A mutant of glutamate dehydrogenase derived from Clostridium symbiosum and its application in the synthesis of L-phosphinothricin

Abstract

The present invention discloses a mutant of glutamate dehydrogenase derived from Clostridium symbiosum ( Clostridium symbiosum ) and its application in the synthesis of L - glufosinate. The present invention uses a rational analysis and design mutation modification method for amino acid residues in the substrate - binding pocket of glutamate dehydrogenase, the geometry in the molecular tunnel, and the interaction between domains in the domain, and performs single - site or multi - site mutations on the wild - type glutamate dehydrogenase derived from Clostridium symbiosum shown in SEQ ID NO.1. The obtained single - site or multi - site mutants significantly improve the catalytic activity towards 2 - oxo - 4 - (hydroxymethylphosphinyl) butyric acid and have application prospects in the biocatalytic preparation of L - glufosinate.

Description

Technical Field

[0001] The present invention belongs to enzyme mutants and their applications, and particularly relates to a mutant of glutamate dehydrogenase derived from Clostridium symbiosum and its application in the synthesis of L-phosphinothricin, belonging to the field of mutants of glutamate dehydrogenase and their applications. Background Art

[0002] L-Phosphinothricin is a class of phosphorus-containing bioamino acid herbicides, and its active ingredient is 2-amino-4-(hydroxymethylphosphinyl)butyric acid. It was first synthesized by the German company Hoechst by chemical method and has good herbicidal activity. Phosphinothricin has low toxicity, is relatively safe, is easily degraded in the soil, is safe for crops, has a wide herbicidal spectrum, requires less dosage, causes less environmental pressure, kills weeds quickly, can use water as a base agent, and is safe and convenient to use. These characteristics superior to other herbicides make phosphinothricin a large-tonnage pesticide variety in the world.

[0003] Biocatalysis is an advantageous method for preparing L-phosphinothricin due to its advantages such as strong specificity, mild reaction conditions, high yield, and less pollution. Among them, the process of preparing L-phosphinothricin by catalyzing 2-oxo-4-(hydroxymethylphosphinyl)butyric acid with L-amino acid dehydrogenase and nucleoside coenzyme regeneration enzyme is relatively simple and economical. L-amino acid dehydrogenases mainly include leucine dehydrogenase, alanine dehydrogenase, glutamate dehydrogenase, etc. Among them, glutamate dehydrogenase (EC 1.4.1.2–1.4.1.4) has rich sources and diversity, and has significant advantages such as strict stereoselectivity, high yield, and easy separation in the field of preparing chiral L-amino acids, and has a very broad application prospect. Therefore, it is of great significance to utilize glutamate dehydrogenase to biocatalyze 2-oxo-4-(hydroxymethylphosphinyl)butyric acid in the process development of asymmetric amination for preparing L-phosphinothricin.

[0004] Chinese Patent CN108588045A uses single-site mutants A164G and V378A of glutamate dehydrogenase derived from Clostridium symbiosum (NCBI accession number WP-003497202.1) to catalyze 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to prepare L-phosphinothricin respectively. From the perspective of industrial application, if the catalytic activity of this glutamate dehydrogenase can be further improved, the preparation cost of the enzyme can be reduced, the conversion cycle can be shortened, and the substrate feeding concentration can be increased, etc.

[0005] Therefore, it is the key to reducing the production cost of L-phosphinothricin to mutate and transform glutamate dehydrogenase by enzyme engineering technology and genetic engineering technology to improve the catalytic activity of glutamate dehydrogenase for the asymmetric amination of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. Summary of the Invention

[0006] One of the objectives of the present invention is to provide Clostridium symbiosum (NCBI accession number CAA77805.1, EC 1.4.1.2) A mutant of wild-type glutamate dehydrogenase CsGluDH derived from, wherein the enzyme activity of the mutant is significantly improved compared to that of wild-type glutamate dehydrogenase CsGluDH, and it is used as a catalyst to catalyze the bioconversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid as a substrate to prepare L-glufosinate, and both the substrate conversion rate and the product yield are greatly improved compared to wild-type glutamate dehydrogenase CsGluDH;

[0007] Another objective of the present invention is to provide a coding gene for the mutant of wild-type glutamate dehydrogenase CsGluDH;

[0008] A third objective of the present invention is to provide an expression cassette, a recombinant expression vector containing the coding gene for the mutant of wild-type glutamate dehydrogenase CsGluDH, or a recombinant host cell containing the recombinant expression vector;

[0009] A fourth objective of the present invention is to apply the mutant of wild-type glutamate dehydrogenase CsGluDH, its coding gene, the expression cassette containing the coding gene for the mutant, the recombinant expression vector, or the recombinant host cell containing the recombinant expression vector, etc. to the synthesis or bioconversion of L-glufosinate.

