CN112280760A - Glutamic dehydrogenase mutant and application thereof - Google Patents

Glutamic dehydrogenase mutant and application thereof Download PDF

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CN112280760A
CN112280760A CN202011353159.3A CN202011353159A CN112280760A CN 112280760 A CN112280760 A CN 112280760A CN 202011353159 A CN202011353159 A CN 202011353159A CN 112280760 A CN112280760 A CN 112280760A
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穆晓清
徐岩
周峰
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SUQIAN JIANGNAN UNIVERSITY INDUSTRY TECHNOLOGY INSTITUTE
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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: will be provided with0.5U/mL of the purified enzyme solution of the mutant is added 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:
Figure BDA0002801872820000041
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:
Figure BDA0002801872820000042
TABLE 3 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 3:
Figure BDA0002801872820000043
TABLE 4 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 4:
Figure BDA0002801872820000051
TABLE 5 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 5:
Figure BDA0002801872820000052
TABLE 6 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 6:
Figure BDA0002801872820000053
Figure BDA0002801872820000061
TABLE 7 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 7:
Figure BDA0002801872820000062
TABLE 8 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 8:
Figure BDA0002801872820000063
TABLE 9 primer sequences required for the construction of enzyme mutants having the amino acid sequence shown in SEQ ID NO. 9:
Figure BDA0002801872820000064
Figure BDA0002801872820000071
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
Figure BDA0002801872820000072
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 ratio in 5 minutes)10/90 to 50/50, then ratio 50/50 to 100/0 in 2 minutes), flow 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 liquor for 5min at 4 ℃ and 8000rpm, discarding the supernatant, taking thalli, washing the thalli twice with 9% physiological saline, suspending in a buffer solution A (100mmol/L Tris, 150mmol/L NaCl, 20mmol/L imidazole pH7.5) to obtain a bacterial suspension, carrying out ultrasonic disruption on the bacterial suspension to obtain a cell disruption solution, centrifuging the cell disruption solution at 10000rpm and 4 ℃ for 30min, and taking the supernatant 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
Figure BDA0002801872820000111
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
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Arg Val Phe Gln Glu Ala Lys Ile Leu Tyr Ala Pro Gly Lys Ala Ala
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Ile Lys Leu Gly Trp Ser Gln Glu Glu Val Asp Glu Lys Leu Lys Ser
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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 (10)

1. A glutamate dehydrogenase mutant, wherein said 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.
2. The glutamate dehydrogenase mutant according to claim 1, wherein the mutant is any one of the following (1) to (9):
(1) mutating the 116 th amino acid of glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.1 from lysine to serine, and mutating the 348 th amino acid 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 into glutamine from lysine, and the 348 th amino acid is mutated into methionine from asparagine;
(2) mutating 114 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.2 from lysine to serine, and mutating 348 th amino acid from asparagine to leucine;
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;
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;
(3) mutating 114 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.3 from lysine to serine, and mutating 349 th amino acid from asparagine to leucine;
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;
or the 114 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.3 is mutated into glutamine from lysine, and the 349 th amino acid is mutated into methionine from asparagine;
(4) mutating 112 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.4 from lysine to serine, and mutating 344 th amino acid from asparagine to leucine;
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;
or the 112 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.4 is mutated into glutamine from lysine, and the 344 th amino acid is mutated into methionine from asparagine;
(5) mutating 104 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.5 from lysine to serine, and mutating 326 th amino acid from asparagine to leucine;
or the 104 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence 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;
or the 104 th amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.5 is mutated into glutamine from lysine, and the 326 th amino acid is mutated into methionine from asparagine;
(6) mutating 102 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.6 from lysine to serine, and mutating 338 th amino acid from asparagine to leucine;
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;
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;
(7) mutating 101 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.7 from lysine to serine, and mutating 340 th amino acid from asparagine to leucine;
or the 101 st amino acid of the glutamate dehydrogenase with the starting amino acid sequence 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;
or the 101 st amino acid of the glutamate dehydrogenase with the starting amino acid sequence shown as SEQ ID NO.7 is mutated into glutamine from lysine, and the 340 st amino acid is mutated into methionine from asparagine;
(8) mutating 102 th amino acid of glutamate dehydrogenase with starting amino acid sequence shown as SEQ ID NO.8 from lysine to serine, and mutating 346 th amino acid from asparagine to leucine;
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;
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;
(9) mutating 113 th amino acid of glutamate dehydrogenase with an original amino acid sequence shown as SEQ ID NO.9 from lysine to serine, and mutating 349 th amino acid from asparagine to leucine;
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;
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.
3. A gene encoding the glutamate dehydrogenase mutant of claim 1 or 2.
4. A recombinant plasmid carrying the gene of claim 3.
5. The recombinant plasmid of claim 4, wherein pET-28a is used as the starting plasmid.
6. A recombinant cell carrying the gene of claim 3 or the recombinant plasmid of claim 4 or 5.
7. The recombinant cell of claim 6, wherein the recombinant cell is a bacterial or fungal host cell.
8. The method for preparing the glutamate dehydrogenase mutant according to claim 1 or 2, which comprises: inoculating the recombinant cell of claim 6 or 7 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.
9. A method for synthesizing (R) -4-aminopentanoic acid is characterized by comprising the following steps: adding the mutant of claim 1 or 2 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.
10. Use of the glutamate dehydrogenase mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant plasmid according to claim 4 or 5, or the recombinant cell according to claim 6 or 7, for the preparation of (R) -4-aminopentanoate.
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