CN112280760B

CN112280760B - Glutamic dehydrogenase mutant and application thereof

Info

Publication number: CN112280760B
Application number: CN202011353159.3A
Authority: CN
Inventors: 穆晓清; 徐岩; 周峰
Original assignee: SUQIAN JIANGNAN UNIVERSITY INDUSTRY TECHNOLOGY INSTITUTE
Current assignee: SUQIAN JIANGNAN UNIVERSITY INDUSTRY TECHNOLOGY INSTITUTE
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-04-15
Anticipated expiration: 2040-11-26
Also published as: CN112280760A

Abstract

The invention discloses a glutamate dehydrogenase mutant and application thereof, belonging to the technical field of enzyme engineering and microbial engineering. The method provided by the invention provides a brand-new method for synthesizing (R) -4-aminopentanoic acid by a biological method; the wild-type glutamate dehydrogenase has no measurable catalytic activity, the mutants of the invention have catalytic activity, and the catalytic activity of the mutants K116S/N348L, K116E/N348M and K116Q/N348M can respectively reach 1.87U/mg, 3.16U/mg and 4.55U/mg, which proves that the technical scheme of the invention realizes the process that the catalytic activity of 4-oxovaleric acid is improved from nothing to nothing, and the biological method provided by the invention for synthesizing (R) -4-aminopentanoic acid is green, environment-friendly and efficient, and has wide industrial application prospect.

Description

Glutamic dehydrogenase mutant and application thereof

Technical Field

The invention relates to a glutamate dehydrogenase mutant and application thereof, belonging to the technical field of enzyme engineering and microbial engineering.

Background

The SacubitriI is an effective component medicine of an anti-heart failure medicine enterento, an enkephalinase inhibitor, and a compound preparation consisting of valsartan and an angiotensin II receptor blocker, and is used for reducing cardiovascular death of patients at risk of heart failure and hospitalizing chronic heart failure (NYHAII-IV grade) and reducing ejection fraction. Optically pure (R) -4-aminopentanoic acid is a chiral building block of a compound Sacubitril; in addition, (R) -4-aminopentanoic acid is a gamma-amino acid and can be used for the research of synthesizing hybrid peptide with physiological activity or special structure; therefore, the (R) -4-aminopentanoic acid has important application value. Asymmetric reductive amination of 4-oxopentanoic acid using an engineered glutamate dehydrogenase is a potentially green reaction for the production of (R) -4-aminopentanoic acid. The glutamate dehydrogenase can catalyze 4-oxo-valeric acid to generate (R) -4-amino-valeric acid in one step only by consuming cheap reducing agents, the byproduct is only water, and the ee value of the product reaches over 95 percent.

To date, no biosynthetic pathway for (R) -4-aminopentanoic acid has been reported. In the chemical synthesis, the problems of complicated process and low optical purity of the product are often existed. Therefore, it is urgently needed to find a method for synthesizing (R) -4-aminopentanoic acid by a high-efficiency biological method.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a glutamate dehydrogenase mutant, wherein the mutant is any one of the following (1) to (9):

(1) obtained by mutating 116 th amino acid lysine and 348 th amino acid asparagine of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO. 1;

(2) obtained by mutating 114 th amino acid lysine and 348 th amino acid asparagine of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO. 2;

(3) obtained by mutating 114 th amino acid lysine and 349 th amino acid asparagine of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO. 3;

(4) mutation is carried out on 112 th amino acid lysine and 344 th amino acid asparagine of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO. 4;

(5) mutation is carried out on 104 th amino acid lysine and 326 th amino acid asparagine of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO. 5;

(6) mutation is carried out on 102 th amino acid lysine and 338 th amino acid asparagine of glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO. 6;

(7) mutation is carried out on 101 th amino acid lysine and 340 th amino acid asparagine of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO. 7;

(8) mutation is carried out on 102 th amino acid lysine and 346 th amino acid asparagine of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO. 8;

(9) the amino acid sequence of the amino acid is shown as SEQ ID NO.9, and the amino acid is obtained by mutating 113 th amino acid lysine and 349 th amino acid asparagine of glutamate dehydrogenase.

In one embodiment of the present invention, the mutant is any one of the following (1) to (9):

(1) the 116 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.1 is mutated into serine from lysine, and the 348 th amino acid is mutated into leucine from asparagine, which is named as K116S/N348L;

or the 116 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.1 is mutated into glutamic acid from lysine, and the 348 th amino acid is mutated into methionine from asparagine, which is named as K116E/N348M;

or the 116 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.1 is mutated into glutamine from lysine, and the 348 th amino acid is mutated into methionine from asparagine, which is named as K116Q/N348M;

(2) the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.2 is mutated into serine from lysine, and the 348 th amino acid is mutated into leucine from asparagine, which is named as K114S/N348L;

or the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.2 is mutated into glutamic acid from lysine, and the 348 th amino acid is mutated into methionine from asparagine, which is named as K114E/N348M;

or the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.2 is mutated into glutamine from lysine, and the 348 th amino acid is mutated into methionine from asparagine, which is named as K116Q/N348M;

(3) the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.3 is mutated into serine from lysine, and the 349 th amino acid is mutated into leucine from asparagine, which is named as K114S/N349L;

or the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.3 is mutated into glutamic acid from lysine, and the 349 th amino acid is mutated into methionine from asparagine, which is named as K114E/N349M;

or the 114 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.3 is mutated into glutamine from lysine, and the 349 th amino acid is mutated into methionine from asparagine, which is named as K116Q/N349M;

(4) the 112 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.4 is mutated into serine from lysine, and the 344 th amino acid is mutated into leucine from asparagine, which is named as K112S/N344L;

or the 112 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.4 is mutated into glutamic acid from lysine, and the 344 th amino acid is mutated into methionine from asparagine, which is named as K112E/N344M;

or the 112 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.4 is mutated into glutamine from lysine, and the 344 th amino acid is mutated into methionine from asparagine, which is named as K112Q/N344M;

(5) the 104 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.5 is mutated into serine from lysine, and the 326 th amino acid is mutated into leucine from asparagine, which is named as K104S/N326L;

or the 104 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.5 is mutated into glutamic acid from lysine, and the 326 th amino acid is mutated into methionine from asparagine, which is named as K104E/N326M;

or the 104 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.5 is mutated into glutamine from lysine, and the 326 th amino acid is mutated into methionine from asparagine, which is named as K104Q/N326M;

(6) the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.6 is mutated into serine from lysine, and the 338 th amino acid is mutated into leucine from asparagine, which is named as K102S/N338L;

or the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.6 is mutated into glutamic acid from lysine, and the 338 th amino acid is mutated into methionine from asparagine, which is named as K102E/N338M;

or the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.6 is mutated into glutamine from lysine, and the 338 th amino acid is mutated into methionine from asparagine, which is named as K102Q/N338M;

(7) the 101 st amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.7 is mutated into serine from lysine, and the 340 st amino acid is mutated into leucine from asparagine, which is named as K101S/N340L;

or the 101 st amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.7 is mutated into glutamic acid from lysine, and the 340 st amino acid is mutated into methionine from asparagine, which is named as K101E/N340M;

or the 101 st amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.7 is mutated into glutamine from lysine, and the 340 st amino acid is mutated into methionine from asparagine, which is named as K101Q/N340M;

(8) the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.8 is mutated into serine from lysine, and the 346 th amino acid is mutated into leucine from asparagine, which is named as K102S/N346L;

or the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.8 is mutated into glutamic acid from lysine, and the 346 th amino acid is mutated into methionine from asparagine, which is named as K102E/N346M;

or the 102 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.8 is mutated into glutamine from lysine, and the 346 th amino acid is mutated into methionine from asparagine, which is named as K102Q/N346M;

(9) the 113 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.9 is mutated into serine from lysine, and the 349 th amino acid is mutated into leucine from asparagine, which is named as K113S/N349L;

or the 113 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.9 is mutated into glutamic acid from lysine, and the 349 th amino acid is mutated into methionine from asparagine, which is named as K113E/N349M;

or the 113 th amino acid of the glutamate dehydrogenase of which the starting amino acid sequence is shown as SEQ ID NO.9 is mutated from lysine to glutamine, and the 349 th amino acid is mutated from asparagine to methionine, which is named as K113Q/N349M.

In one embodiment of the present invention, the nucleotide sequences of the genes encoding glutamate dehydrogenase whose amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively, are shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively.

The invention also provides a recombinant plasmid carrying the gene.

In one embodiment of the invention, the recombinant plasmid uses pET-28a as an expression vector.

The invention also provides a recombinant cell, which carries the gene or carries the recombinant plasmid.

In one embodiment of the invention, the recombinant cell is a bacterial or fungal host cell.

In one embodiment of the present invention, the recombinant cell is an E.coli host cell.

The invention also provides a preparation method of the glutamate dehydrogenase mutant, which comprises the following steps: the recombinant cells are inoculated into an LB culture medium, the recombinant cells are cultured at 37 ℃ until the OD of the cell density reaches 0.5-0.7, IPTG inducer is added, the cell density is induced at 17 ℃ for 15-17h, then bacterial liquid is collected, the obtained bacterial liquid is centrifuged to collect the cells, the obtained cells are resuspended and crushed to obtain cell crushing liquid, the cell crushing liquid is centrifuged to collect supernatant, and glutamate dehydrogenase mutant in the supernatant is separated.

The invention also provides a synthesis method of (R) -4-aminopentanoic acid, which comprises the following steps: adding the above mutant to a solution containing glucose dehydrogenase, 4-oxopentanoic acid, glucose and NH₄Cl-NH₃·H₂And reacting in a reaction system of O buffer solution to obtain the compound.

In one embodiment of the present invention, the method is: adding 0.3-1.0U/mL of the purified enzyme solution of the mutant to a solution containing 20-50 mmol/L4-oxopentanoic acid, 30-75 mmol/L glucose, 0.5-1.5mmol/L NADPH, and 0.5-3M NH₄Cl-NH₃·H₂O (pH 8.5) reaction liquid to obtain a reaction system; the reaction system is placed at 30 ℃ and 200rpm for reaction to prepare (R) -4-aminopentanoic acid.

In one embodiment of the present invention, the method is: adding 0.5U/mL of the purified enzyme solution of the mutant to a solution containing 20 to 50 mmol/L4-oxopentanoic acid, 30 to 75mmol/L glucose, 1mmol/L NADPH, and 1M NH₄Cl-NH₃·H₂O (pH 8.5) reaction liquid to obtain a reaction system; the reaction system is placed at 30 ℃ and 200rpm for reaction to prepare (R) -4-aminopentanoic acid.

The invention also provides the application of the glutamate dehydrogenase mutant, the gene, the recombinant plasmid or the recombinant cell in preparing (R) -4-aminopentanoate.

Advantageous effects

(1) The method provided by the invention provides a brand-new method for synthesizing (R) -4-aminopentanoic acid by a biological method; the activity determination is carried out by taking 4-oxopentanoic acid with the final concentration of 0.8mol/L, wherein wild-type glutamate dehydrogenase has no measurable catalytic activity, the mutants disclosed by the invention have catalytic activity, and the catalytic activities of the mutants K116S/N348L, K116E/N348M and K116Q/N348M can respectively reach 1.87U/mg, 3.16U/mg and 4.55U/mg, so that the technical scheme disclosed by the invention realizes the process that the catalytic activity of the 4-oxopentanoic acid is improved from zero.

