Glutamate dehydrogenase mutant with high thermal stability and method for efficiently preparing l-glufosinate using the same

Abstract

The present application relates to the field of bioengineering, and particularly relates to glutamate dehydrogenase mutant with high thermal stability and method for efficiently preparing L-glufosinate using the same. The present application provides a mutant of glutamate dehydrogenase, which is obtained after single-point mutation or multi-point mutation of positions 8, 61, 62, 67, 142, 161, 179, 237, 238, 252, 275, 281, 335, 345, 354, 395, 397, 412 and 421 of glutamate dehydrogenase. The mutant obtained by the present application significantly improves the enzyme activity and thermal stability of the process for converting substrate 2-carbonyl-4-(hydroxymethyl phosphine) butyric acid to produce L-glufosinate. When the glutamate dehydrogenase mutant of the present application is used to prepare L-glufosinate, the catalytic efficiency of the enzyme is improved, the reaction cost is reduced, and it is conducive to industrial production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a glutamate dehydrogenase mutant with high thermal stability and a method for efficiently preparing L-glufosinate using it. Background Technology

[0002] Glufosinate (also known as 2-amino-4-[hydroxy(methyl)phosphono]butyric acid) works by competitively occupying the active site of glutamine synthase, inhibiting its activity, and thus interfering with the plant's nitrogen metabolism. This leads to the accumulation of free ammonium ions and ultimately plant death. Due to its low toxicity, high efficiency, broad spectrum of herbicides, and long-lasting effect, it has become the world's second best-selling broad-spectrum contact herbicide.

[0003] Glufosinate contains two optical isomers, D- and L-, but only L-glufosinate (also known as refined glufosinate) possesses herbicidal activity. Although there are numerous reports on the chemical synthesis of L-glufosinate, these methods generally suffer from lengthy synthetic steps, complex processes, low yields, and high costs of chiral resolution reagents. In contrast, biosynthesis offers advantages such as strong stereoselectivity, mild reaction conditions, high product yields, and ease of separation and purification, demonstrating significant commercial potential and social benefits.

[0004] Glutamate dehydrogenase (EC 1.4.1.2–4) is a key enzyme catalyzing the synthesis and breakdown of glutamate, widely distributed in various living organisms. Based on coenzyme specificity, this enzyme can be classified into three types: NAD(H)-dependent, NADP(H)-dependent, and dual-coenzyme-dependent. Due to its wide availability and high stereoselectivity, glutamate dehydrogenase shows significant potential for application in the synthesis of chiral amino acids. However, naturally occurring glutamate dehydrogenases typically exhibit strict substrate specificity, showing extremely low catalytic activity for keto acids other than α-ketoglutarate. Furthermore, under non-physiological conditions, these enzymes often exhibit low stability, easily leading to inactivation, which limits their industrial applications. Enhancing the thermostability of proteins helps accelerate reaction rates and shorten reaction times; simultaneously, proteins with improved stability can be stored and transported at room temperature, reducing costs. Therefore, obtaining thermostable alcohol dehydrogenases has significant industrial application value.

[0005] Several biosynthetic routes for L-glufosinate have been reported in the prior art. For example, patent CN108588045B discloses a method for preparing L-glufosinate by catalytic reduction of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) using a glutamate dehydrogenase mutant. However, there is still a need in this field to further improve the catalytic activity and thermal stability of glutamate dehydrogenases for PPO. Therefore, improving the catalytic efficiency of this enzyme for such non-natural substrates through molecular modification will help promote its practical application in the industrial synthesis of L-glufosinate. Summary of the Invention

[0006] In view of this, the present invention provides a mutant of glutamate dehydrogenase and its application in the synthesis of L-glufosinate. The glutamate dehydrogenase mutant obtained by the present invention significantly improves the enzyme activity and thermostability in the process of converting the substrate 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to L-glufosinate. When using the glutamate dehydrogenase mutant of the present invention to prepare L-glufosinate, the enzyme's catalytic efficiency is improved, the reaction cost is reduced, and it is beneficial for industrial production.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides mutants of glutamate dehydrogenase, obtained by single-point or multi-point mutations at positions 8, 61, 62, 67, 142, 161, 179, 237, 238, 252, 275, 281, 335, 345, 354, 395, 397, 412, and 421 of the glutamate dehydrogenase.