[0010] The above objectives of the present invention are achieved by the following technical solutions:

[0011] One aspect of the present invention is to provide Clostridium symbiosum A mutant of wild-type glutamate dehydrogenase CsGluDH derived from, wherein the Clostridium symbiosum Amino acid sequence of wild-type glutamate dehydrogenase CsGluDH derived from is shown as SEQ ID NO.1; the mutant is a single-site mutant obtained by performing a single-site mutation on any one of the amino acids of V162L, A164G, I279A, A372V, V378P, V378G or V378A in the amino acid sequence shown as SEQ ID NO.1; or a multi-site mutant obtained by performing a multi-site mutation on any one of the amino acids of V162L / A372V, A164G / V378A, V162L / I279A / A372V, A164G / I279A / V378A or V162L / A164G / I279A / A372V / V378A in the amino acid sequence shown as SEQ ID NO.1. The meanings of the English abbreviations of the amino acids are as follows: V refers to valine, L refers to leucine, A refers to alanine, G refers to glycine, I refers to isoleucine, and P refers to proline.

[0012] The single-site mutant "V162L" in the present invention means that the amino acid sequence is as shown in SEQ ID NO. Clostridium symbiosum The 162nd amino acid of the wild-type glutamate dehydrogenase CsGluDH of the source is mutated from valine (Val, V) to leucine (Leu, L); the description of the remaining single-site mutants of the present invention is similar.

[0013] The multi-site mutant "V162L / A372V" in the present invention means that the amino acid sequence is as shown in SEQ ID NO. Clostridium symbiosum The 162nd amino acid of the wild-type glutamate dehydrogenase CsGluDH of the source is mutated from valine (Val, V) to leucine (Leu, L), and the 372nd alanine (Ala, A) is mutated to valine (Val, V); the description of the remaining multi-site mutants of the present invention is similar.

[0014] In order to solve the problem that the wild-type glutamate dehydrogenase has low catalytic activity towards 2-carbonyl-4-(hydroxymethylphosphonyl)butyrate, the present invention first targets Clostridium symbiosum The protein three-dimensional structure of the wild-type glutamate dehydrogenase (CsGluDH) of (SEQ ID 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 amino acid residues that form important interactions with the substrate and the important amino acid residues that constitute the cavity structure of the active pocket were found in the enzyme catalytic center of the CsGluDH wild-type protein, and the candidate mutant sequences were screened out. 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 inventors applied the molecular simulation method to explore the morphology of the channel for the substrate to enter and exit the active pocket in the wild-type enzyme structure, and quantitatively described the morphological characteristics of the channel with indicators such as average diameter, bottleneck diameter, and length, and determined the key sites of the channel, and screened out mutants that can improve the substrate permeability. In addition to the modification of the active pocket and the substrate channel, the inventors speculate that the domain-domain interaction of the two domains constituting glutamate dehydrogenase will not only determine the static structural stability of the enzyme, but also regulate the displacement between the two domains, thereby affecting the activity of the enzyme. Therefore, for the domain interaction, the inventors applied the method of molecular simulation, calculated the domain-domain interaction surface, delineated the core area and the peripheral area, and determined the list of important sites based on the interaction contribution of the residues in the peripheral area, carried out virtual mutation and energy optimization, statistically analyzed the structure and interaction characteristics of the domain-domain interaction surface, and screened out sequences with no significant changes in characteristics.

[0015] Another aspect of the present invention is to provide Clostridium symbiosum The genes encoding the mutants of wild-type glutamate dehydrogenase CsGluDH from the source.

[0016] Another aspect of the present invention is to provide an expression cassette containing the gene encoding the mutant, a recombinant expression vector 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.