(2) The invention constructs a method for preparing (R) -4-aminopentanoic acid by a biological method, and the mutant K116Q/N348M can still effectively catalyze the substrate 4-oxopentanoic acid to produce (R) -4-aminopentanoic acid under a higher substrate concentration (1.2mol/L), and the conversion rate can reach 99%.

Drawings

FIG. 1: the crystal structure (PDB: 6dhd) and key action point analysis of glutamate dehydrogenase derived from bovine liver.

FIG. 2: sequence alignment of glutamate dehydrogenase from different sources and key site conservation analysis.

FIG. 3: schematic representation of mutation library was constructed by overlap extension PCR.

FIG. 4: electrophoresis picture of protein of each dominant mutant strain of Ec-GDH.

FIG. 5: and (3) re-screening the dominant mutant strains by partial high-throughput screening, and comparing relative enzyme activity.

Detailed Description

The invention will be further illustrated with reference to specific examples.

The media involved in the following examples are as follows:

LB liquid medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of NaCl.

LB solid medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of NaCl and 15g/L of agar powder.

The primer sequences involved in the following examples are as follows:

TABLE 1 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 1:

remarking: underlined bases correspond to the corresponding mutation sites, bold font is the enzyme site.

TABLE 2 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 2:

TABLE 3 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 3:

TABLE 4 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 4:

TABLE 5 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 5:

TABLE 6 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 6:

TABLE 7 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 7:

TABLE 8 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 8:

TABLE 9 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 9:

the detection method according to the following example is as follows:

detection of glutamate dehydrogenase enzyme activity:

the reaction volume for determining the activity is 200 mu L, and the total reaction system is as follows: 0.08mg/ml of pure enzyme solution was added to a solution containing Tris-HCl (100mmol/L, pH 9.5), NH₄The reaction was carried out in a reaction solution of Cl (1mol/L), 4-oxopentanoic acid (5mmol/L) and NADH (0.2mmol/L), and the control contained no enzyme solution and was otherwise identical.

The reaction components are respectively kept at 30 ℃ for 2min, and the reaction is initiated by adding enzyme liquid. The activity assay was performed at 30 ℃ for 5min and the change in absorbance at 340nm was recorded every 10 s.

The enzyme activity unit (U) is defined as: the amount of enzyme required to catalytically oxidize 1. mu. mol NADH per minute under the above conditions. Specific activity is defined as: the number of units of enzyme activity per mg of enzyme protein (U/mg) was determined by Coomassie blue assay for protein concentration, all following standard assay procedures and performed in triplicate.

The activity calculation formula is as follows: the enzyme activity (U) is EW multiplied by V multiplied by 1000/6220/L. Wherein: EW is the absorbance change value at 340nm within 1 min; v is the volume of the enzyme activity determination reaction system, the unit is mL, and the volume is 0.2 in the example; 6200 is molar extinction coefficient, unit is l/mol/cm; l is the path length in cm, which is 0.6 in this example.

And (3) calculating specific enzyme activity: specific activity (U/mg) enzyme activity (U)/protein amount (mg).

Detection of 4-oxopentanoic acid content:

determining the content of 4-oxo-valeric acid by a UHPLC method, wherein the determination conditions are as follows: use of

UHPLC analysis was performed on a (Phenomenex) column (100X 2.1 mm; 1.7 μm); condition a: MeCN/H₂A mixture of O + 0.1% formic acid as eluent with a linear gradient (ratio 30/70 in 2 minutes, then ratio 30/70 to 80/20 in 2.5 minutes, then ratio 80/20 in 0.5 minutes); flow 0.5mL min^-1At 25 deg.C, the sample size was 1 μ L, and λ is 360 nm. Condition B: MeCN/H₂The mixture of O + 0.1% formic acid as eluent has a linear gradient (ratio 10/90 in 2 minutes, then 10/90 to 50/50 in 5 minutes, then 50/50 to 100/0 in 2 minutes) with a flow rate of 0.5mL min^-1At 25 deg.C, the sample size was 1 μ L, and λ 340 nm.

Example 1: construction of glutamate dehydrogenase mutation library

FIG. 1 is a crystal structure (PDB: 6dhd) of glutamic acid dehydrogenase derived from bovine liver, which shows the mode of action of glutamic acid as a substrate with the enzyme; wherein the amino acid residues 115K and 350N exhibited by the stick structure interact with the two oxygens of the main chain carboxyl of the substrate glutamic acid, anchoring the substrate main chain; these two amino acid residues are conserved in glutamate dehydrogenase as shown in FIG. 2, and thus the two sites are targeted for modification.

A recombinant plasmid pET-28a-GluDH carrying a glutamate dehydrogenase gene with a nucleotide sequence shown as SEQ ID NO.10 and a recombinant strain E.coli BL21(DE3)/pET-28a-GluDH are synthesized by Shanghai biochemistry, the recombinant strain E.coli BL21(DE3)/pET-28a-GluDH is named as E.coli BL21(DE3)/pET-28a-WT1, and the recombinant strain E.coli BL 21/pET-28 a-GluDH is inoculated into an LB solid culture medium for culture.

Primer pairs (shown in table 1) required by experiments are designed according to target gene sequences at the upper and lower positions of two mutation sites, and are sent to the company of biological engineering (Shanghai) to be synthesized, and site-directed mutation is carried out according to the method shown in the instruction book of STAR Primer GXL kit of TaKaRa. The mutation library is constructed by adopting an overlap extension PCR method.

The PCR reaction system is as follows: mu.L of ultrapure water, 25. mu.L of Prime STAR MAX DNA Polymerase, 1. mu.L of both forward and reverse primers (10pM), 1. mu.L of plasmid template (10ng) containing the glutamate dehydrogenase gene of interest.

The overlap extension method is shown in FIG. 3, first round PCR: amplifying gene fragments A, B and C by using the primer pair sequences shown in the table 1 respectively, and extracting amplification products from agarose gel after agarose gel electrophoresis respectively to obtain: fragment a, fragment B and fragment C.

Second round PCR: and respectively fusing and amplifying the fragment B obtained by the first round of PCR with the fragments A and C, extracting the amplified product from agarose gel after agarose gel electrophoresis, and respectively obtaining purified fusion fragments AB and BC.

Third PCR: and fusing the fragments AB and BC obtained in the second round, and purifying by agarose gel electrophoresis and gel extraction to obtain the final fusion gene.

Inserting the final fusion gene into pET-28a by adopting BamH I and Xho I enzyme digestion to obtain recombinant plasmid, transforming the recombinant plasmid into Escherichia coli DH5 alpha competent cells to obtain recombinant Escherichia coli, inoculating the recombinant Escherichia coli into LB solid culture medium to culture to obtain bacterial colony, extracting plasmid from the obtained recombinant Escherichia coli DH5 alpha cells to obtain recombinant plasmid pET-28 a-fusion protein containing mutant gene, transforming the pET-28 a-fusion protein into Escherichia coli BL21(DE3) cells to obtain recombinant bacterium BL21(DE3)/pET-28 a-fusion protein, and inoculating the recombinant bacterium BL21(DE3)/pET-28 a-fusion protein into LB solid culture medium to culture.

Example 2: preliminary screening of glutamate dehydrogenase mutant libraries

The method comprises the following specific steps:

(1) the recombinant strain BL21(DE3)/pET-28 a-fusion protein single colony obtained in example 1 was picked up, transferred to a 96-well deep-well plate (containing 300. mu.L of LB liquid medium and 50. mu.g/mL of kanamycin per well), and incubated at 37 ℃ and 200rpm for 10-12 hours.

(2) mu.L of each of the wells obtained after the incubation in step (1) was transferred to a corresponding well in a second 96-deep-well plate (each well contained 400. mu.L of LB liquid medium and 50. mu.g/mL kanamycin), and the second 96-deep-well plate was incubated at 37 ℃ and 250rpm for 3-4 hours, respectively. Thereafter, 50. mu.L of LB liquid medium containing 2mM IPTG was added to each well of the second 96-deep well plate to induce protein expression, and then the second 96-deep well plate was further incubated at 20 ℃ and 250rpm for 16-18 h.

(3) The product obtained in step (2) was centrifuged, the cells were collected in a 96-well plate and stored overnight at-80 ℃ to give cell bodies, the 96-well plate carrying the cell bodies was thawed at 37 ℃, 200. mu.L of Tris-HCl buffer (5mM, pH 8.0) containing 0.75mg/mL of lysozyme and 2U/mL of DNase I was added to each well of the plate, and the plate was left at 37 ℃ for 1 hour. Subsequently, each well was supplemented with 200. mu.L of Tris-HCl buffer (5mM, pH 8.0), and the supernatant containing the crude enzyme solution was obtained after centrifugation of each well.

(4) Initial rate of change of absorbance at 340nm (corresponding to the amount of NADH (. epsilon.). 6220M) of the supernatant obtained in each well in step (3) was measured using a microplate reader^-1cm^-1) The reductive amination activity of the enzyme in each well of the microplate was determined at 30 ℃. Reaction system: 200 μ L, containing 20 μ L of cell lysis supernatant, 40mM 4-oxopentanoic acid, 0.2mM NADPH and 2M NH₄Cl/NH₄OH buffer (pH 9.5).

Through the steps, 3066 mutant strains are screened in total, the corresponding coverage rate of the library is more than 95 percent according to a fractional library integrity equation developed by Patrick et al, the enzyme activity data of the mutant K116S/N348L is used as a reference, and the mutant strains with the activity of more than 40mU/mL are screened out; finally obtaining 9 mutants, respectively purifying and testing enzyme activity, obtaining three mutant strains (shown in figure 5) with substrate activity by sequencing, and finding that the

numbers

1, 4, 5, 7 and 9 are K116S/N348L by sequencing;

numbers

2, 3 and 8 are K116E/N348M; the number 6 is K116Q/N348M. Thus, BL21(DE3)/pET-28a-K116S/N348L, BL21(DE3)/pET-28a-K116E/N348M, BL21(DE3)/pET-28a-K116Q/N348M are obtained; wherein, the gene for coding the mutant K116S/N348L is obtained by mutating the codon for coding the 116 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.10 into TCT and mutating the codon for coding the 348 th amino acid into CTG; the gene coding the mutant K116E/N348M is characterized in that the codon coding the 116 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.10 is mutated into GAG, and the codon coding the 348 th amino acid is mutated into ATG; the gene for coding the mutant K116Q/N348M is characterized in that the codon for coding the 116 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.10 is mutated into CAG, and the codon for coding the 348 th amino acid is mutated into ATG.

Example 3: preparation of crude glutamate dehydrogenase

The method comprises the following specific steps:

(1) activating strains: coli BL21(DE3)/pET-28a-WT1 obtained in example 1 and the genetically engineered bacterium containing the glutamate dehydrogenase mutant gene obtained in example 2 were separately: BL21(DE3)/pET-28a-K116S/N348L, BL21(DE3)/pET-28a-K116E/N348M, BL21(DE3)/pET-28a-K116Q/N348M were inoculated into a test tube containing 5mL of LB liquid medium, and cultured at 37 ℃ and 200rpm for 6 to 8 hours to obtain a seed solution.