[0009] In some embodiments of the present invention, in the above-mentioned mutants, the valine at position 8 is replaced by methionine; and / or the alanine at position 61 is replaced by isoleucine; and / or the valine at position 62 is replaced by isoleucine; and / or the serine at position 67 is replaced by proline; and / or the alanine at position 142 is replaced by serine; and / or the alanine at position 161 is replaced by proline; and / or the phenylalanine at position 179 is replaced by tyrosine; and / or the alanine at position 237 is replaced by valine; and / or the valine at position 238 is replaced by isoleucine; and / or the... Valine at position 252 is replaced by leucine; and / or leucine at position 275 is replaced by phenylalanine; and / or aspartic acid at position 281 is replaced by alanine; and / or threonine at position 335 is replaced by valine or methionine; and / or alanine at position 345 is replaced by valine; and / or leucine at position 354 is replaced by proline; and / or threonine at position 395 is replaced by proline; and / or glycine at position 397 is replaced by glutamine; and / or histidine at position 412 is replaced by lysine; and / or alanine at position 421 is replaced by glycine.

[0010] In some embodiments of the present invention, the above-mentioned mutants include:

[0011] (1) The glutamate dehydrogenase is derived from *Pseudomonas putida*; and / or

[0012] (2) The glutamate dehydrogenase has a specific amino acid sequence; and / or

[0013] (3) The glutamate dehydrogenase has a specific nucleotide sequence;

[0014] The specific amino acid sequence has:

[0015] (4) An amino acid sequence as shown in SEQ ID NO:3; or

[0016] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0017] (6) An amino acid sequence that is at least 70% homologous to the amino acid sequence shown in (4) or (5);

[0018] The specific nucleotide sequence has:

[0019] (7) A nucleotide sequence as shown in SEQ ID NO:4; or

[0020] (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and whose function is the same as or similar to that of (7); or

[0021] (9) A nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in (7) or (8).

[0022] In some embodiments of the present invention, the above-mentioned mutant has:

[0023] (10) An amino acid sequence as shown in any of SEQ ID NO:5 to SEQ ID NO:30; or

[0024] (11) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (10), and whose function is the same as or similar to that of (10); or

[0025] (12) An amino acid sequence that is at least 70% homologous to the amino acid sequence shown in (10) or (11).

[0026] The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant, having:

[0027] (13) A nucleotide sequence as shown in any of SEQ ID NO:31 to SEQ ID NO:56; or

[0028] (14) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (13), and whose function is the same as or similar to that of (13); or

[0029] (15) A nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in (13) or (14).

[0030] The present invention also provides an expression vector comprising: the above-described nucleic acid molecule and an acceptable gene element.

[0031] The present invention also provides a host cell for transforming or transfecting the above-mentioned nucleic acid molecules and / or the above-mentioned expression vectors.

[0032] The present invention also provides products comprising: the above-described mutant, the above-described nucleic acid molecule, the above-described expression vector, and / or the above-described host cell.

[0033] This invention also provides the use of the above-described mutants, nucleic acid molecules, expression vectors, host cells, and / or products in any of the following:

[0034] (a) Improving the conversion rate of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to L-glufosinate; and / or

[0035] (b) Improve the thermostability of glutamate dehydrogenase; and / or

[0036] (c) Synthesis of L-glufosinate.

[0037] The present invention also provides a method for synthesizing L-glufosinate, comprising: converting raw materials into L-glufosinate by any of the following conversions;

[0038] ( The above mutants; or

[0039] ( ), or the above nucleic acid molecules; or

[0040] ( ), or the above-mentioned expression carriers; or

[0041] ( ), the aforementioned host cells; or

[0042] ( The above-mentioned products;

[0043] The raw materials include: PPO and NADP. + NH4Cl, isopropanol, and isopropanol dehydrogenase.

[0044] The beneficial effects of this invention include:

[0045] (1) This invention is based on glutamate dehydrogenase (PpGluDH, NCBI accession number: WP_010951932.1) from Pseudomonas putida, and performs combined mutations to obtain mutant strains with high activity and high thermal stability in catalyzing PPO.

[0046] (2) The glutamate dehydrogenase mutant of the present invention significantly improves the catalytic activity and thermal stability of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO). The optimal combination of mutants increases enzyme activity by 2.33 times, and the enzyme activity increases by 2.61 times after heat treatment.

[0047] (3) This invention uses 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate and utilizes glutamate dehydrogenase mutant and alcohol dehydrogenase to synthesize L-glufosinate. The substrate conversion rate is >99.9%, which successfully solves the problem of low activity of wild-type glutamate dehydrogenase for non-natural substrates. It is an ideal solution for preparing L-glufosinate and can be widely used in the market. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0049] Figure 1 The high-performance liquid chromatography (HPLC) spectrum of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) standard is shown.