[0017] Another aspect of the present invention is to Clostridium symbiosum The mutant of wild-type glutamate dehydrogenase CsGluDH from the source is applied to the biosynthesis of L-phosphinothricin ammonium, comprising: using the single-site mutant or multi-site mutant as a catalytic enzyme and 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate to carry out an enzymatic reaction to obtain L-phosphinothricin ammonium.

[0018] 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; the reaction process of the enzymatic reaction is controlled at a pH value of 6.5-7.5.

[0019] 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 CsGluDH can be used as catalysts for the catalytic reaction.

[0020] Compared with the prior art, the main beneficial effects of the present invention include:

[0021] 1. The present invention is based on a rational analysis of the active pocket, channel and domain interaction of glutamate dehydrogenase, adopts a rational enzyme design strategy based on protein and substrate structure, and uses molecular simulation calculation methods to Clostridium symbiosum The active pocket and channel wall of glutamate dehydrogenase were mutated and modified to obtain various enzyme mutants with significantly improved catalytic activity. These mutants were used as catalysts to significantly improve the substrate conversion rate and L-phosphinothricin yield of 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate, effectively solving the problems of inactivity or low activity of glutamate dehydrogenase in the biocatalytic conversion of 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate.

[0022] 2. Compared with wild-type glutamate dehydrogenase, the mutant of glutamate dehydrogenase obtained in the present invention has a maximum increase in catalytic activity towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid by 998 times. When the substrate feeding concentration is 500 mM and the reaction proceeds for 2 h, the substrate conversion rate reaches 100%, the production concentration of L-glufosinate is 498 mM, and the ee value > 99.9%, indicating that the mutant of glutamate dehydrogenase provided by the present invention has good industrial application value. Detailed implementation manners

[0023] The present invention will be further described below in conjunction with specific embodiments, and 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 the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0024] Term definitions involved in the present invention

[0025] 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.

[0026] 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 specific 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)).

[0027] 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, in which the amino acid residues are linked via covalent peptide bonds.

[0028] 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, and having the meanings commonly known to those skilled in the art.

[0029] The term "recombinant host cell line" or "host cell" means a cell that contains 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 remain as a non-integrating vector such as a plasmid or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0030] 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.

[0031] Example 1 Construction and Screening of Glutamate Dehydrogenase Mutants

[0032] In the present invention, using the method of molecular simulation calculation, based on the characteristics of the substrate binding site, channel characteristics, and domain interaction characteristics, Clostridium symbiosum the binding pocket, molecular tunnel, and domain-domain interaction of the wild-type glutamate dehydrogenase CsGluDH from the source with the substrate were analyzed and scanned. The results of the substrate binding pocket analysis showed that there were certain spatial limitations in the binding pocket of the wild-type CsGluDH 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; the results of the domain scanning showed that some domain interactions might affect the stability of the static structure of the enzyme and also regulate the displacement between the two domains, thereby affecting the enzyme activity. Therefore, a series of mutants were designed by rationally designing the amino acid residues in the binding pocket of the wild-type glutamate dehydrogenase CsGluDH from the source, the geometry in the molecular tunnel of the glutamate dehydrogenase CsGluDH, and the interaction of the domains, among which, Clostridium symbiosum the wild-type glutamate dehydrogenase CsGluDH from the source was rationally designed with respect to the amino acid residues in the substrate binding pocket, the geometry in the molecular tunnel of the glutamate dehydrogenase CsGluDH, and the interaction of the domains to obtain a series of mutants, among which, Clostridium symbiosum The amino acid sequence of the wild-type glutamate dehydrogenase CsGluDH (NCBI accession number CAA77805.1, EC 1.4.1.2) from the source is as shown in SEQ ID NO.1 below:

[0033] 1 MSKYVDRVIA EVEKKYADEP EFVQTVEEVL SSLGPVVDAH PEYEEVALLE RMVIPERVIE

[0034] 61 FRVPWEDDNG KVHVNTGYRV QFNGAIGPYK GGLRFAPSVN LSIMKFLGFE QAFKDSLTTL

[0035] 121 PMGGAKGGSD FDPNGKSDRE VMRFCQAFMT ELYRHIGPDI DVPAGDLGVG AREIGYMYGQ

[0036] 181 YRKIVGGFYN GVLTGKARSF GGSLVRPEAT GYGSVYYVEA VMKHENDTLV GKTVALAGFG

[0037] 241 NVAWGAAKKL AELGAKAVTL SGPDGYIYDP EGITTEEKIN YMLEMRASGR NKVQDYADKF

[0038] 301 GVQFFPGEKP WGQKVDIIMP CATQNDVDLE QAKKIVANNV KYYIEVANMP TTNEALRFLM

[0039] 361 QQPNMVVAPS KAVNAGGVLV SGFEMSQNSE RLSWTAEEVD SKLHQVMTDI HDGSAAAAER

[0040] 421 YGLGYNLVAG ANIVGFQKIA DAMMAQGIAW (SEQ ID NO.1).

[0041] The series of mutants obtained by single-site or multi-site mutations of the amino acid sequence shown in SEQ ID NO.1 are shown in Table 1.

[0042] Table 1 Glutamate dehydrogenase CsGluDH mutation sites

[0043]

[0044] Note: The various mutants in Table 1 are various mutants obtained by mutating the wild-type glutamate dehydrogenase CsGluDH with the amino acid sequence shown in SEQ ID NO.1. The meanings of the English abbreviations of amino acids are as follows: V refers to valine, L refers to leucine, A refers to alanine, G refers to glycine, I refers to isoleucine, and P refers to proline.

[0045] Test Example 1 Enzyme Activity Detection Test of Glutamate Dehydrogenase CsGluDH Mutants

[0046] The coding genes of the wild-type glutamate dehydrogenase (NCBI accession number CAA77805.1, EC 1.4.1.2) and its mutants derived from SEQ ID NO.1 in Example 1 were sent to a gene synthesis company for total gene synthesis and cloned into the pET28a vector. The recombinant vector containing the target gene was expressed in Escherichia coli BL21 (DE3). After verification by PCR and sequencing, positive colonies were picked for culture and induction to prepare wet bacterial cells expressing glutamate dehydrogenase shown in SEQ ID NO.1 or its single-site and multi-site mutants.

[0047] In addition, the full synthetic genes of the wild-type glutamate dehydrogenase (NCBI accession number WP_003497202.1) gene and its single-site mutants A164G and V378A derived from Clostridium symbiosum in the publication number CN108588045A (invention name: Glutamate Dehydrogenase Mutants and Their Application in the Preparation of L-Glyphosate) were cloned into the pET28a vector and expressed in Escherichia coli BL21 (DE3). After verification by PCR and sequencing, positive colonies were picked for culture and induction to prepare wet bacterial cells expressing Clostridium symbiosum glutamate dehydrogenase (NCBI accession number WP_003497202.1) or its single-site mutants.

[0048] The amino acid sequence of the glutamate dehydrogenase (NCBI accession number WP_003497202.1) derived from Clostridium symbiosum in the publication number CN108588045A (invention name: Glutamate Dehydrogenase Mutants and Their Application in the Preparation of L-Glyphosate) is shown as SEQ ID NO.2 as follows:

[0049] 1 MSKYVDRVIA EVEKKYADEP EFVQTVEEVL SSLGPVVDAH PEYEEVALLE RMVIPERVIE

[0050] 61 FRVPWEDDNG KVHVNTGYRV QFNGAIGPYK GGLRFAPSVN LSIMKFLGFE QAFKDSLTTL

[0051] 121 PMGGAKGGSD FDPNGKSDRE VMRFCQAFMT ELYRHIGPDI DVPAGDLGVG AREIGYMYGQ

[0052] 181 YRKIVGGFYN GVLTGKARSF GGSLVRPEAT GYGSVYYVEA VMKHENDTLV GKTVALAGFG

[0053] 241 NVAWGAAKKL AELGAKAVTL SGPDGYIYDP EGITTEEKIN YMLEMRASGR NKVQDYADKF

[0054] 301 GVQFFPGEKP WGQKVDIIMP CATQNDVDLE QAKKIVANNI KYYIEVANMP TTNEALRFLM

[0055] 361 QQPNMVVAPS KAVNAGGVLV SGFEMSQNSE RLSWTAEEVD SKLHQVMTDI HDGSAAAAER

[0056] 421 YGLGYNLVAG ANIVGFQKIA DAMMAQGIAW (SEQ ID NO.2).