(2) Preparation of crude enzyme solution: inoculating the activated seed solution into a triangular flask containing 100mL LB liquid medium at an inoculation amount of 2% (v/v), and culturing at 37 deg.C and 200rpm for 2-3 hr to OD₆₀₀0.6-0.8, adding IPTG to a final concentration of 0.1mmol/L, and continuously culturing at 17 deg.C and 200rpm for 12-17h to obtain fermentation liquor; respectively centrifuging the obtained fermentation liquid at 4 deg.C and 8000rpm for 5min, discarding supernatant, collecting thallus, washing thallus with 9% physiological saline twice, and suspending in buffer solution A (100mmol/L Tris, 150mmol/L NaCl, 20mmol/L imidazole pH7.5) to obtain bacterial suspension, and ultrasonically crushing the bacterial suspensionObtaining cell disruption solution, centrifuging the cell disruption solution for 30min at 10000rpm and 4 ℃, and taking supernatant fluid to obtain: the crude enzyme solution contains wild type glutamate dehydrogenase, the crude enzyme solution contains glutamate dehydrogenase mutant K116S/N348L, the crude enzyme solution contains glutamate dehydrogenase mutant K116E/N348M and the crude enzyme solution contains glutamate dehydrogenase mutant K116Q/N348M.

Example 4: purification of glutamate dehydrogenase

The crude enzyme solution containing wild-type glutamate dehydrogenase, the crude enzyme solution containing glutamate dehydrogenase mutant K116S/N348L, the crude enzyme solution containing glutamate dehydrogenase mutant K116E/N348M and the crude enzyme solution containing glutamate dehydrogenase mutant K116Q/N348M, which are obtained in example 3, are respectively filtered by a 0.22um water-based filter membrane, and then are slowly loaded to a Ni-NAT affinity chromatography column, after loading, the column is washed by buffer A, and then is subjected to gradient elution by buffer B (100mmol/L Tris, 150mmol/L NaCl, 500mmol/L imidazole, pH7.5), and an elution peak corresponding to 300mmol/L imidazole is collected.

The identification by SDS-PAGE gave, respectively: the purified wild-type glutamate dehydrogenase, designated WT1 (corresponding to EC-GDH in FIG. 4); mutant K116S/N348L, mutant K116E/N348M and mutant K116Q/N348M (the results are shown in FIG. 4), and the target protein band is 48 kDa; the imidazole in the pure enzyme was subsequently removed by desalting column and concentrated by centrifugation at 4000rpm using ultrafiltration tubes (molecules with a molecular weight cut-off of more than 30 kDa). Adding 10% glycerol, and storing at-80 deg.C.

Example 5: obtaining glutamate dehydrogenase

According to the method of examples 1 to 4, in which the primer sequences involved are as shown in tables 2 to 9, respectively:

wild-type glutamate dehydrogenase (nucleotide sequence shown as SEQ ID NO.11), named WT 2; mutant K114S/N348L, mutant K114E/N348M, mutant K114Q/N348M; wherein, the gene for coding the mutant K114S/N348L is obtained by mutating the codon for coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.11 into TCT and mutating the codon for coding the 348 th amino acid into CTG; the gene coding the mutant K114E/N348M is characterized in that the codon coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.11 is mutated into GAG, and the codon coding the 348 th amino acid is mutated into ATG; the gene for coding the mutant K114Q/N348M is characterized in that the codon for coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.11 is mutated into CAG, and the codon for coding the 348 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence as shown in SEQ ID NO.12), designated WT 3; mutant K114S/N349L; mutant pure enzyme K114E/N349M; mutant K114Q/N349M; wherein, the gene coding the mutant K114S/N349L is obtained by mutating the codon coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.12 into TCT and mutating the codon coding the 348 th amino acid into CTG; the gene coding the mutant K114E/N349M is characterized in that the codon coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.12 is mutated into GAG, and the codon coding the 348 th amino acid is mutated into ATG; the gene for coding the mutant K114Q/N349M is characterized in that the codon for coding the 114 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.12 is mutated into CAG, and the codon for coding the 348 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence shown as SEQ ID NO.13), named WT 4; mutant K112S/N344L; mutant K112E/N344M; mutant K112Q/N344M; wherein, the gene for coding the mutant K112S/N344L is obtained by mutating the codon for coding the 112 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.13 into TCT and mutating the codon for coding the 344 th amino acid into CTG; the gene coding the mutant K112E/N344M is characterized in that the codon coding the 112 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.13 is mutated into GAG, and the codon coding the 344 th amino acid is mutated into ATG; the gene for coding the mutant K112Q/N344M is characterized in that the codon for coding the 112 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.13 is mutated into CAG, and the codon for coding the 344 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence is shown as SEQ ID NO.14), named WT5 mutant K104S/N326L; mutant K104E/N326M mutant K104Q/N326M; wherein, the gene for coding the mutant K104S/N326L is obtained by mutating the codon for coding the 104 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.14 into TCT and mutating the codon for coding the 326 th amino acid into CTG; the gene coding the mutant K104E/N326M is characterized in that the codon coding the 104 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.14 is mutated into GAG, and the codon coding the 326 th amino acid is mutated into ATG; the gene for coding the mutant K104Q/N326M is characterized in that the codon for coding the 104 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.14 is mutated into CAG, and the codon for coding the 326 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence shown as SEQ ID NO.15), named WT 6; mutant K102S/N338L; mutant K102E/N338M; mutant K102Q/N338M; wherein, the gene for coding the mutant K102S/N338L is obtained by mutating the codon for coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.15 into TCT and mutating the codon for coding the 338 th amino acid into CTG; the gene coding the mutant K102E/N338M is characterized in that the codon coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.15 is mutated into GAG, and the codon coding the 338 th amino acid is mutated into ATG; the gene for coding the mutant K102Q/N338M is characterized in that the codon for coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.15 is mutated into CAG, and the codon for coding the 338 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence shown as SEQ ID NO.16), named WT 7; mutant K101S/N340L; mutant K101E/N340M; mutant K101Q/N340M; wherein, the gene for coding the mutant K101S/N340L is obtained by mutating the codon for coding the 101 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.16 into TCT and mutating the codon for coding the 340 th amino acid into CTG; the gene of the coded mutant K101E/N340M is characterized in that the codon of the coded 101 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.16 is mutated into GAG, and the codon of the coded 340 th amino acid is mutated into ATG; the gene for coding the mutant K101Q/N340M is characterized in that the codon for coding the 101 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.16 is mutated into CAG, and the codon for coding the 340 th amino acid is mutated into ATG;

the wild enzyme glutamate dehydrogenase (nucleotide sequence is shown as SEQ ID NO.17) is named WT 8; mutant K102S/N346L; mutant K102E/N346M; mutant K102Q/N346M; wherein, the gene for coding the mutant K102S/N346L is obtained by mutating the codon for coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.17 into TCT and mutating the codon for coding the 346 th amino acid into CTG; the gene coding the mutant K102E/N346M is characterized in that the codon coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.17 is mutated into GAG, and the codon coding the 346 th amino acid is mutated into ATG; the gene for coding the mutant K102Q/N346M is characterized in that the codon for coding the 102 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.17 is mutated into CAG, and the codon for coding the 346 th amino acid is mutated into ATG;

wild-type glutamate dehydrogenase (nucleotide sequence shown as SEQ ID NO.18), named WT 9; mutant K113S/N349L; mutant K113E/N349M; mutant K113Q/N349M. Wherein, the gene coding the mutant K113S/N349L is obtained by mutating the codon coding the 113 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.18 into TCT and mutating the codon coding the 349 th amino acid into CTG; the gene coding the mutant K113S/N349L is characterized in that the codon coding the 113 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.18 is mutated into GAG, and the codon coding the 349 th amino acid is mutated into ATG; the gene for coding the mutant K113S/N349L is characterized in that the codon for coding the 113 th amino acid of the glutamate dehydrogenase gene with the nucleotide sequence shown as SEQ ID NO.18 is mutated into CAG, and the codon for coding the 349 th amino acid is mutated into ATG.

Example 6: activity determination of glutamate dehydrogenase mutant for 4-oxopentanoic acid

The activities of the purified wild-type enzyme and the purified glutamate dehydrogenase mutant obtained in examples 1 to 5 on 4-oxopentanoic acid were measured by a microplate reader, and the results are shown in Table 10.

The results show that: as is clear from Table 10, no measurable catalytic activity was detected for 4-oxopentanoic acid by the wild-type glutamate dehydrogenase, whereas the mutants obtained according to the invention all had catalytic activity. In the key two-point mutation, the combination of KQ/NM can show the highest enzyme activity.

TABLE 10 specific enzyme activities of various glutamate dehydrogenases

Type (B)	Specific activity (U/g)	Type (B)	Specific activity (U/g)
				WT1	0	WT2	0
K116S/N348L	1.87	K114S/N348L	0.38
				K116E/N348M	3.16	K114E/N348M	0.88
K116Q/N348M	4.55	K114Q/N348M	1.35
				WT3	0	WT4	0
K114S/N349L	1.56	K112S/N344L	0.24
				K114E/N349M	2.13	K112E/N344M	0.65
K114Q/N349M	2.56	K112Q/N344M	0.97
				WT5	0	WT6	0
K104S/N326L	0.33	K102S/N338L	0.89
				K104E/N326M	0.89	K102E/N338M	1.22
K104Q/N326M	1.33	K102Q/N338M	1.58
				WT7	0	WT8	0
K101S/N340L	0.67	K102S/N346L	0.25
				K101E/N340M	1.21	K102E/N346M	0.77
K101Q/N340M	1.77	K102Q/N346M	1.21
				WT9	0
K113S/N349L	0.63
				K113E/N349M	1.21
K113Q/N349M	1.89

Example 7: synthesis of (R) -4-aminopentanoic acid

The present invention is directed to the synthesis of (R) -4-aminopentanoate by coupling with Glucose Dehydrogenase (GDH), taking the glutamate dehydrogenase mutant K116Q/N348M as an example.

The volume of the reaction system was 1mL, and the glutamate dehydrogenase mutant K116Q/N348M pure enzyme (1mg/mL) was added to the reaction system containing GluDH pure enzyme (1mg/mL), 4-oxopentanoic acid (50mmol/L), glucose (75mmol/L), NADP⁺(1mmol/L)，NH₄Cl-NH₃·H₂In the reaction solution of O buffer solution (1M, pH 8.5), obtaining a reaction system; the reaction system was reacted at 30 ℃ for 6h at 200 rpm. Samples were taken periodically and the samples were quenched with aqueous NaOH (200. mu.L, 10M) and extracted with methyl tert-butyl ether (500uL, twice).

