CN114736880A

CN114736880A - Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof

Info

Publication number: CN114736880A
Application number: CN202210648724.1A
Authority: CN
Inventors: 涂涛; 黄火清; 闫亚茹; 张伟; 姚斌; 罗会颖; 柏映国; 王苑; 苏小运; 王亚茹; 张�杰; 秦星; 王晓璐; 张红莲; 于会民
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2022-07-12
Anticipated expiration: 2042-06-09
Also published as: CN114736880B

Abstract

The invention belongs to the technical field of feed biology, and particularly relates to a mutant D497N with improved acid stability and glucose oxidase GoxM10, and a derivative mutant and application thereof. The invention takes glucose oxidase GoxM10 with high thermal stability as a research material, and improves and researches the acid stability of the glucose oxidase GoxM10 by adopting a protein engineering technology. Finally, the mutant with improved acid stability is obtained, thereby promoting the application of Gox in the feed industry.

Description

Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof

Technical Field

The invention belongs to the technical field of feed biology, and particularly relates to a mutant D497N with improved acid stability and glucose oxidase GoxM10, and a derivative mutant and application thereof.

Background

Glucose oxidase (Gox; EC 1.1.3.4) is a well-characterized aerobic dehydrogenase that catalyzes the production of d-gluconolactone and hydrogen peroxide from β -d-glucose using an oxygen molecule as an electron acceptor under aerobic conditions, followed by hydrolysis of the d-gluconolactone into gluconic acid and water. In recent years, Gox is generally concerned by the feed industry as a novel antibiotic substitute additive. Gox is used as a substitute of antibiotics and growth promoters and is vital to ensuring the health and production performance of animals on the premise of not damaging the health of human beings. A large number of application studies show that Gox as a green feed additive can be used for preventing gastrointestinal tract infection and diarrhea of livestock and has the effect of promoting animal growth.

It is crucial that Gox is able to maintain high activity in an acidic environment in order to exert its maximum effect in the gastrointestinal tract of animals. Therefore, in order to meet the application of Gox in the feed industry, Gox has good acid stability. The prior art has improved Aspergillus nigerAspergillus niger) Stability of the source Gox, obtainedT ₅₀Mutant GoxM4 increased by 7.5 ℃ and higher than GoxM4T _mThe mutant GoxM10 with the temperature being increased by 9 ℃.

Disclosure of Invention

The purpose of the present invention is to provide a mutant of glucose oxidase GoxM10 with improved acid stability.

It is still another object of the present invention to provide a gene encoding the above mutant.

It is still another object of the present invention to provide a recombinant vector comprising the above mutant gene.

It is still another object of the present invention to provide a recombinant strain comprising the above mutant gene.

It is a further object of the present invention to provide the use of the above mutants.

It is a further object of the present invention to provide a method for preparing glucose oxidase with improved stability.

It is a further object of the present invention to provide a method for increasing the glucose oxidase GoxM 10.

According to a specific embodiment of the present invention, site-directed mutagenesis is performed on glucose oxidase having an amino acid sequence shown in SEQ ID NO. 1.

According to the specific embodiment of the invention, the glucose oxidase with the amino acid sequence shown as SEQ ID NO. 1 is subjected to mutation at amino acid positions 113, 193, 362, 497, 535, 113/497, 193/497, 362/497 and 497/535, so as to obtain the glucose oxidase mutant with improved acid stability.

According to the specific embodiment of the invention, H113S mutation is carried out on glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO. 1, so as to obtain a mutant with an amino acid sequence shown as SEQ ID NO. 2 and improved acid stability.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant H113S with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

According to the specific embodiment of the invention, D193N mutation is carried out on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with the amino acid sequence shown as SEQ ID NO. 4 and improved acid stability.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant D193N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 5.

According to the specific implementation mode of the invention, K362Q mutation is carried out on glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with improved acid stability, with an amino acid sequence shown as SEQ ID NO. 6.

According to the specific embodiment of the invention, the invention also provides a gene for coding the glucose oxidase mutant K362Q with improved acid stability, and the nucleotide sequence of the gene is shown as SEQ ID NO. 7.

According to the specific embodiment of the invention, D497N mutation is carried out on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with the amino acid sequence shown as SEQ ID NO. 8 and improved acid stability.

According to a specific embodiment of the invention, the gene of the glucose oxidase mutant D497N with improved acid stability is encoded, and the nucleotide sequence is shown as SEQ ID NO. 9.