[0057] All kinds of wet bacterial cells used in the present invention can be prepared according to the following general preparation method: The recombinant strain E. coli BL21(DE3) glycerol bacterial liquid is inoculated into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL, cultured at 37 °C for 15 h, inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1% (v / v), cultured at 37 °C and 180 rpm for 3 h, then IPTG with a final concentration of 0.1 mM is added to the culture solution for induction, cultured at 25 °C and 180 rpm for 16 h, and then centrifuged at 4000 rpm for 15 min. The precipitate collected is the wet bacterial cells containing the target enzyme.

[0058] After resuspending 40 g / L of the wet cells containing glutamate dehydrogenase and 10 g / L of the wet cells containing glucose dehydrogenase evenly, cooling and disrupting the cells, the crude enzyme solution thus prepared was used as a catalyst to produce L-glufosinate. In addition to the crude enzyme solution, the catalytic reaction system also contained 100 mM 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 120 mM glucose, 0.5 mM NAD, 200 mM (NH4)2SO4. The reaction was controlled at pH 7.5, the reaction temperature was 35 °C, and the rotation speed was 180 rpm. After reacting for 10 min, after terminating the reaction, HPLC was used to analyze and detect the production amount and chirality of the product L-glufosinate.

[0059] According to 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 and its mutants (as shown in Table 1), the wild-type glutamate dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 and its single-site mutants A164G and V378A towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid were measured respectively.

[0060] The specific results of the enzyme activity detection are shown in Table 2.

[0061] Table 2 Detection results of the enzyme activities of glutamate dehydrogenase CsGluDH and its mutants

[0062]

[0063] Note: SEQ ID NO.2 - A164G is a mutant obtained by performing a single-site mutation of A164G on the glutamate dehydrogenase shown in SEQ ID NO.2; SEQ ID NO.2 - V378A is a mutant obtained by performing a single-site mutation of V378A on the glutamate dehydrogenase shown in SEQ ID NO.2.

[0064] The enzyme activity detection results in Table 2 show that by using the method of molecular simulation calculation for rational design based on the characteristics of the substrate binding site, channel characteristics and domain interactions, the glutamate dehydrogenase CsGluDH shown in SEQ ID NO.1 was modified and mutated, which could significantly improve its catalytic activity towards 2-oxo-4-(hydroxymethylphosphinyl)butyric acid. The combination mutant V162L / A164G / I279A / A372V / V378A of the binding pocket site, molecular tunnel and domain had the most significant increase in enzyme activity, and its enzyme activity was 998 times that of the wild-type enzyme activity. At the same time, it was 3 times that of the mutant A164G (CN108588045A) of the glutamate dehydrogenase from the source shown in SEQ ID NO.2. Clostridium symbiosum of the glutamate dehydrogenase.

[0065] Test Example 2: Preparation of L-glufosinate by catalytic reaction using the cells containing wild-type glutamate dehydrogenase CsGluDH shown in SEQ ID NO.1 as catalyst

[0066] The reaction system was 30 mL, containing 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of wild-type glutamate dehydrogenase CsGluDH shown in SEQ ID NO.1 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH during the reaction was controlled at 7.5. During the reaction process, the residual amount of the substrate was detected by HPLC, and at the same time, the production amount and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0067] After 12 h of conversion, 490 mM of the substrate remained, the conversion rate was 2.0%, the production concentration of the prepared L-glufosinate was 9.6 mM, and the ee value > 99.9%.

[0068] The amino acid sequence of glucose dehydrogenase is as shown in SEQ ID NO.3 below:

[0069] 1 MYPDLKGKVV AITGAASGLG KAMAIRFGKE QAKVVINYYS NKQDPNEVKE EVIKAGGEAV

[0070] 61 VVQGDVTKEE DVKNIVQTAI KEFGTLDIMI NNAGLENPVP SHEMPLKDWD KVIGTNLTGA

[0071] 121 FLGSREAIKY FVENDIKGNV INMSSVHEVI PWPLFVHYAA SKGGIKLMTR TLALEYAPKG

[0072] 181 IRVNNIGPGA INTPINAEKF ADPKQKADVE SMIPMGYIGE PEEIAAVAAW LASKEASYVT

[0073] 241 GITLFADGGM TLYPSFQAGR G (SEQ ID NO.3).