[0050] Figure 2 The high-performance liquid chromatography (HPLC) spectrum of L-glufosinate standard is shown;

[0051] Figure 3 High-performance liquid chromatography (HPLC) chromatograms of samples after the double-enzyme coupling preparation of L-glufosinate were completed to detect PPO content.

[0052] Figure 4 High-performance liquid chromatography (HPLC) chromatograms of L-glufosinate content in samples after the double-enzyme coupling preparation of L-glufosinate are shown. Detailed Implementation

[0053] This invention discloses a glutamate dehydrogenase mutant with high thermal stability and a method for efficiently preparing L-glufosinate using it.

[0054] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0055] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0056] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0057] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0058] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0059] This invention provides a thermostable variant of glutamate dehydrogenase, wherein the glutamate dehydrogenase mutant has the 167th amino acid of the amino acid sequence shown in SEQ ID NO:3 replaced with glycine, and contains one or more of the following mutation sites: valine at position 8, alanine at position 61, valine at position 62, serine at position 67, alanine at position 142, alanine at position 161, phenylalanine at position 179, alanine at position 237, valine at position 238, valine at position 252, leucine at position 275, aspartic acid at position 281, threonine at position 335, alanine at position 345, leucine at position 354, threonine at position 395, glycine at position 397, histidine at position 412, and alanine at position 421.

[0060] This invention involves combinatorial mutation of the NADP(H)-specific glutamate dehydrogenase (PpGluDH, NCBI accession number: WP_010951932.1, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) derived from Pseudomonas putida to obtain mutant strains with high activity and high thermal stability in catalyzing PPO.

[0061] Furthermore, the thermostability-enhanced glutamate dehydrogenase mutant is one of the following combinations of mutations:

[0062] This invention provides an amino acid sequence encoding glutamate dehydrogenase, having at least one of the following sequences:

[0063] V8M / A167G, A61I / A167G, V62I / A167G, S67P / A167G, A142S / A167G, A161P / A167G, A167G / F179Y, A167G / A2 37V, A167G / V238I, A167G / V252L, A167G / L275F, A167G / D281A, A167G / T335V, A167G / T335M, A167G / A345V, A167G / L354P, A167G / T395P, A167G / G397Q, A167G / H412K, A167G / A421G, V62I / A161P / A167G, V62I / A167G / V238I, V62I / A167G / T335M, V62I / A167G / T395P, V62I / A161P / A167G / V238I, V62I / A167G / V238I / T335M3.

[0064] The " / " in the above text means "and", that is, the two sites before and after the " / " are mutated at the same time; for example, V62I / A167G means that the valine at position 62 is mutated to isoleucine, and the amino acid at position 167 is mutated from alanine to glycine.

[0065] The present invention also provides the encoding gene for the glutamate dehydrogenase variant as described in any of the preceding claims.

[0066] The present invention also provides an expression vector comprising the coding gene as described above. Preferably, the original expression vector is pET-28a(+).

[0067] The present invention also provides a genetically engineered bacterium containing the coding gene as described above. Preferably, the host cell of the genetically engineered bacterium is E. coli BL21(DE3).

[0068] The present invention also provides the application of the thermostable glutamate dehydrogenase mutant described above in the preparation of L-glufosinate.

[0069] The present invention also provides the application of the genetically engineered bacteria described above in the preparation of L-glufosinate.

[0070] The thermostability-enhanced glutamate dehydrogenase of the present invention is obtained by host fermentation culture of a genome integrating the nucleic acid or expression unit of the glutamate dehydrogenase gene of the present invention, or by host fermentation culture of a transfected or transformed plasmid vector or plasmid combination of the present invention.

[0071] This invention also provides a method for preparing L-glufosinate, comprising using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate, and in the presence of an amino donor and a coenzyme regeneration system, carrying out a catalytic reaction in a buffer solution using a biocatalyst to obtain L-glufosinate. The biocatalyst is a glutamate dehydrogenase mutant or its immobilized enzyme; or, as described above, the genetically engineered bacteria.

[0072] The coenzyme regeneration system uses alcohol dehydrogenase as the coenzyme regeneration enzyme and isopropanol as the coenzyme regeneration substrate.

[0073] Specifically, the temperature of the catalytic reaction is 30~50℃, the reaction time is 6~12h, and the pH value of the reaction solution is 6~9.