The results are shown in table 11 below:

table 11: yield of (R) -4-aminopentanoate synthesized by glutamate dehydrogenase wild enzyme and mutant K116Q/N348M

The results showed that the wild enzyme was unable to synthesize (R) -4-aminopentanoic acid, while the mutant K116Q/N348M after mutation was able to synthesize (R) -4-aminopentanoic acid with a yield of 49mmol/L and a substrate conversion of: 98 percent.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Industrial and technical research institute of south Jiangnan university in Suqian City

<120> glutamate dehydrogenase mutant and application thereof

<130> BAA200843A

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 447

<212> PRT

<213> Artificial sequence

<400> 1

Met Asp Gln Thr Tyr Ser Leu Glu Ser Phe Leu Asn His Val Gln Lys

1 5 10 15

Arg Asp Pro Asn Gln Thr Glu Phe Ala Gln Ala Val Arg Glu Val Met

20 25 30

Thr Thr Leu Trp Pro Phe Leu Glu Gln Asn Pro Lys Tyr Arg Gln Met

35 40 45

Ser Leu Leu Glu Arg Leu Val Glu Pro Glu Arg Val Ile Gln Phe Arg

50 55 60

Val Val Trp Val Asp Asp Arg Asn Gln Ile Gln Val Asn Arg Ala Trp

65 70 75 80

Arg Val Gln Phe Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Met Arg

85 90 95

Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe Glu

100 105 110

Gln Thr Phe Lys Asn Ala Leu Thr Thr Leu Pro Met Gly Gly Gly Lys

115 120 125

Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Glu Gly Glu Val Met

130 135 140

Arg Phe Cys Gln Ala Leu Met Thr Glu Leu Tyr Arg His Leu Gly Ala

145 150 155 160

Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Gly Gly Arg Glu Val

165 170 175

Gly Phe Met Ala Gly Met Met Lys Lys Leu Ser Asn Asn Thr Ala Cys

180 185 190

Val Phe Thr Gly Lys Gly Leu Ser Phe Gly Gly Ser Leu Ile Arg Pro

195 200 205

Glu Ala Thr Gly Tyr Gly Leu Val Tyr Phe Thr Glu Ala Met Leu Lys

210 215 220

Arg His Gly Met Gly Phe Glu Gly Met Arg Val Ser Val Ser Gly Ser

225 230 235 240

Gly Asn Val Ala Gln Tyr Ala Ile Glu Lys Ala Met Glu Phe Gly Ala

245 250 255

Arg Val Ile Thr Ala Ser Asp Ser Ser Gly Thr Val Val Asp Glu Ser

260 265 270

Gly Phe Thr Lys Glu Lys Leu Ala Arg Leu Ile Glu Ile Lys Ala Ser

275 280 285

Arg Asp Gly Arg Val Ala Asp Tyr Ala Lys Glu Phe Gly Leu Val Tyr

290 295 300

Leu Glu Gly Gln Gln Pro Trp Ser Leu Pro Val Asp Ile Ala Leu Pro

305 310 315 320

Cys Ala Thr Gln Asn Glu Leu Asp Val Asp Ala Ala His Gln Leu Ile

325 330 335

Ala Asn Gly Val Lys Ala Val Ala Glu Gly Ala Asn Met Pro Thr Thr

340 345 350

Ile Glu Ala Thr Glu Leu Phe Gln Gln Ala Gly Val Leu Phe Ala Pro

355 360 365

Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Thr Ser Gly Leu Glu Met

370 375 380

Ala Gln Asn Ala Ala Arg Leu Gly Trp Lys Ala Glu Lys Val Asp Ala

385 390 395 400

Arg Leu His His Ile Met Leu Asp Ile His His Ala Cys Val Glu His

405 410 415

Gly Gly Glu Gly Glu Gln Thr Asn Tyr Val Gln Gly Ala Asn Ile Ala

420 425 430

Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Ile

435 440 445

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Ser Lys Tyr Val Asp Arg Val Ile Ala Glu Val Glu Lys Lys Tyr Ala

1 5 10 15

Asp Glu Pro Glu Phe Val Gln Thr Val Glu Glu Val Leu Ser Ser Leu

20 25 30

Gly Pro Val Val Asp Ala His Pro Glu Tyr Glu Glu Val Ala Leu Leu

35 40 45

Glu Arg Met Val Ile Pro Glu Arg Val Ile Glu Phe Arg Val Pro Trp

50 55 60

Glu Asp Asp Asn Gly Lys Val His Val Asn Thr Gly Tyr Arg Val Gln

65 70 75 80

Phe Asn Gly Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe Ala Pro

85 90 95

Ser Val Asn Leu Ser Ile Met Lys Phe Leu Gly Phe Glu Gln Ala Phe

100 105 110

Lys Asp Ser Leu Thr Thr Leu Pro Met Gly Gly Ala Lys Gly Gly Ser

115 120 125

Asp Phe Asp Pro Asn Gly Lys Ser Asp Arg Glu Val Met Arg Phe Cys

130 135 140

Gln Ala Phe Met Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Ile Asp

145 150 155 160

Val Pro Ala Gly Asp Leu Gly Val Gly Ala Arg Glu Ile Gly Tyr Met

165 170 175

Tyr Gly Gln Tyr Arg Lys Ile Val Gly Gly Phe Tyr Asn Gly Val Leu

180 185 190

Thr Gly Lys Ala Arg Ser Phe Gly Gly Ser Leu Val Arg Pro Glu Ala

195 200 205

Thr Gly Tyr Gly Ser Val Tyr Tyr Val Glu Ala Val Met Lys His Glu

210 215 220

Asn Asp Thr Leu Val Gly Lys Thr Val Ala Leu Ala Gly Phe Gly Asn

225 230 235 240

Val Ala Trp Gly Ala Ala Lys Lys Leu Ala Glu Leu Gly Ala Lys Ala

245 250 255

Val Thr Leu Ser Gly Pro Asp Gly Tyr Ile Tyr Asp Pro Glu Gly Ile

260 265 270

Thr Thr Glu Glu Lys Ile Asn Tyr Met Leu Glu Met Arg Ala Ser Gly

275 280 285

Arg Asn Lys Val Gln Asp Tyr Ala Asp Lys Phe Gly Val Gln Phe Phe

290 295 300

Pro Gly Glu Lys Pro Trp Gly Gln Lys Val Asp Ile Ile Met Pro Cys

305 310 315 320

Ala Thr Gln Asn Asp Val Asp Leu Glu Gln Ala Lys Lys Ile Val Ala

325 330 335

Asn Asn Val Lys Tyr Tyr Ile Glu Val Ala Asn Met Pro Thr Thr Asn

340 345 350

Glu Ala Leu Arg Phe Leu Met Gln Gln Pro Asn Met Val Val Ala Pro

355 360 365

Ser Lys Ala Val Asn Ala Gly Gly Val Leu Val Ser Gly Phe Glu Met

370 375 380

Ser Gln Asn Ser Glu Arg Leu Ser Trp Thr Ala Glu Glu Val Asp Ser

385 390 395 400

Lys Leu His Gln Val Met Thr Asp Ile His Asp Gly Ser Ala Ala Ala

405 410 415

Ala Glu Arg Tyr Gly Leu Gly Tyr Asn Leu Val Ala Gly Ala Asn Ile

420 425 430

Val Gly Phe Gln Lys Ile Ala Asp Ala Met Met Ala Gln Gly Ile Ala

435 440 445

Trp

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Ala Asp Arg Glu Asp Asp Pro Asn Phe Phe Lys Met Val Glu Gly Phe

1 5 10 15

Phe Asp Arg Gly Ala Ser Ile Val Glu Asp Lys Leu Val Glu Asp Leu

20 25 30

Lys Thr Arg Glu Thr Glu Glu Gln Lys Arg Asn Arg Val Arg Gly Ile

35 40 45

Leu Arg Ile Ile Lys Pro Cys Asn His Val Leu Ser Leu Ser Phe Pro

50 55 60

Ile Arg Arg Asp Asp Gly Ser Trp Glu Val Ile Glu Gly Tyr Arg Ala

65 70 75 80

Gln His Ser Gln His Arg Thr Pro Cys Lys Gly Gly Ile Arg Tyr Ser

85 90 95

Thr Asp Val Ser Val Asp Glu Val Lys Ala Leu Ala Ser Leu Met Thr

100 105 110

Tyr Lys Cys Ala Val Val Asp Val Pro Phe Gly Gly Ala Lys Ala Gly

115 120 125

Val Lys Ile Asn Pro Lys Asn Tyr Thr Asp Asn Glu Leu Glu Lys Ile

130 135 140

Thr Arg Arg Phe Thr Met Glu Leu Ala Lys Lys Gly Phe Ile Gly Pro

145 150 155 160

Gly Val Asp Val Pro Ala Pro Asp Met Ser Thr Gly Glu Arg Glu Met

165 170 175

Ser Trp Ile Ala Asp Thr Tyr Ala Ser Thr Ile Gly His Tyr Asp Ile

180 185 190

Asn Ala His Ala Cys Val Thr Gly Lys Pro Ile Ser Gln Gly Gly Ile

195 200 205

His Gly Arg Ile Ser Ala Thr Gly Arg Gly Val Phe His Gly Ile Glu

210 215 220

Asn Phe Ile Asn Glu Ala Ser Tyr Met Ser Ile Leu Gly Met Thr Pro

225 230 235 240

Gly Phe Gly Asp Lys Thr Phe Ala Val Gln Gly Phe Gly Asn Val Gly

245 250 255

Leu His Ser Met Arg Tyr Leu His Arg Phe Gly Ala Lys Cys Val Ala

260 265 270

Val Gly Glu Ser Asp Gly Ser Ile Trp Asn Pro Asp Gly Ile Asp Pro

275 280 285

Lys Glu Leu Glu Asp Phe Lys Leu Gln His Gly Thr Ile Leu Gly Phe

290 295 300

Pro Lys Ala Lys Ile Tyr Glu Gly Ser Ile Leu Glu Val Asp Cys Asp

305 310 315 320

Ile Leu Ile Pro Ala Ala Ser Glu Lys Gln Leu Thr Lys Ser Asn Ala

325 330 335

Pro Arg Val Lys Ala Lys Ile Ile Ala Glu Gly Ala Asn Gly Pro Thr

340 345 350

Thr Pro Glu Ala Asp Lys Ile Phe Leu Glu Arg Asn Ile Met Val Ile

355 360 365

Pro Asp Leu Tyr Leu Asn Ala Gly Gly Val Thr Val Ser Tyr Phe Glu

370 375 380

Trp Leu Lys Asn Leu Asn His Val Ser Tyr Gly Arg Leu Thr Phe Lys

385 390 395 400

Tyr Glu Arg Asp Ser Asn Tyr His Leu Leu Met Ser Val Gln Glu Ser

405 410 415

Leu Glu Arg Lys Phe Gly Lys His Gly Gly Thr Ile Pro Ile Val Pro

420 425 430

Thr Ala Glu Phe Gln Asp Arg Ile Ser Gly Ala Ser Glu Lys Asp Ile

435 440 445

Val His Ser Gly Leu Ala Tyr Thr Met Glu Arg Ser Ala Arg Gln Ile

450 455 460

Met Arg Thr Ala Met Lys Tyr Asn Leu Gly Leu Asp Leu Arg Thr Ala

465 470 475 480

Ala Tyr Val Asn Ala Ile Glu Lys Val Phe Arg Val Tyr Asn Glu Ala

485 490 495

Gly Val Thr Phe Thr

500

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Met Asn Ala Ala Lys Val Leu Glu Asp Leu Lys Arg Arg Phe Pro Asn