[0074] Test Example 3: Preparation of L-glufosinate by catalytic reaction using the cells containing wild-type glutamate dehydrogenase CsGluDH shown in SEQ ID NO.2 as catalyst

[0075] The reaction system was 30 mL, containing 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the wild-type glutamate dehydrogenase CsGluDH shown in SEQ ID NO.2 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction process. The residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected by chiral derivatizing reagents.

[0076] After 12 h of conversion, the remaining substrate was 491.2 mM, the conversion rate was 1.8%, the production concentration of the prepared L-glufosinate was 7.5 mM, and the ee value > 99.9%.

[0077] Test Example 4: Preparation of L-glufosinate using the cells containing the mutant A164G of glutamate dehydrogenase shown in SEQ ID NO.1 as a catalyst

[0078] The reaction system was 30 mL, containing 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the mutant A164G of glutamate dehydrogenase shown in SEQ ID NO.1 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction process. The residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected by chiral derivatizing reagents.

[0079] After 5.6 h of conversion, the remaining substrate was 0.5 mM, the conversion rate was 99.9%, the production concentration of L-glufosinate was 493 mM, and the ee value > 99.9%.

[0080] Test Example 5: Preparation of L-glufosinate using the cells containing the mutant A164G of glutamate dehydrogenase shown in SEQ ID NO.2 as a catalyst

[0081] The reaction system is 30 mL and contains 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant A164G shown in SEQ ID NO.2 is 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) is 1.25 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.5 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate are detected by using a chiral derivatizing reagent.

[0082] After 6 h of conversion, 0.5 mM of the substrate remained, the conversion rate was 99.9%, the production concentration of the prepared L-glufosinate was 436 mM, and the ee value > 99.9%.

[0083] Test Example 6: Test for catalytic preparation of L-glufosinate using the cells containing the glutamate dehydrogenase mutant I279A shown in SEQ ID NO.1 as a catalyst

[0084] The reaction system is 30 mL and contains 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant I279A shown in SEQ ID NO.1 is 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) is 1.25 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.5 during the reaction process. The residual amount of the substrate is detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate are detected by using a chiral derivatizing reagent.

[0085] After 5 h of conversion, 0 mM of the substrate remained, the conversion rate was 100%, the production concentration of the prepared L-glufosinate was 495 mM, and the ee value > 99.9%.

[0086] Test Example 7: Test for catalytic preparation of L-glufosinate using the cells containing the glutamate dehydrogenase mutant A372V shown in SEQ ID NO.1 as a catalyst

[0087] The reaction system was 30 mL and contained 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant V372A shown in SEQ ID NO.1 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction process, the residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected by using a chiral derivatizing reagent.

[0088] After 4.6 h of conversion, the remaining substrate was 0 mM, the conversion rate was 100%, the production concentration of the prepared L-glufosinate was 496 mM, and the ee value > 99.9%

[0089] Test Example 8: Test for catalytic preparation of L-glufosinate using the cells containing the glutamate dehydrogenase mutant V378A shown in SEQ ID NO.1 as a catalyst

[0090] The reaction system was 30 mL and contained 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant V378A shown in SEQ ID NO.1 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction process, the residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected by using a chiral derivatizing reagent.

[0091] After 10.9 h of conversion, the remaining substrate was 0 mM, the conversion rate was 100%, the production concentration of the prepared L-glufosinate was 491 mM, and the ee value > 99.9%.

[0092] Test Example 9: Test for catalytic preparation of L-glufosinate using the cells containing the glutamate dehydrogenase mutant V378A shown in SEQ ID NO.2 as a catalyst

[0093] The reaction system was 30 mL and contained 500 mM of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant V378A shown in SEQ ID NO.2 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction, the residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0094] After 12 h of conversion, 1.5 mM of the substrate remained, the conversion rate was 99.7%, the production concentration of the prepared L-glufosinate was 434 mM, and the ee value > 99.9%.