[0074] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0075] Reagents used in upstream genetic engineering: DPNI used in these embodiments was purchased from TaKaRa, Takara Bio Engineering (Dalian) Co., Ltd.; plasmid extraction kit and DNA recovery and purification kit were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21(DE3), plasmid pET-28a(+), etc., were purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose gel electrophoresis reagent were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and sequencing were performed by Qingke Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0076] This invention relates to recombinant Escherichia coli carrying the glutamate dehydrogenase gene, all of which were constructed and preserved in our laboratory. The vector used was PET28a(+), and the host used was Escherichia coli E. coli BL21(DE3).

[0077] The reagents used in the downstream catalytic process: 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) was artificially synthesized. D,L-glufosinate standards were purchased from Sigma-Aldrich; NADP... + NADPH was purchased from Bangtai Biotechnology (Shenzhen) Co., Ltd.; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The three-letter or single-letter expressions for amino acids used in this application follow the amino acid codes specified by IUPAC (Eur. J. Biochem., 138:9-37, 1984).

[0078] This invention uses high-performance liquid chromatography (HPLC) to analyze the concentration of the substrate PPO in the reaction solution and monitor the reaction process. The specific method is as follows: Column type: Pntulips® QS-C18, 5µm, 4.6×250mm. Mobile phase: 50mM (NH4)2HPO4, with 1% of 10% tetrabutylammonium hydroxide aqueous solution added, pH adjusted to 3.6 with 50% phosphoric acid (mass fraction), and 8% acetonitrile added. Detection wavelength: 205nm. Flow rate: 1mL / min. Column temperature: 40℃. See attached figure for substrate peak characteristics. Figure 1 .

[0079] The concentration of L-glufosinate was determined by high performance liquid chromatography (HPLC), specifically using pre-column derivatization. Chromatographic conditions: column: Pntulips® QS-C18; detection wavelength: 338 nm; column temperature: 30℃; injection volume: 20 µL; mobile phase: 50 mM sodium acetate aqueous solution: acetonitrile = 9:0.5; flow rate: 1 mL / min.

[0080] Derivatization reagent: Weigh 0.03 g of phthalaldehyde and 0.1 g of N-acetyl-L-cysteine, add 400 µL of anhydrous ethanol and 4 mL of 0.2 M (pH 9.8) borate buffer, sonicate to dissolve completely, and use immediately.

[0081] Derivatization reaction and determination: Take 100 µL of sample, add 100 µL of derivatization reagent, mix well, and incubate at 25℃ for 5 min.

[0082] L-glufosinate peak elution is as follows: Figure 2 As shown.

[0083] The amino acid sequence of PpGluDH derived from Pseudomonas putida is as follows:

[0084] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 1).

[0085] The nucleic acid sequence of said PpGluDH derived from Pseudomonas putida is:

[0086]

[0087] The amino acid sequence of the mutant A167G is:

[0088] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 3).

[0089] The amino acid sequence of the mutant V8M / A167G is:

[0090] MSTMIESMDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 5).

[0091] The amino acid sequence of the mutant A61I / A167G is:

[0092] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERIVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 6).

[0093] The amino acid sequence of the mutant V62I / A167G is as follows:

[0094] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFE QVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKR QDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRN GCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO.7).

[0095] The amino acid sequence of the mutant S67P / A167G is as follows:

[0096] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVPWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV(SEQ ID NO.8).

[0097] The amino acid sequence of the mutant A142S / A167G is:

[0098] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDSEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV(SEQ ID NO.9).

[0099] The amino acid sequence of said mutant A161P / A167G is:

[0100] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGPDCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 10).

[0101] The amino acid sequence of the mutant A167G / F179Y is:

[0102] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGYMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 11).

[0103] The amino acid sequence of said mutant A167G / A237V is

[0104] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVVVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 12).

[0105] The amino acid sequence of the mutant A167G / V238I is:

[0106] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAISGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 13).

[0107] The amino acid sequence of the mutant A167G / V252L is:

[0108] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKLMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 14).

[0109] The amino acid sequence of the mutant A167G / L275F is:

[0110] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGFTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV(SEQ ID NO. 15).

[0111] The amino acid sequence of the mutant A167G / D281A is:

[0112] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWAALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 16).

[0113] The amino acid sequence of the mutant A167G / T335V is:

[0114] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARVLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 17).

[0115] The amino acid sequence of the mutant A167G / T335M is:

[0116] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARMLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 18).

[0117] The amino acid sequence of said mutant A167G / A345V is:

[0118] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVVEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV(SEQ ID NO. 19).

[0119] The amino acid sequence of the mutant A167G / L354P is:

[0120] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTPEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 20).

[0121] The amino acid sequence of the mutant A167G / T395P is:

[0122] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWPAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 21).