1 5 10 15

Glu Pro Glu Tyr His Gln Ala Val Glu Glu Val Leu Ser Thr Ile Glu

20 25 30

Glu Glu Tyr Asn Lys His Pro Glu Phe Asp Lys Val Asn Leu Ile Glu

35 40 45

Arg Leu Cys Ile Pro Asp Arg Val Tyr Gln Phe Arg Val Thr Trp Met

50 55 60

Asp Asp Lys Gly Asn Ile Gln Thr Asn Met Gly Tyr Arg Val Gln His

65 70 75 80

Asn Asn Ala Ile Gly Pro Tyr Lys Gly Gly Ile Arg Phe His Ser Ser

85 90 95

Val Asn Leu Gly Ile Leu Lys Phe Leu Ala Phe Glu Gln Thr Phe Lys

100 105 110

Asn Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser Asp

115 120 125

Phe Ser Pro Arg Gly Lys Ser Asn Ala Glu Val Met Arg Phe Cys Gln

130 135 140

Ala Phe Met Leu Glu Leu Trp Arg His Ile Gly Pro Glu Thr Asp Val

145 150 155 160

Pro Ala Gly Asp Ile Gly Val Gly Gly Arg Glu Val Gly Phe Met Phe

165 170 175

Gly Met Tyr Lys Lys Leu Ser His Glu Phe Ser Gly Val Leu Thr Gly

180 185 190

Lys Gly Arg Glu Phe Gly Gly Ser Leu Ile Arg Pro Glu Ala Thr Gly

195 200 205

Tyr Gly Asn Ile Tyr Phe Leu Met Glu Met Leu Lys Thr Lys Gly Thr

210 215 220

Asp Leu Lys Gly Lys Thr Cys Leu Val Ser Gly Ser Gly Asn Val Ala

225 230 235 240

Gln Tyr Thr Val Glu Lys Val Ile Glu Leu Gly Gly Lys Val Val Thr

245 250 255

Met Ser Asp Ser Asp Gly Tyr Ile Tyr Asp Pro Asp Gly Ile Asp Arg

260 265 270

Glu Lys Leu Asp Phe Ile Met Glu Leu Lys Asn Leu Tyr Arg Gly Arg

275 280 285

Ile Arg Glu Tyr Ala Glu Lys Tyr Gly Cys Lys Tyr Val Ala Gly Ala

290 295 300

Arg Pro Trp Gly Glu Lys Ala Asp Ile Ala Leu Pro Ser Ala Thr Gln

305 310 315 320

Asn Glu Leu Asn Gly Asp Glu Ala Arg Gln Leu Val Ala Asn Gly Val

325 330 335

Met Ala Val Ser Glu Gly Ala Asn Met Pro Ser Thr Pro Glu Ala Ile

340 345 350

Arg Val Phe Gln Glu Ala Lys Ile Leu Tyr Ala Pro Gly Lys Ala Ala

355 360 365

Asn Ala Gly Gly Val Ser Val Ser Gly Leu Glu Met Thr Gln Asn Ser

370 375 380

Ile Lys Leu Gly Trp Ser Gln Glu Glu Val Asp Glu Lys Leu Lys Ser

385 390 395 400

Ile Met Lys Asn Ile His Glu Ala Cys Val Gln Tyr Gly Thr Glu Ala

405 410 415

Asp Gly Tyr Val Asn Tyr Val Lys Gly Ala Asn Val Ala Gly Phe Met

420 425 430

Lys Val Ala Lys Ala Met Met Ala Gln Gly Ile Val

435 440

<210> 5

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<400> 5

Met Ser Ala Met Gln Val Ser Lys Asp Glu Glu Lys Glu Ala Leu Asn

1 5 10 15

Leu Phe Leu Ser Thr Gln Thr Ile Ile Lys Glu Ala Leu Arg Lys Leu

20 25 30

Gly Tyr Pro Gly Asp Met Tyr Glu Leu Met Lys Glu Pro Gln Arg Met

35 40 45

Leu Thr Val Arg Ile Pro Val Lys Met Asp Asn Gly Ser Val Asn Val

50 55 60

Phe Thr Gly Tyr Arg Ser Gln His Asn Asp Ala Val Gly Pro Thr Lys

65 70 75 80

Gly Gly Val Arg Phe His Pro Glu Val Asn Glu Glu Glu Val Lys Ala

85 90 95

Leu Ser Ile Trp Met Thr Leu Lys Cys Gly Ile Ala Asn Leu Pro Tyr

100 105 110

Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp Pro Arg Thr Met Ser Phe

115 120 125

Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln

130 135 140

Ile Val Gly Pro Thr Lys Asp Ile Pro Ala Pro Asp Val Tyr Thr Asn

145 150 155 160

Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Leu Arg Glu

165 170 175

Phe Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly

180 185 190

Ser Gln Gly Arg Glu Thr Ala Thr Ala Gln Gly Val Thr Ile Cys Ile

195 200 205

Glu Glu Ala Val Lys Lys Lys Gly Ile Lys Leu Gln Asn Ala Arg Ile

210 215 220

Ile Ile Gln Gly Phe Gly Asn Ala Gly Ser Phe Leu Ala Lys Phe Met

225 230 235 240

His Asp Ala Gly Ala Lys Val Ile Gly Ile Ser Asp Ala Asn Gly Gly

245 250 255

Leu Tyr Asn Pro Asp Gly Leu Asp Ile Pro Tyr Leu Leu Asp Lys Arg

260 265 270

Asp Ser Phe Gly Met Val Thr Asn Leu Phe Thr Asp Val Ile Thr Asn

275 280 285

Glu Glu Leu Leu Glu Lys Asp Cys Asp Ile Leu Val Pro Ala Ala Ile

290 295 300

Ser Asn Gln Ile Thr Ala Lys Asn Ala His Asn Ile Gln Ala Ser Ile

305 310 315 320

Val Val Glu Ala Ala Asn Gly Pro Thr Thr Ile Asp Ala Thr Lys Ile

325 330 335

Leu Asn Glu Arg Gly Val Leu Leu Val Pro Asp Ile Leu Ala Ser Ala

340 345 350

Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly

355 360 365

Tyr Tyr Trp Ser Glu Glu Glu Val Ala Glu Lys Leu Arg Ser Val Met

370 375 380

Val Arg Ser Phe Glu Thr Ile Tyr Gln Thr Ala Ala Thr His Lys Val

385 390 395 400

Asp Met Arg Leu Ala Ala Tyr Met Thr Gly Ile Arg Lys Ser Ala Glu

405 410 415

Ala Ser Arg Phe Arg Gly Trp Val

420

<210> 6

<211> 454

<212> PRT

<213> Artificial sequence

<400> 6

Met Ser Asn Leu Pro Ser Glu Pro Glu Phe Glu Gln Ala Tyr Lys Glu

1 5 10 15

Leu Ala Tyr Thr Leu Glu Asn Ser Ser Leu Phe Gln Lys His Pro Glu

20 25 30

Tyr Arg Thr Ala Leu Thr Val Ala Ser Ile Pro Glu Arg Val Ile Gln

35 40 45

Phe Arg Val Val Trp Glu Asp Asp Asn Gly Asn Val Gln Val Asn Arg

50 55 60

Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly

65 70 75 80

Leu Arg Leu His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly

85 90 95

Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Ser Met Gly Gly

100 105 110

Gly Lys Gly Gly Ala Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu

115 120 125

Ile Arg Arg Phe Cys Cys Ala Phe Met Ala Glu Leu His Lys His Ile

130 135 140

Gly Ala Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Gly Gly Arg

145 150 155 160

Glu Ile Gly Tyr Met Phe Gly Ala Tyr Arg Lys Ala Ala Asn Arg Phe

165 170 175

Glu Gly Val Leu Thr Gly Lys Gly Leu Ser Trp Gly Gly Ser Leu Ile

180 185 190

Arg Pro Glu Ala Thr Gly Tyr Gly Leu Val Tyr Tyr Val Gly His Met

195 200 205

Leu Glu Tyr Ser Gly Ala Gly Ser Tyr Ala Gly Lys Arg Val Ala Leu

210 215 220

Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Leu Ile Glu

225 230 235 240

Leu Gly Ala Thr Val Val Ser Leu Ser Asp Ser Lys Gly Ala Leu Val

245 250 255

Ala Thr Gly Glu Ser Gly Ile Thr Val Glu Asp Ile Asn Ala Ile Met

260 265 270

Ala Ile Lys Glu Ala Arg Gln Ser Leu Thr Thr Phe Gln His Ala Gly

275 280 285

His Val Lys Trp Ile Glu Gly Ala Arg Pro Trp Leu His Val Gly Lys

290 295 300

Val Asp Ile Ala Phe Pro Cys Ala Thr Gln Asn Glu Val Ser Lys Glu

305 310 315 320

Glu Ala Glu Gly Leu Leu Ala Ala Gly Cys Lys Phe Val Ala Glu Gly

325 330 335

Ser Asn Met Gly Cys Thr Leu Glu Ala Ile Glu Val Phe Glu Asn Asn

340 345 350

Arg Lys Glu Lys Lys Gly Glu Ala Val Trp Tyr Ala Pro Gly Lys Ala

355 360 365

Ala Asn Cys Gly Gly Val Ala Val Ser Gly Leu Glu Met Ala Gln Asn

370 375 380

Ser Gln Arg Leu Asn Trp Thr Gln Ala Glu Val Asp Glu Lys Leu Lys

385 390 395 400

Asp Ile Met Lys Asn Ala Phe Phe Asn Gly Leu Asn Thr Ala Lys Thr

405 410 415

Tyr Val Glu Ala Ala Glu Gly Glu Leu Pro Ser Leu Val Ala Gly Ser

420 425 430

Asn Ile Ala Gly Phe Val Lys Val Ala Gln Ala Met His Asp Gln Gly

435 440 445

Asp Trp Trp Ser Lys Asn

450

<210> 7

<211> 451

<212> PRT

<213> Artificial sequence

<400> 7

Met Ser Thr Pro Tyr Glu Pro Glu Phe Gln Gln Ala Tyr Lys Glu Ile

1 5 10 15

Val Gly Ser Ile Glu Ser Ser Lys Leu Phe Glu Val His Pro Glu Leu

20 25 30

Lys Arg Val Leu Pro Ile Ile Ser Ile Pro Glu Arg Val Leu Glu Phe

35 40 45

Arg Val Thr Trp Glu Asp Asp Lys Gly Asn Cys Arg Val Asn Thr Gly

50 55 60

Tyr Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly Leu

65 70 75 80

Arg Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe

85 90 95

Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Pro Met Gly Gly Gly

100 105 110

Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Asn Glu Ile

115 120 125

Arg Arg Phe Ser Gln Ala Phe Met Arg Gln Leu Phe Arg Tyr Ile Gly

130 135 140

Pro Gln Thr Asp Val Pro Ala Gly Asp Ile Gly Val Thr Gly Phe Val

145 150 155 160

Val Met His Met Phe Gly Glu Tyr Lys Arg Leu Arg Asn Glu Tyr Ser

165 170 175

Gly Val Val Thr Gly Lys His Met Leu Thr Gly Gly Ser Asn Ile Arg

180 185 190

Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Lys His Met Ile

195 200 205

Glu His Arg Thr Lys Gly Ala Glu Thr Leu Lys Gly Lys Arg Val Ala

