl)butyric acid in the process development of asymmetric amination to prepare L-glufosinate.

[0003] Chinese Patent CN107630052A discloses that the whole cells of an engineered bacterium co-expressing the glutamate dehydrogenase gene and the formate dehydrogenase gene derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), under the combined action of the additives apple extract powder and spirulina powder, catalyze 30 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to produce L-glufosinate acid, with the highest conversion rate of L-glufosinate acid being 95.8% and the conversion time being 16.8 h; in the absence of the additives apple extract powder and spirulina powder, catalyze 30 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to produce L-glufosinate acid, with the conversion rate of L-glufosinate acid being 82.3% and the conversion time being 26.1 h. From the perspective of industrial application, the catalytic activity of glutamate dehydrogenase in this patent is relatively low, which will bring a series of problems such as high cost of preparing enzyme catalysts and heavy burden on the post-treatment of the conversion solution in production. In addition, the addition of the additives apple extract powder and spirulina powder not only increases the production cost, but also increases the difficulty of subsequent product purification.

[0004] Therefore, it is the key to reducing the production cost of L-glufosinate to mutate and transform glutamate dehydrogenase by enzyme engineering technology and genetic engineering technology to improve the catalytic activity of glutamate dehydrogenase towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. Summary of the Invention

[0005] One object of the present invention is to provide Saccharomyces cerevisiaeMutants of wild-type glutamate dehydrogenase ScGluDH from the [source], wherein the enzyme activity of the mutants is significantly improved compared to that of wild-type glutamate dehydrogenase ScGluDH, and it is used as a catalyst to catalyze the bioconversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as a substrate, and both the substrate conversion rate and the yield of L-glufosinate are greatly improved compared to wild-type glutamate dehydrogenase ScGluDH;

[0006] The second object of the present invention is to provide the coding gene of the mutant of the wild-type glutamate dehydrogenase ScGluDH;

[0007] The third object of the present invention is to provide an expression cassette or chimeric gene containing the coding gene of the mutant of the wild-type glutamate dehydrogenase ScGluDH, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector;

[0008] The fourth object of the present invention is to apply the mutant of the wild-type glutamate dehydrogenase ScGluDH, its coding gene, an expression cassette or chimeric gene containing the coding gene of the mutant, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector, etc. to the synthesis or bioconversion of L-glufosinate.

[0009] The above objects of the present invention are achieved by the following technical solutions:

[0010] One aspect of the present invention is to provide Saccharomyces cerevisiae Mutants of wild-type glutamate dehydrogenase ScGluDH from the [source], wherein the Saccharomyces cerevisiae Amino acid sequence of wild-type glutamate dehydrogenase ScGluDH from the [source] is shown as SEQ ID NO.1; the mutant is a single-site mutant obtained by performing any one of the amino acid single-site mutations A148G, V378P, V378G, or V378A on the amino acid sequence shown as SEQ ID NO.1; or a multi-site mutant obtained by performing any one of the amino acid multi-site mutations A148G / V378A, A148G / V272R / L375A / V378A, or A148G / I151L / V272R / L375A / V378A / A379L on the amino acid sequence shown as SEQ ID NO.1, where the meanings of the amino acid abbreviations are as follows: A represents alanine, G represents glycine, V represents valine, P represents proline, R represents arginine, L represents leucine, and I represents isoleucine.

[0011] The single-site mutant "A148G" in the present invention means that the amino acid sequence shown as SEQ ID NO.1 Saccharomyces cerevisiaeThe 148th amino acid of the wild-type glutamate dehydrogenase ScGluDH from the source is mutated from alanine (Ala, A) to glycine (Gly, G); the description of the remaining single-site mutants of the present invention is similar.

[0012] The multi-site mutant "A148G / V378A" 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 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.