[0123] The amino acid sequence of said mutant A167G / G397Q is:

[0124] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAQEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 22).

[0125] The amino acid sequence of the mutant A167G / H412K is:

[0126] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHKACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 23).

[0127] The amino acid sequence of said mutant A167G / A421G is:

[0128] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEGDGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 24).

[0129] The amino acid sequence of said mutant V62I / A161P / A167G is:

[0130] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGPDCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 25).

[0131] The amino acid sequence of the mutant V62I / A167G / V238I is:

[0132] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAISGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 26).

[0133] The amino acid sequence of the mutant V62I / A167G / T335M is:

[0134] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARMLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 27).

[0135] The amino acid sequence of the mutant V62I / A167G / T395P is:

[0136] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAVSGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWPAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 28).

[0137] The amino acid sequence of said mutant V62I / A161P / A167G / V238I is:

[0138] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGPDCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAISGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARTLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV (SEQ ID NO. 29).

[0139] The amino acid sequence of the mutant V62I / A167G / V238I / T335M is:

[0140] MSTMIESVDNFLARLKQRDPGQPEFHQAVEEVLRTLWPFLEANPHYLQSGILERMVEPERAILFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPGGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQDKRIDGRRVAISGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEAGLTDAQWDALMELKNVKRGRISELAGQFGLEFRKGQTPWSLPCDIALPCATQNELGAEDARMLLRNGCICVAEGANMPTTLEAVDIFLDAGILYAPGKASNAGGVAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGRINYVKGANIAGFVKVADAMLAQGVV(SEQ ID NO. 30).

[0141] The nucleotide sequence of said mutant A167G is:

[0142]

[0143] The nucleotide sequence of the mutant V8M / A167G is as follows:

[0144]

[0145] The nucleotide sequence of the mutant A61I / A167G is as follows:

[0146]

[0147] The nucleotide sequence of the mutant V62I / A167G is as follows:

[0148]

[0149] The nucleotide sequence of the mutant S67P / A167G is as follows:

[0150]

[0151] The nucleotide sequence of the mutant A142S / A167G is as follows:

[0152]

[0153] The nucleotide sequence of the mutant A161P / A167G is as follows:

[0154]

[0155] The nucleotide sequence of the mutant A167G / F179Y is as follows:

[0156]

[0157] The nucleotide sequence of the mutant A167G / A237V is as follows:

[0158]

[0159] The nucleotide sequence of the mutant A167G / V238I is as follows:

[0160]

[0161] The nucleotide sequence of the mutant A167G / V252L is as follows:

[0162]

[0163] The nucleotide sequence of the mutant A167G / L275F is as follows:

[0164]

[0165] The nucleotide sequence of the mutant A167G / D281A is as follows:

[0166]

[0167] The nucleotide sequence of the mutant A167G / T335V is as follows:

[0168]

[0169] The nucleotide sequence of the mutant A167G / T335M is as follows:

[0170]

[0171] The nucleotide sequence of the mutant A167G / A345V is as follows:

[0172]

[0173] The nucleotide sequence of the mutant A167G / L354P is as follows:

[0174]

[0175] The nucleotide sequence of the mutant A167G / T395P is as follows:

[0176]

[0177] The nucleotide sequence of the mutant A167G / G397Q is as follows:

[0178]

[0179] The nucleotide sequence of the mutant A167G / H412K is as follows:

[0180]

[0181] The nucleotide sequence of the mutant A167G / A421G is as follows:

[0182]

[0183] The nucleotide sequence of the mutant V62I / A161P / A167G is as follows:

[0184]

[0185] The nucleotide sequence of the mutant V62I / A167G / V238I is as follows:

[0186]

[0187] The nucleotide sequence of the mutant V62I / A167G / T335M is as follows:

[0188]

[0189] The nucleotide sequence of the mutant V62I / A167G / T395P is as follows:

[0190]

[0191] The nucleotide sequence of the mutant V62I / A161P / A167G / V238I is as follows:

[0192]

[0193] The nucleotide sequence of the mutant V62I / A167G / V238I / T335M is as follows:

[0194]

[0195] In Examples 1 to 3 of this invention, all raw materials and reagents used can be purchased from the market.