[0095] Test Example 10: Test for catalytic preparation of L-glufosinate using the cells containing the multi-site mutant V162L / I279A / A372V of glutamate dehydrogenase shown in SEQ ID NO.1 as the catalyst

[0096] The reaction system was 30 mL and contained 500 mM of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant V162L / I279A / A372V shown in SEQ ID NO.1 was 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) was 1.25 g / L. The reaction temperature was controlled at 35 °C, the pH was controlled at 7.5 during the reaction, the residual amount of the substrate was detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate were detected using a chiral derivatizing reagent.

[0097] After 2.5 h of conversion, 0 mM of the substrate remained, the conversion rate was 100%, the production concentration of the prepared L-glufosinate was 498 mM, and the ee value > 99.9%.

[0098] Test Example 11: Test for catalytic preparation of L-glufosinate using the cells containing the multi-site mutant A164G / V378A of glutamate dehydrogenase shown in SEQ ID NO.1 as the catalyst

[0099] The reaction system is 30 mL, containing 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant A164G / V378A shown in SEQ ID NO.1 is 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) is 1.25 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.5 during the reaction process. The residual situation of the substrate is detected by HPLC during the reaction process, and at the same time, the production amount and ee value of L-glufosinate are detected by using a chiral derivatizing reagent.

[0100] After 3.5 h of conversion, the remaining substrate is 0 mM, the conversion rate is 100%, the production concentration of the prepared L-glufosinate is 497 mM, and the ee value > 99.9%.

[0101] Test Example 12: Test for catalytic preparation of L-glufosinate using the cells containing the multi-site mutant A164G / I279A / V378A of glutamate dehydrogenase shown in SEQ ID NO.1 as a catalyst

[0102] The reaction system is 30 mL, containing 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant A164G / I279A / V378A shown in SEQ ID NO.1 is 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) is 1.25 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.5 during the reaction process. The residual situation of the substrate is detected by HPLC during the reaction process, and at the same time, the production amount and ee value of L-glufosinate are detected by using a chiral derivatizing reagent.

[0103] After 3 h of conversion, the remaining substrate is 0 mM, the conversion rate is 100%, the production concentration of the prepared L-glufosinate is 498 mM, and the ee value > 99.9%.

[0104] Test Example 13: Test for catalytic preparation of L-glufosinate using the cells containing the multi-site mutant V162L / A164G / I279A / A372V / V378A of glutamate dehydrogenase shown in SEQ ID NO.1 as a catalyst

[0105] The reaction system is 30 mL and contains 500 mM of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid substrate, 0.5 mM NAD, 600 mM glucose, 250 mM (NH4)2SO4. The dry cell weight concentration of the glutamate dehydrogenase mutant V162L / A164G / I279A / A372V / V378A shown in SEQ ID NO.1 is 1.25 g / L, and the dry cell weight concentration of glucose dehydrogenase (SEQ ID NO.3) is 1.25 g / L. The reaction temperature is controlled at 35 °C, the pH is controlled at 7.5 during the reaction process, the residual amount of the substrate is detected by HPLC during the reaction process, and the production amount and ee value of L-glufosinate are detected by a chiral derivatizing reagent.

[0106] After 2 h of conversion, the remaining substrate is 0 mM, the conversion rate is 100%, the production concentration of the prepared L-glufosinate is 498 mM, and the ee value > 99.9%.

Claims

1. A mutant of glutamate dehydrogenase, characterized in that, The mutant is obtained by performing any one of the mutations of I279A, V162L / I279A / A372V, A164G / I279A / V378A, or V162L / A164G / I279A / A372V / V378A on the amino acid sequence of glutamate dehydrogenase shown in SEQ ID NO. 1, where the meanings of the English abbreviations of amino acids are as follows: V represents valine, L represents leucine, A represents alanine, G represents glycine, and I represents isoleucine.

2. A coding gene for the mutant according to claim 1.

3. An expression cassette or chimeric gene containing 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. Including:

6. A method for preparing the mutant as described in claim 1, characterized in that, (1) A recombinant expression vector is constructed by operably connecting the coding gene of the mutant according to 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 according to claim 1, the coding gene according to claim 2, the recombinant expression vector according to claim 4, or the recombinant host cell according to claim 5 in the synthesis of L-glufosinate. Including:

8. A method for the biosynthesis of L-glufosinate, characterized in that, Using the mutant according to claim 1 as a catalytic enzyme and 2-oxo-4-(hydroxymethylphosphonyl)butyric acid as a substrate to perform 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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