210 215 220

Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Cys Ile

225 230 235 240

Gln Glu Gly Ala Ile Val Lys Ser Ile Ser Asp Ser Lys Gly Val Leu

245 250 255

Ile Ala Lys Thr Ala Glu Gly Leu Val Pro Glu Glu Ile His Glu Ile

260 265 270

Met Ala Leu Lys Glu Lys Arg Ala Ser Ile Ala Asp Ser Ala Ser Leu

275 280 285

Cys Lys Lys His His Tyr Ile Ala Gly Ala Arg Pro Trp Thr Asn Val

290 295 300

Gly Glu Ile Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Val Ser

305 310 315 320

Gly Glu Glu Ala Ala Ala Leu Ile Lys Gln Gly Cys Arg Tyr Val Ala

325 330 335

Glu Gly Ser Asn Met Gly Ser Ser Ala Glu Ala Val Glu Val Phe Glu

340 345 350

Lys Ser Arg Ala Ser Gly Glu Gly Cys Trp Leu Ala Pro Gly Lys Ala

355 360 365

Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ala Gln Asn

370 375 380

Ala Gln Phe Ser Thr Trp Thr His Ala Glu Val Asp Ala Lys Leu Ala

385 390 395 400

Gly Ile Met Gln Asn Ile Phe Glu Gln Ser Thr Asp Val Ala Ser Lys

405 410 415

Tyr Cys Asp Ser Gly Ser Asn Asn Ile Pro Ser Leu Val Asp Gly Ala

420 425 430

Asn Ile Ala Gly Phe Leu Lys Val Ala Thr Ala Met Gln Ala Val Gly

435 440 445

Asp Trp Trp

450

<210> 8

<211> 459

<212> PRT

<213> Artificial sequence

<400> 8

Met Ser Asn Leu Pro Val Glu Pro Glu Phe Glu Gln Ala Tyr Lys Glu

1 5 10 15

Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Glu Gln His Pro Glu

20 25 30

Tyr Arg Arg Ala Leu Gln Val Val Ser Val Pro Glu Arg Val Ile Gln

35 40 45

Phe Arg Val Val Trp Glu Asn Asp Lys Gly Glu Val Gln Ile Asn Arg

50 55 60

Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly

65 70 75 80

Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly

85 90 95

Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Asn Met Gly Gly

100 105 110

Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ser Glu

115 120 125

Ile Arg Arg Phe Cys Thr Ala Phe Met Thr Glu Leu Cys Lys His Ile

130 135 140

Gly Ala Asp Thr Asp Leu Pro Ala Gly Asp Ile Gly Val Thr Gly Arg

145 150 155 160

Glu Val Gly Phe Leu Phe Gly Gln Tyr Arg Arg Ile Arg Asn Gln Trp

165 170 175

Glu Gly Val Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser Leu Ile

180 185 190

Arg Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Gln His Met

195 200 205

Ile Lys His Val Thr Gly Gly Lys Glu Ser Phe Ala Gly Lys Arg Val

210 215 220

Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys Val

225 230 235 240

Ile Glu Leu Gly Gly Ser Val Val Ser Leu Ser Asp Ser Lys Gly Ser

245 250 255

Leu Ile Val Lys Asp Glu Ser Ala Ser Phe Thr Pro Glu Glu Ile Ala

260 265 270

Leu Ile Ala Asp Leu Lys Val Ala Arg Lys Gln Leu Ser Glu Leu Ala

275 280 285

Thr Ser Ser Ala Phe Ala Gly Lys Phe Thr Tyr Ile Pro Asp Ala Arg

290 295 300

Pro Trp Thr Asn Ile Pro Gly Lys Phe Glu Val Ala Leu Pro Ser Ala

305 310 315 320

Thr Gln Asn Glu Val Ser Gly Glu Glu Ala Glu His Leu Ile Lys Ser

325 330 335

Gly Val Arg Tyr Ile Ala Glu Gly Ser Asn Met Gly Cys Thr Gln Ala

340 345 350

Ala Ile Asp Ile Phe Glu Ala His Arg Asn Ala Asn Pro Gly Asp Ala

355 360 365

Ile Trp Tyr Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val

370 375 380

Ser Gly Leu Glu Met Ala Gln Asn Ser Ala Arg Leu Ser Trp Thr Ser

385 390 395 400

Glu Glu Val Asp Ala Arg Leu Lys Gly Ile Met Glu Asp Cys Phe Lys

405 410 415

Asn Gly Leu Glu Thr Ala Gln Lys Phe Ala Thr Pro Ala Lys Gly Val

420 425 430

Leu Pro Ser Leu Val Thr Gly Ser Asn Ile Ala Gly Phe Thr Lys Val

435 440 445

Ala Glu Ala Met Lys Asp Gln Gly Asp Trp Trp

450 455

<210> 9

<211> 449

<212> PRT

<213> Artificial sequence

<400> 9

Met Pro Ala Gln Thr Ile Glu Glu Leu Ile Ala Val Ile Lys Gln Arg

1 5 10 15

Asp Gly His Met Thr Glu Phe Arg Gln Ala Val Glu Glu Val Val Asp

20 25 30

Ser Leu Lys Val Ile Phe Glu Arg Glu Pro Lys Tyr Ile Pro Ile Phe

35 40 45

Glu Arg Met Leu Glu Pro Glu Arg Val Ile Ile Phe Arg Val Pro Trp

50 55 60

Met Asp Asp Ala Gly Arg Ile Asn Val Asn Arg Gly Phe Arg Val Gln

65 70 75 80

Tyr Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro

85 90 95

Ser Val Asn Leu Ser Ile Leu Lys Phe Leu Gly Phe Glu Gln Ile Leu

100 105 110

Lys Asn Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser

115 120 125

Asp Phe Asp Pro Lys Gly Lys Ser Asp Asn Glu Val Met Arg Phe Cys

130 135 140

Gln Ser Phe Met Thr Glu Leu Gln Arg His Val Gly Ala Asp Thr Asp

145 150 155 160

Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu

165 170 175

Tyr Gly Gln Tyr Lys Arg Leu Arg Asn Glu Phe Thr Gly Val Leu Thr

180 185 190

Gly Lys Asn Val Lys Trp Gly Gly Ser Phe Ile Arg Pro Glu Ala Thr

195 200 205

Gly Tyr Gly Ala Val Tyr Phe Leu Glu Glu Met Cys Lys Asp Asn Asn

210 215 220

Thr Val Ile Arg Gly Lys Asn Val Leu Leu Ser Gly Ser Gly Asn Val

225 230 235 240

Ala Gln Phe Ala Cys Glu Lys Leu Ile Gln Leu Gly Ala Lys Val Leu

245 250 255

Thr Phe Ser Asp Ser Asn Gly Thr Ile Val Asp Lys Asp Gly Phe Asn

260 265 270

Glu Glu Lys Leu Ala His Leu Met Tyr Leu Lys Asn Glu Lys Arg Gly

275 280 285

Arg Val Ser Glu Phe Lys Asp Lys Tyr Pro Ser Val Ala Tyr Tyr Glu

290 295 300

Gly Lys Lys Pro Trp Glu Cys Phe Glu Gly Gln Val Asp Cys Ile Met

305 310 315 320

Pro Cys Ala Thr Gln Asn Glu Val Ser Gly Asp Asp Ala Thr Arg Leu

325 330 335

Val Gly Leu Gly Leu Lys Phe Val Ala Glu Gly Ala Asn Met Pro Ser

340 345 350

Thr Ala Glu Ala Val His Val Tyr His Ala Lys Gly Val Met Tyr Gly

355 360 365

Pro Ala Lys Ala Ser Asn Ala Gly Gly Val Ser Val Ser Gly Leu Glu

370 375 380

Met Ser Gln Asn Ser Val Arg Leu Gln Trp Thr Ala Glu Glu Val Asp

385 390 395 400

Gln Lys Leu Arg Gly Ile Met Arg Gly Ile Phe Val Ala Cys Arg Asp

405 410 415

Thr Ala Lys Lys Tyr Gly His Pro Lys Asn Tyr Gln Met Gly Ala Asn

420 425 430

Ile Ala Gly Phe Leu Lys Val Ala Asp Ser Met Ile Glu Gln Gly Cys

435 440 445

Val

<210> 10

<211> 1344

<212> DNA

<213> Artificial sequence

<400> 10

atggatcaga catattctct ggagtcattc ctcaaccatg tccaaaagcg cgacccgaat 60

caaaccgagt tcgcgcaagc cgttcgtgaa gtaatgacca cactctggcc ttttcttgaa 120

caaaatccaa aatatcgcca gatgtcatta ctggagcgtc tggttgaacc ggagcgcgtg 180

atccagtttc gcgtggtatg ggttgatgat cgcaaccaga tacaggtcaa ccgtgcatgg 240

cgtgtgcagt tcagctctgc catcggcccg tacaaaggcg gtatgcgctt ccatccgtca 300

gttaaccttt ccattctcaa attcctcggc tttgaacaaa ccttcaaaaa tgccctgact 360

actctgccga tgggcggtgg taaaggcggc agcgatttcg atccgaaagg aaaaagcgaa 420

ggtgaagtga tgcgtttttg ccaggcgctg atgactgaac tgtatcgcca cctgggcgcg 480

gataccgacg ttccggcagg tgatatcggg gttggtggtc gtgaagtcgg ctttatggcg 540

gggatgatga aaaagctctc caacaatacc gcctgcgtct tcaccggtaa gggcctttca 600

tttggcggca gtcttattcg cccggaagct accggctacg gtctggttta tttcacagaa 660

gcaatgctaa aacgccacgg tatgggtttt gaagggatgc gcgtttccgt ttctggctcc 720

ggcaacgtcg cccagtacgc tatcgaaaaa gcgatggaat ttggtgctcg tgtgatcact 780

gcgtcagact ccagcggcac tgtagttgat gaaagcggat tcacgaaaga gaaactggca 840

cgtcttatcg aaatcaaagc cagccgcgat ggtcgagtgg cagattacgc caaagaattt 900

ggtctggtct atctcgaagg ccaacagccg tggtctctac cggttgatat cgccctgcct 960

tgcgccaccc agaatgaact ggatgttgac gccgcgcatc agcttatcgc taatggcgtt 1020

aaagccgtcg ccgaaggggc aaatatgccg accaccatcg aagcgactga actgttccag 1080

caggcaggcg tactatttgc accgggtaaa gcggctaatg ctggtggcgt cgctacatcg 1140

ggcctggaaa tggcacaaaa cgctgcgcgc ctgggctgga aagccgagaa agttgacgca 1200

cgtttgcatc acatcatgct ggatatccac catgcctgtg ttgagcatgg tggtgaaggt 1260

gagcaaacca actacgtgca gggcgcgaac attgccggtt ttgtgaaggt tgccgatgcg 1320

atgctggcgc agggtgtgat ttaa 1344

<210> 11

<211> 1353

<212> DNA

<213> Artificial sequence

<400> 11

atgagcaagt acgttgatcg tgtgatcgcc gaagtggaga agaagtacgc cgatgaacca 60

gagttcgtgc agaccgtgga ggaagtgctg agtagtctgg gtccagtggt tgatgcgcac 120

ccagaatacg aggaagtggc gctgctcgaa cgcatggtta tcccggagcg cgttatcgaa 180