[0196] The present invention will be further illustrated below with reference to the embodiments:

[0197] Example 1: Construction of recombinant bacteria and detection of enzyme activity

[0198] 1. Enzyme source and recombinant bacteria construction

[0199] Glutamate dehydrogenase, derived from *Pseudomonas putida* and named PpGluDH, was obtained from the NCBI database. Based on the amino acid sequence and codon optimization according to *E. coli* codon bias, the nucleotide sequence was synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 2. The amino acid sequence encoding the enzyme is shown in SEQ ID NO. 1. A 6×His-tag was added to the end of the nucleotide sequence, and restriction enzyme sites NdeI and XhoI were added to both ends. The gene was cloned into the NdeI and XhoI sites corresponding to pET28a(+), obtaining the recombinant expression plasmid pET28a(+)-PpGluDH. These three plasmids were transformed into *E. coli* BL21(DE3) competent cells to obtain recombinant bacteria *E. coli* BL21(DE3) / pET28a(+)-PpGluDH.

[0200] 2. Plasmid extraction

[0201] The recombinant strain E. coli BL21(DE3) / pET28a(+)-PpGluDH was activated and cultured using LB medium.

[0202] The specific LB medium formula is as follows: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, dissolved in deionized water and brought to a final volume. Sterilize at 121°C for 30 minutes and set aside for use. The solid medium is LB medium with 1.5% agar powder added.

[0203] Glycerol-containing PpGluDH engineered bacteria were streaked onto LB agar plates containing 50 µg / mL kanamycin and incubated at 37°C for 12 h. Single colonies were picked from the plates and inoculated into 5 mL LB agar plates containing 50 µg / mL kanamycin and incubated at 37°C and 200 rpm for 12 h. After obtaining the culture medium, plasmids were extracted according to the instructions of the plasmid extraction kit. The obtained plasmids can be used directly for subsequent operations or stored at -20°C for long-term preservation.

[0204] 3. Site-directed mutation

[0205] (1) Whole plasmid PCR

[0206] Using pET-28a(+)-PpGluDH plasmid as a template, and A167G-F and A167G-R (Table 1) as upstream and downstream primers, full plasmid PCR was performed to obtain the mutant pET-28a(+)-PpGluDH-A167G.

[0207] Using pET-28a(+)-PpGluDH-A167G plasmid as a template, upstream and downstream primers (Table 1) were designed for whole-plasmid PCR to obtain a two-point mutant:

[0208] Table 1 Primers for PpGluDH site-directed mutagenesis

[0209]

[0210]

[0211] PCR amplification system:

[0212] 25 µL of DNA polymerase;

[0213] 1.5 µL of upstream primer (10 pmol / µL);

[0214] 1.5 µL of downstream primer (10 pmol / µL);

[0215] Template 1.0µL;

[0216] ddH2O 21µL.

[0217] PCR amplification conditions:

[0218] 1) Pre-denaturation: 95℃ for 5 min;

[0219] 2) Denaturation: 98℃ for 10s; Annealing: 58℃ for 15s; Extension: 72℃ for 90s; 30 cycles in total;

[0220] 3) Post-extension: 72℃ for 10 minutes;

[0221] 4) Store at 4℃.

[0222] (2) Template digestion:

[0223] The PCR products were subjected to agarose gel electrophoresis, and the plasmid template was recovered and digested with Dpn I enzyme. Digestion of the template was completed at 37°C for 2 hours.

[0224] (3) Transformation and verification:

[0225] After the digestion products were verified by nucleic acid agarose gel electrophoresis, they were transformed into E. coli BL21(DE3) competent cells using the 42℃ heat shock method. The cells were plated, and single colonies were picked and transferred to LB medium for sequencing to verify the correctness of the mutation. Positive mutants that were verified were stored at -80℃.

[0226] 4. Cultivation of bacterial cells and preparation of crude enzyme solution

[0227] After activation by streak plating, single colonies of the preserved mutant strain were inoculated into 5 mL of LB broth containing 100 µg / mL kanamycin and cultured at 37°C with shaking for 12 h. A 2% inoculum was then transferred to 50 mL of LB broth containing 100 µg / mL kanamycin and cultured at 37°C with shaking until the OD600 reached approximately 0.6–0.8. IPTG was then added to a final concentration of 0.5 mM, and the culture was induced at 18°C ​​for 16 h. After culturing, the culture medium was centrifuged at 4000 g at 4°C for 10 min, the supernatant was discarded, and the cells were collected. The collected cells were washed twice with 100 mM pH 8.0 phosphate buffer, resuspended in phosphate buffer, and sonicated at 400 W for 30 cycles, with each cycle lasting 3 seconds and a 7-second interval. The cell lysate was centrifuged at 12000 g at 4°C for 10 min to remove the precipitate; the resulting supernatant was the crude enzyme solution.