[0013] In order to solve the problem of low catalytic activity of glutamate dehydrogenase to 2-carbonyl-4-(hydroxymethylphosphonyl)butyrate, the present invention first targets Saccharomyces cerevisiae The protein three-dimensional structure of the wild-type glutamate dehydrogenase (ScGluDH, SEQID NO.1) was used, and 2-carbonyl-4-(hydroxymethylphosphono)butyric acid was used as the substrate to carry out molecular simulation calculations to obtain a reasonable three-dimensional structure of the complex between the two. Based on the three-dimensional structure of the complex, the key residues that form important interactions with the substrate and the important residues that constitute the cavity structure of the active pocket 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 speculate that the channel for the substrate to enter and exit the active pocket may also have an important influence on the catalytic activity and substrate selectivity of the enzyme. Therefore, for the channel for the substrate to enter and exit the active pocket, the molecular simulation method was applied to explore the morphology of the channel for the substrate to enter and exit the active pocket in the wild-type enzyme structure, and the average diameter, bottleneck diameter, length and other indicators were used to quantitatively describe the morphological characteristics of the channel and determine the key sites of the channel, and screen out mutants that can improve the substrate permeability.

[0014] Another aspect of the present invention is to provide Saccharomyces cerevisiae The genes encoding the mutants of the wild-type glutamate dehydrogenase ScGluDH were derived from .

[0015] 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.

[0016] The present invention further provides a method for preparing any of the mutants, comprising:

[0017] (1) operably connecting the coding gene of the mutant to an expression regulatory element to construct a recombinant expression vector;

[0018] (2) Transform the recombinant expression vector into a host cell, culture the host cell, induce the expression of the recombinant protein, and purify it to obtain the product.

[0019] A preferred specific embodiment is that the expression regulatory elements are selected from one or more of a promoter, a terminator, an enhancer, a transposon, a leader sequence, or a marker gene.

[0020] Another aspect of the present invention applies the mutant of the wild-type glutamate dehydrogenase ScGluDH from the Saccharomyces cerevisiae to the biosynthesis of L-glufosinate, including: using the single-site mutant or multi-site mutant as the catalytic enzyme, and performing an enzymatic reaction with 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as the substrate to obtain L-glufosinate.

[0021] A preferred specific scheme of the present invention is that 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; the pH value during the reaction process of the enzymatic reaction is controlled at 6.5 - 7.5.

[0022] A preferred specific scheme of the present invention is that in order to achieve a better catalytic effect, formate dehydrogenase can be added to the enzymatic reaction, and it and the mutant of glutamate dehydrogenase ScGluDH are used together as the catalyst for the catalytic reaction.

[0023] Compared with the prior art, the main beneficial effects of the present invention include:

[0024] 1. Based on the rational design of the active pocket and channel of glutamate dehydrogenase, and the rational design strategy of enzymes based on the protein and substrate structures, the present invention uses the method of molecular simulation calculation to mutate and transform the active pocket and channel wall of glutamate dehydrogenase from Saccharomyces cerevisiae to obtain various mutants with significantly improved enzyme activity. Using these mutants as catalysts significantly improves the substrate conversion rate of catalyzing the formation of L-glufosinate with 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as the substrate and the production concentration of L-glufosinate, and effectively solves the problems of inactivity or low activity of glutamate dehydrogenase in biocatalysis with 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as the substrate.

[0025] 2. Compared with wild-type glutamate dehydrogenase, the mutant of glutamate dehydrogenase obtained in the present invention has a catalytic activity towards the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid increased by up to 256 times. When the substrate feeding concentration is 180 g / L and the conversion is carried out for 10 h, the substrate conversion rate can reach up to 100%, the production concentration of L-glufosinate can reach up to 177.3 g / L, and the ee value > 99.9%, indicating that the mutant of glutamate dehydrogenase provided by the present invention has good industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 are the residues of the active pocket binding site of the glutamate dehydrogenase shown in SEQ ID NO.1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings of the specification. The advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0028] Term Definitions Related to the Present Invention

[0029] 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.

[0030] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (such as phosphorothioates, phosphoroamidates, etc.) used in antisense technology. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue ( Mol Cell. Probes 8:91-98 (1994)).

[0031] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. 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 joined by covalent peptide bonds.

[0032] The terms "mutation" and "mutant" have their ordinary meanings herein, referring to genetic, naturally occurring, or introduced changes in a nucleic acid or polypeptide sequence, which have the same meaning as is commonly understood by one of ordinary skill in the art.

[0033] The term "recombinant host cell line" or "host cell" means a cell that contains the 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-integrated 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.