ttccgcgtgc cgtgggaaga cgacaacggt aaggtgcatg ttaacaccgg ttaccgcgtg 240

cagttcaacg gtgccatcgg tccatacaaa ggtggtctgc gtttcgcgcc gagcgtgaat 300

ctgagtatca tgaagttcct cggcttcgag caagccttta aggacagcct caccacgctg 360

ccgatgggcg gtgcgaaagg tggcagcgac ttcgatccga acggtaagag cgaccgtgag 420

gttatgcgct tttgccaagc cttcatgacc gaactgtatc gccacatcgg tccagacatt 480

gacgttccgg ccggtgatct gggtgttggt gcgcgcgaga ttggctacat gtacggccag 540

tatcgcaaga tcgttggcgg cttctacaac ggcgttctga ccggcaaggc gcgcagcttt 600

ggtggcagtc tggttcgtcc ggaagccacg ggctacggca gtgtgtacta cgtggaggcg 660

gtgatgaagc atgagaacga caccctcgtt ggcaagaccg tggcgctggc gggttttggt 720

aacgttgcgt ggggcgccgc caagaaactg gccgaactgg gcgccaaagc cgttacgctg 780

agcggcccag atggctacat ttacgacccg gaaggcatca ccacggagga gaaaatcaac 840

tacatgctgg aaatgcgcgc gagtggccgc aacaaggtgc aagattacgc ggacaaattc 900

ggcgtgcagt tctttccggg cgagaaaccg tggggccaga aggttgatat catcatgccg 960

tgcgccaccc agaatgatgt tgatctggag caagccaaga agatcgtggc gaacaacgtg 1020

aagtactaca tcgaggtggc caacatgccg accaccaatg aggcgctgcg ctttctgatg 1080

cagcagccga atatggttgt tgccccgagc aaagcggtga atgcgggtgg tgtgctggtt 1140

agcggctttg agatgagcca gaacagcgaa cgtctgagct ggaccgcgga agaagtggac 1200

agcaaactgc accaagttat gaccgacatt catgatggca gtgccgcggc cgcggaacgt 1260

tatggtctgg gctacaatct ggttgcgggt gccaacatcg tgggctttca gaaaatcgcc 1320

gatgccatga tggcccaagg cattgcgtgg taa 1353

<210> 12

<211> 1506

<212> DNA

<213> Artificial sequence

<400> 12

atggctgacc gtgaagacga cccgaacttc tttaaaatgg ttgaaggctt cttcgaccgt 60

ggcgcttcta tcgttgaaga caaactggtt gaagacctga aaacccgtga aaccgaagaa 120

cagaaacgta accgtgttcg tggtatcctg cgtatcatca aaccgtgcaa ccacgttctg 180

tctctgtctt tcccgatccg tcgtgacgac ggttcttggg aagttatcga aggttaccgt 240

gctcagcact ctcagcaccg taccccgtgc aaaggtggta tccgttactc taccgacgtt 300

tctgttgacg aagttaaagc tctggcttct ctgatgacct acaaatgcgc tgttgttgac 360

gttccgttcg gtggtgctaa agctggtgtt aaaatcaacc cgaaaaacta caccgacaac 420

gaactggaaa aaatcacccg tcgtttcacc atggaactgg ctaaaaaagg tttcatcggt 480

ccgggtgttg acgttccggc tccggacatg tctaccggtg aacgtgaaat gtcttggatc 540

gctgacacct acgcttctac catcggtcac tacgacatca acgctcacgc ttgcgttacc 600

ggtaaaccga tctctcaggg tggtatccac ggtcgtatct ctgctaccgg tcgtggcgtg 660

ttccacggga tcgaaaactt catcaacgaa gcttcttaca tgtctatcct gggcatgact 720

ccgggcttcg gtgacaaaac tttcgctgtt cagggtttcg gtaacgttgg tctgcactct 780

atgcgttacc tgcaccgttt cggtgctaaa tgcgttgctg ttggtgaatc tgacggttct 840

atctggaacc cggacggtat cgacccgaaa gaactggaag acttcaaact gcagcacggt 900

accatcctgg gtttcccgaa agctaaaatc tacgaaggtt ctatcctgga agttgactgc 960

gacatcctga tcccggctgc ttctgaaaaa cagctgacca aatctaacgc tccgcgtgtt 1020

aaagctaaaa tcatcgctga aggtgctaac ggtccgacca ccccggaagc tgacaaaatc 1080

ttcctggaac gtaacatcat ggttatcccg gacctgtacc tgaacgctgg tggtgttacc 1140

gtttcttact tcgaatggct gaaaaacctg aaccacgttt cttacggtcg tctgaccttc 1200

aaatacgaac gtgactctaa ctaccacctg ctgatgtctg ttcaggaatc tctggaacgt 1260

aaattcggta aacacggtgg taccatcccg atcgttccga ccgctgaatt ccaggaccgt 1320

atctctggtg cttctgaaaa agacatcgtt cactctggtc tggcttacac catggaacgt 1380

tctgctcgtc agatcatgcg taccgctatg aaatacaacc tgggtctgga cctgcgtacc 1440

gctgcttacg ttaacgctat cgagaaagtt ttccgtgtct acaacgaagc gggtgttacc 1500

ttcacc 1506

<210> 13

<211> 1332

<212> DNA

<213> Artificial sequence

<400> 13

atgaacgctg ctaaagttct ggaagacctg aaacgtcgtt tcccgaacga accggaatac 60

caccaggctg ttgaagaagt tctgtctacc atcgaagaag aatacaacaa acacccggaa 120

ttcgacaaag ttaacctgat cgaacgtctg tgcataccag accgtgtcta ccagttccga 180

gttacctgga tggacgacaa aggtaacatc cagaccaaca tgggttaccg tgttcagcac 240

aacaacgcta tcggtccgta caaaggtggt atccgtttcc actcttctgt taacctgggt 300

atcctgaaat tcctggcttt cgaacagacc ttcaaaaact ctctgaccac cctgccgatg 360

ggtggtggta aaggtggttc tgacttctct ccgcgtggta aatctaacgc tgaagttatg 420

cgtttctgcc aggctttcat gctggaactg tggcgtcaca tcggtccgga aaccgacgtt 480

ccggctggtg acatcggtgt tggtggtcgt gaagttggtt tcatgttcgg tatgtacaaa 540

aaactgtctc acgaattctc tggtgttctg accggtaaag gtcgtgaatt cggtggttct 600

ctgatccgtc cggaagctac cggttacggt aacatctact tcctgatgga aatgctgaaa 660

accaaaggta ccgacctgaa aggtaaaacc tgcctggttt ctggttctgg taacgttgct 720

cagtacaccg ttgaaaaagt tatcgaactg ggtggtaaag ttgttaccat gtctgactct 780

gacggttaca tctacgaccc ggacggtatc gaccgtgaaa aactggactt catcatggaa 840

ctgaaaaacc tgtaccgtgg tcgtatccgt gaatacgctg aaaaatacgg ttgcaaatac 900

gttgctggtg ctcgtccgtg gggtgaaaaa gctgacatcg ctctgccgtc tgctacccag 960

aacgaactga acggtgacga agctcgtcag ctggttgcta acggtgttat ggctgtttct 1020

gaaggtgcta acatgccgtc taccccggaa gctatacgtg tgttccagga agccaaaatc 1080

ctgtacgctc cgggtaaagc tgctaacgct ggtggtgttt ctgtttctgg tctggaaatg 1140

acccagaact ctatcaaact gggttggtct caggaagaag ttgacgaaaa actgaaatct 1200

atcatgaaaa acatccacga agcttgcgtt cagtacggta ccgaagctga cggttacgtt 1260

aactacgtta aaggtgctaa cgttgctggt ttcatgaaag ttgctaaagc tatgatggct 1320

cagggtatcg tt 1332

<210> 14

<211> 1272

<212> DNA

<213> Artificial sequence

<400> 14

atgagtgcga tgcaggtaag caaagacgaa gaaaaagaag ctctgaacct gttcctgtct 60

acccagacca tcatcaaaga agctctgcgt aaactgggtt acccgggtga catgtacgaa 120

ctgatgaaag aaccgcagcg tatgctgacc gttcgtatcc cggttaaaat ggacaacggt 180

tctgttaacg ttttcaccgg ttaccgttct cagcacaacg acgctgttgg tccgaccaaa 240

ggtggtgttc gtttccaccc ggaagttaac gaagaagaag ttaaagctct gtctatctgg 300

atgaccctga aatgcggtat cgctaacctg ccgtacggtg gtggtaaagg tggtatcatc 360

tgcgacccgc gtaccatgtc tttcggtgaa ctggaacgtc tgtctcgtgg ttacgttcgt 420

gctatctctc agatcgttgg tccgaccaaa gacatcccgg cgccagacgt atacaccaac 480

agccagatca tggcttggat gatggacgaa tactctcgtc tgcgtgaatt cgactctccg 540

ggtttcatca ccggtaaacc gctggttctg ggtggttctc agggtcgtga aaccgctacc 600

gctcagggtg ttaccatctg catcgaagaa gctgttaaaa aaaaaggtat caaactgcag 660

aacgctcgta tcatcatcca gggtttcggt aacgcgggca gcttcctcgc gaaattcatg 720

cacgacgcag gtgctaaagt tatcggtatc tctgacgcta acggtggtct gtacaacccg 780

gacggtctgg acatcccgta cctgctggac aaacgtgact ctttcggtat ggttaccaac 840

ctgttcaccg acgttatcac caacgaagaa ctgctggaaa aagactgcga catcctggtt 900

ccggctgcta tctctaacca gatcaccgct aaaaacgctc acaacatcca ggcttctatc 960

gttgttgaag ctgctaacgg tccgaccacc atcgacgcta ccaaaatcct gaacgaacgt 1020

ggtgttctgc tggttccgga catcctggct tctgctggtg gtgttaccgt ttcttacttc 1080

gaatgggttc agaacaacca gggttactac tggtctgaag aagaagttgc tgaaaaactg 1140

cgttctgtta tggttcgttc tttcgaaacc atctaccaga ccgctgctac ccacaaagtt 1200

gacatgcgtc tggctgctta catgaccggt atccgtaaat ctgctgaagc ttctcgtttc 1260

cgtggttggg tt 1272

<210> 15

<211> 1362

<212> DNA

<213> Artificial sequence

<400> 15

atgtctaacc tgccgtctga accggaattc gaacaggctt acaaagaact ggcttacacc 60

ctggaaaact cttctctgtt ccagaaacac ccggaatacc gtaccgctct gaccgttgct 120

tctatcccgg aacgtgttat ccagttccgt gttgtttggg aagacgacaa cggtaacgtt 180

caggttaacc gtggttaccg tgttcagttc aactctgctc tgggtccgta caaaggtggt 240

ctgcgtctgc acccgtctgt taacctgtct atcctgaaat tcctgggttt cgaacagatc 300

ttcaaaaacg ctctgaccgg tctgtctatg ggtggtggta aaggtggtgc tgacttcgac 360

ccgaaaggta aatctgacgc tgaaatccgt cgtttctgct gcgctttcat ggctgaactg 420

cacaaacaca tcggtgctga caccgacgtt ccggctggtg acatcggtgt tggtggtcgt 480

gaaatcggtt acatgttcgg tgcttaccgt aaagctgcta accgtttcga aggtgttctg 540

accggtaaag gtctgtcttg gggtggttct ctgatccgtc cggaagctac cggttacggt 600

ctggtttact acgttggtca catgctggaa tactctggtg ctggttctta cgctggtaaa 660

cgtgttgctc tgtctggttc tggtaacgtt gctcagtacg ctgctctgaa actgatcgaa 720

ctgggtgcta ccgttgtttc tctgtctgac tctaaaggtg ctctggttgc taccggtgaa 780

tctggtatca ccgttgaaga catcaacgct atcatggcta tcaaagaagc tcgtcagtct 840

ctgaccacct tccagcacgc tggtcacgtt aaatggatcg aaggtgctcg tccgtggctg 900