[0228] 5. Enzyme activity and heat resistance assay

[0229] The activity of glutamate dehydrogenase was detected using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) as a substrate. The assay system consisted of: appropriate amount of enzyme, 100 mM substrate, 10 mM NADPH coenzyme, 500 mM ammonium sulfate, and a total volume of 400 µL. The reaction medium was phosphate buffer at pH 7.5. The reaction was carried out at 35 °C for 10 min, and 40 µL of 5 M NaOH solution was added to terminate the reaction. The reaction mixture was centrifuged at 4000 rpm for 10 min to remove cells and enzyme proteins. The L-glufosinate generated in the reaction system was determined by high performance liquid chromatography (HPLC). Enzyme activity was defined as the amount of enzyme that can convert the substrate to 1 μmol of L-glufosinate in 1 min at 30 °C, which is 1 U. The activities of wild-type glutamate dehydrogenase and its mutants were determined.

[0230] Using pET-28a(+)-PpGluDH-A167G as the initial control strain, its enzyme activity was set to 100%. The initial relative enzyme activity was the percentage of activity of the untreated mutant enzyme compared to the A167G mutant. The relative enzyme activity after heat treatment was the percentage of mutant enzyme activity compared to the A167G mutant activity after incubation at 50°C for 2 hours.

[0231] Following the steps outlined above, single-point mutagenesis and enzyme activity / thermotility tests were performed on PpGluDH-A167G. The results are shown in Table 2. The enzyme activity of the initial mutant A167G decreased to 93% of its initial activity after heat treatment. The thermotility of all 20 mutants showed varying degrees of improvement. Among them, the enzyme activity and thermotility of five mutants—V62I / A167G, A161P / A167G, A167G / V238I, A167G / T335M, and A167G / T395P—were significantly improved. V62I / A167G exhibited the best enzyme activity and thermotility, with an activity 175% of the initial mutant A167G, and a post-heat treatment activity of 213% of the initial mutant A167G.

[0232] Table 2. Results of enzyme activity and heat resistance of PpGluDH mutant.

[0233]

[0234] Example 2 Construction of combined mutants and detection of enzyme activity

[0235] Using the PpGluDH-V62I / A167G plasmid as a template, superimposed mutations were performed on the remaining four target sites that significantly enhanced enzyme activity. The mutants were constructed and expressed according to steps 1-4 of Example 1, and the enzyme activity and thermostability of the mutant glutamate dehydrogenase were determined according to step 5 of Example 1. The results are shown in Table 3. The V62I / A161P / A167G, V62I / A167G / V238I, and V62I / A167G / T335M mutants showed significantly enhanced thermostability. Among them, V62I / A167G / V238I was the optimal mutant, with an enzyme activity of 215% of the initial mutant A167G, and an enzyme activity of 258% after heat treatment.

[0236] Further stacking mutations were performed using the PpGluDH-V62I / A167G / V238I plasmid as a template. The results showed that V62I / A167G / V238I / T335M reached the highest enzyme activity, which was 233% of the initial mutant A167G. After heat treatment, the enzyme activity was 261% of the initial mutant A167G.

[0237] Table 3. Results of enzyme activity and thermostability of the combined mutants.

[0238]

[0239] Example 3: Preparation of L-Glufosinate by Dual Enzyme Conjugation

[0240] Through site-directed mutagenesis and screening of PpGluDH, we obtained a mutant strain PpGluDH-V62I / A167G / V238I / T335M with optimal enzyme activity and heat resistance, and constructed a two-enzyme coupling system. Following the method described in step 4 of Example 1, we cultured engineered bacteria, including the control group mutant PpGluDH-A167G and the optimal mutant PpGluDH-V62I / A167G / V238I / T335M. Cells were collected by centrifugation and then sonicated to prepare crude enzyme solution.

[0241] Quantitatively weigh PPO and NADP + Add NH4Cl and isopropanol to a 100 mL reactor, adjust the pH to 8 with 25% ammonia solution, add 600 U of crude glutamate dehydrogenase enzyme solution, and 5 mL of crude alcohol dehydrogenase CbADH (derived from Clostridium beijerinckii, NCBI No.: WP_077844196.1) prepared in the same manner. Adjust the volume to 100 mL with 100 mM pH=8 phosphate buffer, making the final PPO concentration 200 mM and the final NADPH concentration 20 mM. NH4Cl and isopropanol are added. + The concentration was 0.5 M. The temperature was controlled at 40°C using a water bath; the mixture was magnetically stirred, and the reaction time was 10 h.