[0034] The term "operably linked" refers to a functional linkage between two or more elements, and the elements that are operably linked may be adjacent or non-adjacent.

[0035] Example 1 Construction and Screening of Glutamate Dehydrogenase Mutants

[0036] In the present invention, based on the substrate binding site characteristics and channel characteristics, a method of molecular simulation calculation was used to analyze and scan the binding pocket and molecular tunnel of glutamate dehydrogenase ScGluDH derived from Saccharomyces cerevisiae with the substrate. The results of the substrate binding pocket analysis ( Figure 1 ) showed that there were certain spatial limitations in the binding pocket of ScGluDH when binding to the substrate, which might lead to a decrease in the binding efficiency of the substrate; the results of the tunnel scanning showed that some channels might be narrow or blocked, which might limit the effective entry of the substrate and the smooth release of the product. Therefore, a series of mutants were designed by rationally designing and analyzing the amino acid residues in the binding pocket of the wild-type glutamate dehydrogenase ScGluDH derived from Saccharomyces cerevisiae and the geometry in the molecular tunnel of the glutamate dehydrogenase ScGluDH. Among them, Saccharomyces cerevisiae the amino acid sequence of the wild-type glutamate dehydrogenase ScGluDH derived from

[0037] 1 MSEPEFQQAY EEVVSSLEDS TLFEQHPEYR KVLPIVSVPE RIIQFRVTWE NDKGEQEVAQ

[0038] 61 GYRVQYNSAK GPYKGGLRFH PSVNLSILKF LGFEQIFKNS LTGLDMGGGK GGLCVDLKGR

[0039] 121 SNNEIRRICY AFMRELSRHI GQDTDVPAGD IGVGGREIGY LFGAYRSYKN SWEGVLTGKG

[0040] 181 LNWGGSLIRP EATGYGLVYY TQAMIDYATN GKESFEGKRV TISGSGNVAQ YAALKVIELG

[0041] 241 GTVVSLSDSK GCIISETGIT SEQVADISSA KVNFKSLEQI VNEYSTFSEN KVQYIAGARP

[0042] 301 WTHVQKVDIA LPCATQNEVS GEEAKALVAQ GVKFIAEGSN MGSTPEAIAV FETARSTATG

[0043] 361 PSEAVWYGPP KAANLGGVAV SGLEMAQNSQ RITWTSERVD QELKRIMINC FNECIDYAKK

[0044] 421 YTKDGKVLPS LVKGANIASF IKVSDAMFDQ GDVF (SEQ ID NO.1).

[0045] A series of mutants obtained by performing single-site or multi-site mutations on the wild-type glutamate dehydrogenase shown in SEQ ID NO.1 are shown in Table 1, where the meanings of the English abbreviations of amino acids are as follows: 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.

[0046] Table 1 Mutation sites of glutamate dehydrogenase ScGluDH

[0047]

[0048] Note: The various mutants in Table 1 are mutants obtained by mutating the wild-type glutamate dehydrogenase ScGluDH with the amino acid sequence shown in SEQ ID NO.1.

[0049] Test Example 1 Detection Test of the Enzyme Activity of Glutamate Dehydrogenase ScGluDH Mutants

[0050] 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 whole gene synthesis and were respectively cloned into the pET28a vector. The recombinant vectors containing the target genes were transformed into Escherichia coli BL21(DE3). After being verified correct by sequencing, positive colonies were picked for culturing and induction to prepare wet bacterial cells expressing the wild-type glutamate dehydrogenase and its mutants with the amino acid sequence shown in SEQ ID NO.1.

[0051] In addition, the full-length synthetic gene of the glufosinate dehydrogenase (Note: the nucleotide sequence of the gene of the glufosinate dehydrogenase derived from Saccharomyces cerevisiae is shown as SEQ ID NO.1 in the specification of CN 107630052A (invention name: Biological Transformation Method of L-Glufosinate)) was cloned into the pET28a vector and transformed into Escherichia coli BL21(DE3). After being verified correct by sequencing, positive colonies were picked for culturing and induction to prepare wet bacterial cells expressing the glutamate dehydrogenase with the amino acid sequence shown in SEQ ID NO.2. Saccharomyces cerevisiae The amino acid sequence of the glufosinate dehydrogenase derived from