cacgttggta aagttgacat cgctttcccg tgcgctaccc agaacgaagt ttctaaagaa 960

gaagctgaag gtctgctggc tgctggttgc aaattcgttg ctgaaggttc taacatgggt 1020

tgcaccctgg aagctatcga agttttcgaa aacaaccgta aagaaaaaaa aggtgaagct 1080

gtttggtacg ctccgggtaa agctgctaac tgcggtggtg ttgctgtttc tggtctggaa 1140

atggctcaga actctcagcg tctgaactgg acccaggctg aagttgacga aaaactgaaa 1200

gacatcatga aaaacgcttt cttcaacggt ctgaacaccg ctaaaaccta cgttgaagct 1260

gctgaaggtg aactgccgtc tctggttgct ggttctaaca tcgctggttt cgttaaagtt 1320

gctcaggcta tgcacgacca gggtgactgg tggtctaaaa ac 1362

<210> 16

<211> 1353

<212> DNA

<213> Artificial sequence

<400> 16

atgtctaccc cgtacgaacc ggaattccag caggcttaca aagaaatcgt tggttctatc 60

gaatcttcta aactgttcga agttcacccg gaactgaaac gtgttctgcc gatcatctct 120

atcccggaac gtgttctgga attccgtgtt acctgggaag acgacaaagg taactgccgt 180

gttaacaccg gttaccgtgt tcagttcaac tctgctctgg gtccgtacaa aggtggtctg 240

cgtttccacc cgtctgttaa cctgtctatc ctgaaattcc tgggtttcga acagatcttc 300

aaaaacgctc tgaccggtct gccgatgggt ggtggtaaag gtggttctga cttcgacccg 360

aaaggtaaat ctgacaacga aatccgtcgt ttctctcagg ctttcatgcg tcagctgttc 420

cgttacatcg gtccgcagac cgacgttccg gctggtgaca tcggtgttac aggcttcgtt 480

gttatgcata tgttcggtga atacaaacgt ctgcgtaacg aatactctgg tgttgttacc 540

ggtaaacaca tgctgaccgg tggttctaac atccgtccgg aagctaccgg ttacggtgtt 600

gtttactacg ttaaacacat gatcgaacac cgtaccaaag gtgctgaaac cctgaaaggt 660

aaacgtgttg ctatctctgg ttctggtaac gttgctcagt acgctgctct gaaatgcatc 720

caggaaggtg ctatcgttaa atctatctct gactctaaag gtgttctgat cgctaaaacc 780

gctgaaggtc tggttccgga agaaatccac gaaatcatgg ctctgaaaga aaaacgtgct 840

tctatcgctg actctgcttc tctgtgcaaa aaacaccact acatcgctgg tgctcgtccg 900

tggaccaacg ttggtgaaat cgacatcgct ctgccgtgcg ctacccagaa cgaagtttct 960

ggtgaagaag ctgctgctct gatcaaacag ggttgccgtt acgttgctga aggttctaac 1020

atgggttctt ctgctgaagc tgttgaagtt ttcgaaaaat ctcgtgcttc tggtgaaggt 1080

tgctggctgg ctccgggtaa agctgctaac gctggtggtg ttgctgtttc tggtctggaa 1140

atggctcaga acgctcagtt ctctacctgg acccacgctg aagttgacgc taaactggct 1200

ggtatcatgc agaacatctt cgaacagtct accgacgttg cttctaaata ctgcgactct 1260

ggttctaaca acatcccgtc tctggttgac ggtgctaata tagctggctt cctgaaagtt 1320

gcgaccgcta tgcaggctgt tggtgactgg tgg 1353

<210> 17

<211> 1377

<212> DNA

<213> Artificial sequence

<400> 17

atgtctaacc tgccggttga accggaattc gaacaggctt acaaagaact ggcttctacc 60

ctggaaaact ctaccctgtt cgaacagcac ccggaatacc gtcgtgctct ccaggttgtg 120

agcgttccgg aacgtgttat ccagttccgt gttgtttggg aaaacgacaa aggtgaagtt 180

cagatcaacc gtggttaccg tgttcagttc aactctgctc tgggtccgta caaaggtggt 240

ctgcgtttcc acccgtctgt taacctgtct atcctgaaat tcctgggttt cgaacagatc 300

ttcaaaaacg ctctgaccgg tctgaacatg ggtggtggta aaggtggttc tgacttcgac 360

ccgaaaggta aatctgactc tgaaatccgt cgtttctgca ccgctttcat gaccgaactg 420

tgcaaacaca tcggtgctga caccgacctg ccggctggtg acatcggtgt taccggtcgt 480

gaggttggct tcctgttcgg tcagtaccgt cgtatccgta accagtggga aggtgttctg 540

accggtaaag gtggttcttg gggtggttct ctgatccgtc cggaagctac cggttacggt 600

gttgtttact acgttcagca catgatcaaa cacgttaccg gtggcaagga aagcttcgct 660

ggtaaacgtg ttgctatctc tggttctggt aacgttgctc agtacgctgc tctgaaagtt 720

atcgaactgg gtggttctgt tgtttctctg tctgactcta aaggttctct gatcgttaaa 780

gacgaatctg cttctttcac cccggaagaa atcgctctga tcgctgacct gaaagttgct 840

cgtaaacagc tgtctgaact ggctacctct tctgctttcg ctggtaaatt cacctacatc 900

ccggacgctc gtccgtggac caacatcccg ggtaaattcg aagttgctct gccgtctgct 960

acccagaacg aagtttctgg tgaagaagct gaacacctga tcaaatctgg tgttcgttac 1020

atcgctgaag gttctaacat gggttgcacc caggctgcta tcgacatctt cgaagctcac 1080

cgtaacgcta acccgggtga cgctatctgg tacgctccgg gtaaagctgc taacgctggt 1140

ggtgttgctg tttctggtct ggaaatggct cagaactctg ctcgtctgtc ttggacctct 1200

gaagaagttg acgctcgtct gaaaggtatc atggaagact gcttcaaaaa cggtctggaa 1260

accgctcaga aattcgctac cccggctaaa ggtgttctgc cgtctctggt taccggttct 1320

aacatcgctg gtttcaccaa agttgctgaa gctatgaaag accagggtga ctggtgg 1377

<210> 18

<211> 1347

<212> DNA

<213> Artificial sequence

<400> 18

atgccggctc agaccatcga agaactgatc gctgttatca aacagcgtga cggtcacatg 60

accgaattcc gtcaggctgt tgaagaagtt gttgactctc tgaaagttat cttcgaacgt 120

gaaccgaaat acatcccgat cttcgaacgt atgctggaac cggaacgtgt tatcatcttc 180

cgtgttccgt ggatggacga cgctggtcgt atcaacgtta accgtggttt ccgtgttcag 240

tacaactctg ctctgggtcc gtacaaaggt ggtctgcgtt tccacccgtc tgttaacctg 300

tctatcctga aattcctggg tttcgaacag atcctgaaaa actctctgac caccctgccg 360

atgggtggtg gtaaaggtgg ttctgacttc gacccgaaag gtaaatctga caacgaagtt 420

atgcgtttct gccagtcttt catgaccgaa ctgcagcgtc acgttggtgc tgacaccgac 480

gttccggctg gtgacatcgg tgttggtgct cgtgaaatcg gttacctgta cggtcagtac 540

aaacgtctgc gtaacgaatt caccggtgtt ctgaccggta aaaacgttaa atggggtggt 600

tctttcatcc gtccggaagc taccggttac ggtgctgttt acttcctgga agaaatgtgc 660

aaagacaaca acaccgttat ccgtggtaaa aacgttctgc tgtctggttc tggtaacgtt 720

gctcagttcg cttgcgaaaa actgatccag ctgggtgcta aagttctgac cttctctgac 780

tctaacggta ccatcgttga caaagacggt ttcaacgaag aaaaactggc tcacctgatg 840

tacctgaaaa acgaaaaacg tggtcgtgtt tctgaattca aagacaaata cccgtctgtt 900

gcttactacg aaggtaaaaa accgtgggaa tgcttcgaag gtcaggttga ctgcatcatg 960

ccgtgcgcta cccagaacga agtttctggt gacgacgcta cccgtctggt tggtctgggt 1020

ctgaaattcg ttgctgaagg tgctaacatg ccgtctaccg ctgaagctgt tcacgtttac 1080

cacgctaaag gtgttatgta cggtccggct aaagcttcta acgctggtgg tgtttctgtt 1140

tctggtctgg aaatgtctca gaactctgtt cgtctgcagt ggaccgctga agaagttgac 1200

cagaaactgc gtggtatcat gcgtggtatc ttcgttgctt gccgtgacac cgctaaaaaa 1260

tacggtcacc cgaaaaacta ccagatgggt gctaacatcg ctggtttcct gaaagttgct 1320

gactctatga tcgaacaggg ttgcgtt 1347

Claims

1. A glutamate dehydrogenase mutant is characterized in that the 116 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.1 is mutated from lysine to serine, and the 348 th amino acid is mutated from asparagine to leucine;

or the 116 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.1 is mutated into glutamic acid from lysine, and the 348 th amino acid is mutated into methionine from asparagine;

or the 116 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.1 is mutated from lysine to glutamine, and the 348 th amino acid is mutated from asparagine to methionine.

2. A gene encoding the glutamate dehydrogenase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. The recombinant plasmid of claim 3, wherein pET-28a is used as the starting plasmid.

5. A recombinant cell carrying the gene of claim 2 or the recombinant plasmid of claim 3 or 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is a bacterial or fungal host cell.

7. The method for preparing the glutamate dehydrogenase mutant according to claim 1, which comprises: inoculating the recombinant cell of claim 5 or 6 into a culture medium, culturing at 37 ℃ until the OD of the cell density reaches 0.5-0.7, adding IPTG inducer, inducing at 17 ℃ for 15-17h, collecting bacterial liquid, centrifuging the obtained bacterial liquid, collecting bacterial body, resuspending and crushing the obtained bacterial body to obtain cell crushing liquid, centrifuging the cell crushing liquid, collecting supernatant, and separating glutamate dehydrogenase mutant in the supernatant.

8. A method for synthesizing (R) -4-aminopentanoic acid is characterized by comprising the following steps: adding the mutant of claim 1 to a medium containing glucose dehydrogenase, 4-oxopentanoic acid, glucose and NH₄Cl-NH₃·H₂And reacting in a reaction system of O buffer solution to obtain the compound.

9. Use of the glutamate dehydrogenase mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to claim 3 or 4, or the recombinant cell according to claim 5 or 6, for the preparation of (R) -4-aminopentanoate.