[0242] L-Glufosinate was prepared using a dual-enzyme coupling reaction of glutamate dehydrogenase and isopropanol dehydrogenase. The contents of PPO and L-glufosinate were measured simultaneously throughout the reaction, and the conversion rate was calculated. After 10 hours of reaction, the PpGluDH-A167G mutant system contained 26.8 mM of PPO, with a conversion rate of 86.6%. Approximately 163.2 mM of L-glufosinate was ultimately produced. The PpGluDH-V62I / A167G / V238I / T335M mutant system contained 0.12 mM of PPO, with a conversion rate of 99.9%. Approximately 194.7 mM of L-glufosinate was ultimately produced. Therefore, the optimal mutant PpGluDH-V62I / A167G / V238I / T335M showed a significantly improved conversion efficiency, making it more suitable for industrial production applications.

[0243] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of glutamate dehydrogenase, characterized in that, The glutamate dehydrogenase was obtained by single-point or multi-point mutation at positions 8, 61, 62, 67, 142, 161, 179, 237, 238, 252, 275, 281, 335, 345, 354, 395, 397, 412, and 421.

2. The mutant as described in claim 1, characterized in that, The valine at position 8 is replaced by methionine; and / or the alanine at position 61 is replaced by isoleucine; and / or the valine at position 62 is replaced by isoleucine; and / or the serine at position 67 is replaced by proline; and / or the alanine at position 142 is replaced by serine; and / or the alanine at position 161 is replaced by proline; and / or the phenylalanine at position 179 is replaced by tyrosine; and / or the alanine at position 237 is replaced by valine; and / or the valine at position 238 is replaced by isoleucine; and / or the valine at position 252 is replaced by... Leucine substitution; and / or the leucine at position 275 is substituted with phenylalanine; and / or the aspartic acid at position 281 is substituted with alanine; and / or the threonine at position 335 is substituted with valine or methionine; and / or the alanine at position 345 is substituted with valine; and / or the leucine at position 354 is substituted with proline; and / or the threonine at position 395 is substituted with proline; and / or the glycine at position 397 is substituted with glutamine; and / or the histidine at position 412 is substituted with lysine; and / or the alanine at position 421 is substituted with glycine.

3. The mutant as described in claim 1 or 2, characterized in that, include: (1) The glutamate dehydrogenase is derived from *Pseudomonas putida*; and / or (2) The glutamate dehydrogenase has a specific amino acid sequence; and / or (3) The glutamate dehydrogenase has a specific nucleotide sequence; The specific amino acid sequence has: (4) An amino acid sequence as shown in SEQ ID NO:3; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or (6) An amino acid sequence that is at least 70% homologous to the amino acid sequence shown in (4) or (5); The specific nucleotide sequence has: (7) A nucleotide sequence as shown in SEQ ID NO:4; or (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and whose function is the same as or similar to that of (7); or (9) A nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in (7) or (8).

4. The mutant according to any one of claims 1 to 3, characterized in that, have: (10) An amino acid sequence as shown in any of SEQ ID NO:5 to SEQ ID NO:30; or (11) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (10), and whose function is the same as or similar to that of (10); or (12) An amino acid sequence that is at least 70% homologous to the amino acid sequence shown in (10) or (11).

5. A nucleic acid molecule encoding a mutant as described in any one of claims 1 to 4, characterized in that, have: (13) A nucleotide sequence as shown in any of SEQ ID NO:31 to SEQ ID NO:56; or (14) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (13), and whose function is the same as or similar to that of (13); or (15) A nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in (13) or (14).

6. An expression carrier, characterized in that, include: The nucleic acid molecule as described in claim 5 and the acceptable gene element.

7. A host cell, characterized in that, Transformation or transfection with the nucleic acid molecule as described in claim 5 and / or the expression vector as described in claim 6.

8. The product, characterized in that, include: The mutant as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, and / or the host cell as described in claim 7.

9. The use of the mutant as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, the host cell as described in claim 7, and / or the product as described in claim 8 in any of the following: (a) Improving the conversion rate of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid to L-glufosinate; and / or (b) Improve the thermostability of glutamate dehydrogenase; and / or (c) Synthesis of L-glufosinate.

10. A method for synthesizing L-glufosinate, characterized in that, include: L-glufosinate was obtained by converting the raw material through any of the following methods; ( (a) the mutant as described in any one of claims 1 to 4; or ( ), or the nucleic acid molecule as described in claim 5; or ( ), or the expression vector as described in claim 6; or ( ( ), the host cell as described in claim 7; or ( ), the product as described in claim 8; The raw materials include: PPO and NADP. + NH4Cl, isopropanol, and isopropanol dehydrogenase.

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