[0052] in CN 107630052A (invention name: Biological Transformation Method of L-Glufosinate) is shown as SEQ ID NO.2: Saccharomyces cerevisiae 1 MSEPEFQQAY EEVVSSLEDS TLFEQHPEYR KVLPIVSVPE RIIQFRVTWE NDKGEQEVAQ

[0053] 1 MSEPEFQQAY EEVVSSLEDS TLFEQHPEYR KVLPIVSVPE RIIQFRVTWE NDKGEQEVAQ

[0054] 61 GYRVQYNSAK GPYKGGLRFH PSVNLSILKF LGFEQIFKNS LTGLDMGGGK GGLCVDLKGR

[0055] 121 SNNEIRRPCY PFMRELSRHI GQDTDVPAGD IGVGGREIGY LFGAYRSYKN SWEGVLTGKG

[0056] 181 LNWGGSLIRP EATGYGLVYY TQAMIDYATN GKESFEGKRV TISGSGNVAQ YAALKVIELG

[0057] 241 GTVVSLSDSK GCVISETGIT SEQVADISSA KVNFKSLEQI VNEYSTFSEN KVQYIAGARP

[0058] 301 WTHVQKVDIA LPCATQNEVS GEEAKALVAQ GVKFIAEGSN MGSTPEAIAV FETARSTATG

[0059] 361 PSEAVWYGPP KAANLGGVAV SGLEMAQNSQ RITWTSERVD QELKRIMINC FNECIDYAKK

[0060] 421 YTKDGKVLPS LVKGANIASF IKVSDAMFDQ GDVF (SEQ ID NO.2).

[0061] All kinds of wet bacterial cells used in the present invention can be prepared according to the following general preparation method: Inoculate the glycerol bacteria of recombinant strain E. coli BL21(DE3) containing the encoding gene of the target enzyme (glutamate dehydrogenase or its various mutants, formate dehydrogenase) into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL, culture at 37 °C for 15 h, inoculate into fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1% by volume, culture at 37 °C and 180 revolutions per minute for 3 h, then add IPTG with a final concentration of 0.1 mM to the culture solution for induction, culture at 25 °C and 180 revolutions per minute for 16 h, centrifuge at 4000 rpm for 15 min, and the collected precipitate is the wet bacterial cells containing the target enzyme.

[0062] After resuspending and mixing 40 g / L of the collected wet bacterial cells containing glutamate dehydrogenase and 10 g / L of the wet bacterial cells containing formate dehydrogenase, cool and disrupt the cells. Use the prepared crude enzyme solution as a catalyst, use 30 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as a substrate, 0.3 g / L of NADP as a coenzyme, 15 g / L of ammonium formate as an amino donor, the reaction medium is 50 mM sodium phosphate buffer at pH 7.5, react at 35 °C and 180 rpm for 10 min. After terminating the reaction, analyze and detect the production concentration and chirality of the product L-phosphinothricin by HPLC.

[0063] Under the above specific reaction conditions, the catalytic activities of the wild-type glutamate dehydrogenase with the amino acid sequence shown in SEQ ID NO.1, its various glutamate dehydrogenase mutants, and the glufosinate dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid were measured respectively.

[0064] The specific results of the enzyme activity detection are shown in Table 2.

[0065] Table 2 Detection results of glutamate dehydrogenase enzyme activity

[0066]

[0067] Note: "-" indicates that the detection was not carried out.

[0068] The enzyme activity detection results in Table 2 show that by using the method of molecular simulation calculation and rational design based on the characteristics of the substrate binding site and the channel, the ScGluDH glutamate dehydrogenase shown in SEQ ID NO.1 was modified and mutated, which can significantly improve its catalytic activity towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. The most significant increase in enzyme activity was the combined mutant A148G / V272R / L375A / V378A of the binding pocket site and the channel site, whose enzyme activity was 256 times that of the wild-type enzyme activity and 10 times that of the glufosinate dehydrogenase from Saccharomyces cerevisiae shown in SEQ ID NO.2 (the glufosinate dehydrogenase described in CN107630052A).

[0069] Experimental Example 2 Experiment on the catalytic preparation of L-glufosinate using the wet cells containing the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0070] The volume of the reaction system was 100 mL, containing 180 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the wild-type glutamate dehydrogenase 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 controlled at 35 °C, the pH was controlled at 7.4 during the reaction process, the residual situation of the substrate was detected by HPLC during the reaction process, and the production concentration and ee value of L-glufosinate were detected using a chiral derivatizing reagent at the same time.

[0071] After 24 h of conversion, the remaining substrate was 178.2 g / L, the conversion rate was 1.0%, the production concentration of L-glufosinate was 1.1 g / L, and the ee value > 99.9%.

[0072] The amino acid sequence of formate dehydrogenase is as shown in SEQ ID NO.3 below:

[0073] 1 MATVLCVLYP DPVDGYPPHY VRDTIPVITR YADGQTAPTP AGPPGFRPGE LVGSVSGALG

[0074] 61 LRGYLEAHGH TLIVTSDKDG PDSEFERRLP DADVVISQPF WPAYLTAERI ARAPKLRLAL

[0075] 121 TAGIGSDHVD LDAAARAHIT VAEVTGSNSI SVAEHVVMTT LALVRNYLPS HAIAQQGGWN

[0076] 181 IADCVSRSYD VEGMHFGTVG AGRIGLAVLR RLKPFGLHLH YTQRHRLDAA IEQELGLTYH

[0077] 241 ADPASLAAAV DIVNLQIPLY PSTEHLFDAA MIARMKRGAY LINTARAKLV DRDAVVRAVT

[0078] 301 SGHLAGYGGD VWFPQPAPAD HPWRAMPFNG MTPHISGTSL SAQARYAAGT LEILQCWFDG

[0079] 361 RPIRNEYLIV DGGTLAGTGA QSYRLT (SEQ ID NO.3).

[0080] Test Example 3: Preparation of L-glufosinate by catalysis using wet cells containing glufosinate dehydrogenase shown in SEQ ID NO.2 as a catalyst

[0081] The reaction system was 100 ml and contained 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The concentration of wet cells of glufosinate dehydrogenase (CN 107630052A) shown in SEQ ID NO.2 was 40 g / L, and the concentration of wet cells of formate dehydrogenase (SEQ ID NO.3) was 10 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.4 during the reaction process, the residual amount of the substrate was detected by HPLC during the reaction process, and the production concentration and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0082] After 24 hours of conversion, the remaining substrate was 155.3 g / L, the conversion rate was 13.7%, the production concentration of L-glufosinate was 24.1 g / L, and the ee value > 99.9%.

[0083] Test Example 4: Preparation of L-glufosinate using the wet cells of the mutant A148G of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0084] The reaction system was 100 ml, containing 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The concentration of the wet cells of the mutant A148G of the glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 was 40 g / L, and the concentration of the wet cells of formate dehydrogenase (SEQ ID NO.3) was 10 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.4 during the reaction process. The residual situation of the substrate was detected by HPLC during the reaction process, and at the same time, the production concentration and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0085] After 21 hours of conversion, the remaining substrate was 1.6 g / L, the conversion rate was 99.1%, the production concentration of L-glufosinate was 175.7 g / L, and the ee value > 99.9%.

[0086] Test Example 5: Preparation of L-glufosinate using the wet cells of the mutant V378P of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0087] The reaction system was 100 ml, containing 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The concentration of the wet cells of the mutant V378P of the glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 was 40 g / L, and the concentration of the wet cells of formate dehydrogenase (SEQ ID NO.3) was 10 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.4 during the reaction process. The residual situation of the substrate was detected by HPLC during the reaction process, and at the same time, the production concentration and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0088] After 24 hours of conversion, the remaining substrate was 143.8 g / L, the conversion rate was 20.1%, the production concentration of L-glufosinate was 35.7 g / L, and the ee value > 99.9%.

[0089] Test Example 6: Preparation of L-glufosinate using the wet cells of the mutant V378G of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0090] The reaction system is 100 ml and contains 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the mutant V378G of glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 is 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO.3) is 10 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.4 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and at the same time, the chiral derivatizing reagent is used to detect the production concentration and ee value of L-glufosinate.

[0091] After 24 h of conversion, the remaining substrate is 93.6 g / L, the conversion rate is 48.0%, the production concentration of L-glufosinate is 85.1 g / L, and the ee value > 99.9%.

[0092] Test Example 7: Preparation of L-glufosinate by catalysis using the wet cells of the mutant V378A of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0093] The reaction system is 100 ml and contains 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the mutant V378A of glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 is 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO.3) is 10 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.4 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and at the same time, the chiral derivatizing reagent is used to detect the production concentration and ee value of L-glufosinate.

[0094] After 24 h of conversion, the remaining substrate is 85.7 g / L, the conversion rate is 52.4%, the production concentration of L-glufosinate is 92.9 g / L, and the ee value > 99.9%.

[0095] Test Example 8: Preparation of L-glufosinate by catalysis using the wet cells of the mutant A148G / V378A of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0096] The reaction system is 100 ml, containing 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the mutant A148G / V378A of glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 is 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO.3) is 10 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.4 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and at the same time, the chiral derivatizing reagent is used to detect the production concentration and ee value of L-glufosinate.

[0097] After 19.5 h of conversion, the remaining substrate is 2.3 g / L, the conversion rate is 98.7%, the production concentration of L-glufosinate is 175.0 g / L, and the ee value > 99.9%.

[0098] Test Example 9: Preparation of L-glufosinate using the wet cells of the mutant A148G / V272R / L375A / V378A of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0099] The reaction system is 100 ml, containing 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the mutant A148G / V272R / L375A / V378A of glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 is 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO.3) is 10 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.4 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and at the same time, the chiral derivatizing reagent is used to detect the production concentration and ee value of L-glufosinate.

[0100] After 10 h of conversion, the remaining substrate is 0.0 g / L, the conversion rate is 100%, the production concentration of L-glufosinate is 177.3 g / L, and the ee value > 99.9%.

[0101] Test Example 10: Preparation of L-glufosinate using the wet cells of the mutant A148G / I151L / V272R / L375A / V378A / A379L of the wild-type glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 as a catalyst

[0102] The reaction system is 100 ml and contains 180 g / L of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.3 g / L of NADP, and 82 g / L of ammonium formate. The wet cell concentration of the mutant A148G / I151L / V272R / L375A / V378A / A379L of glutamate dehydrogenase ScGluDH shown in SEQ ID NO.1 is 40 g / L, and the wet cell concentration of formate dehydrogenase (SEQ ID NO.3) is 10 g / L. The reaction temperature is controlled at 35 °C, the pH during the reaction is controlled at 7.4, the residual amount of the substrate is detected by HPLC during the reaction process, and the production concentration and ee value of L-glufosinate are detected using a chiral derivatizing reagent.

[0103] After 24 h of conversion, the remaining substrate is 134.5 g / L, the conversion rate is 25.3%, the production concentration of L-glufosinate is 44.8 g / L, and the ee value > 99.9%.

Claims

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

2. A coding gene of the mutant as described in claim 1.

3. A chimeric gene containing the coding gene described in claim 2.

4. A recombinant expression vector containing the coding gene described in claim 2.

5. A recombinant host cell containing the recombinant expression vector described in claim 4. Comprising:

6. A method for preparing the mutant according to claim 1, characterized in that, (1) A recombinant expression vector is constructed by operably connecting the coding gene of the mutant described in claim 1 with an expression regulatory element; (2) The recombinant expression vector is transformed into a host cell, the host cell is cultured, the recombinant protein is induced to express, and purified to obtain the product.

7. Use of the mutant as described in claim 1, the coding gene described in claim 2, the chimeric gene described in claim 3, the recombinant expression vector described in claim 4 or the recombinant host cell described in claim 5 in the synthesis of L-glufosinate. Comprising:

8. A method for the biosynthesis of L-glufosinate, characterized in that, Using the mutant described in claim 1 as a catalytic enzyme and 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as a substrate to carry out an enzymatic reaction to obtain L-glufosinate. The reaction temperature of the enzymatic reaction is controlled at 28-38 °C; the pH value during the reaction process of the enzymatic reaction is controlled at 6.5-7.

5.

9. The biosynthetic method according to claim 8, characterized in that, ​

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