According to the specific embodiment of the invention, R535N mutation is carried out on glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO. 1, so as to obtain a mutant with an amino acid sequence shown as SEQ ID NO. 10 and improved acid stability.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant R535N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 11.

According to the specific embodiment of the invention, H113S/D497N mutation is carried out on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with the amino acid sequence shown as SEQ ID NO. 12 and improved acid stability.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant H113S/D497N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 13.

According to the specific embodiment of the invention, D193N/D497N mutation is carried out on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with improved acid stability with the amino acid sequence shown as SEQ ID NO. 14.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant D193N/D497N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 15.

According to the specific embodiment of the invention, K362Q/D497N mutation is carried out on glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO. 1 to obtain the mutant with improved acid stability with the amino acid sequence shown as SEQ ID NO. 16.

According to the specific embodiment of the invention, the gene for coding the glucose oxidase mutant K362Q/D497N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 17.

According to the specific embodiment of the invention, D497N/R535N mutation is carried out on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO. 1, so as to obtain the mutant with the amino acid sequence shown as SEQ ID NO. 18 and improved acid stability.

According to a specific embodiment of the invention, the gene for coding the glucose oxidase mutant D497N/R535N with improved acid stability is also provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 19.

According to a specific embodiment of the invention, the invention also provides a gene containing the glucose oxidase mutantThe starting vector of the recombinant expression vector is pPIC9, and the recombinant expression vector is specifically pPIC9-goxm10-H113S，pPIC9-goxm10-D193N，pPIC9-goxm10-K362Q，pPIC9-goxm10-D497N，pPIC9-goxm10-R535N，pPIC9-goxm10-H113S/D497N，pPIC9-goxm10-D193N/D497N，pPIC9-goxm10-K362Q/D497N，pPIC9-goxm10-D497N/R535N。

According to the specific embodiment of the invention, the recombinant strain containing the glucose oxidase mutant gene is also provided, and the starting strain of the recombinant strain is GS115(pPIC 9)-goxm10)。

The method for preparing glucose oxidase with improved stability comprises the following steps:

1) preparing a recombinant vector containing the mutant gene;

2) the recombinant vector transforms a pichia pastoris GS115 expression host;

3) fermenting and culturing the host, and isolating the glucose oxidase.

The invention provides application of the glucose oxidase mutant with improved acid stability, and particularly can be applied to the fields of food and feed.

Compared with GoxM10, the acid stability of the mutant is obviously improved. The optimum pH of mutants D193N, D497N and D193N/D497N was decreased by 1 unit, the optimum reaction pH of double-site mutants H113S/D497N, K362Q/D497N and D497N/R535N was 5.5, and the optimum reaction pH was decreased by 0.5 unit compared with GoxM10, and the optimum pH of the other mutants and GoxM10 were both 6.0. When treated for 30 min under the condition of pH 2.5, relative residual enzyme activities of mutants H113S, D193N, K362Q, D497N, R535N, H113S/D497N, D193N/D497N, K362Q/D497N and D497N/R535N are respectively 22%, 26%, 34%, 24%, 27%, 35%, 25% and 28% which are higher than GoxM10 (21%); after the mutant is treated for 60 min at the conditions of pH 2.75 and 37 ℃, the relative residual enzyme activities of the mutants H113S, D193N, K362Q, D497N, R535N, H113S/D497N, D193N/D497N, K362Q/D497N and D497N/R535N are respectively about 26%, 28%, 26%, 32%, 29%, 47%, 49%, 32% and 43% and are higher than 24% of the relative residual enzyme activity of GoxM 10.

The glucose oxidase mutant provided by the invention can well meet the application requirements in the fields of food, feed and the like, and has a very wide application prospect.

Drawings

FIG. 1 shows acid stability comparison of GoxM10 and the mutant treated at pH 2.5 for 30 min;

FIG. 2 shows a comparison of acid stability of GoxM10 and the mutant treated at pH 2.75 for 60 min;

figure 3 shows the pH optimum comparison of GoxM10 and the mutant.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: the expression host isPichia pastoris GS115, expression plasmid vector pPIC 9.

2. Enzymes and other biochemical reagents: endonuclease/high fidelity DNA polymerase and recombinase.

3. Coli LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH natural). Pichia pastoris YPD medium (1% yeast extract, 2% peptone, 2% glucose, pH natural); BMGY (1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, pH Natural); BMMY (1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, 1% methanol, pH natural).

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1 recombinant StrainGS115(pPIC9-goxm10) Preparation of

1. Amplification of nucleic acid sequence of high-thermal stability glucose oxidase GoxM10goxm10

Amplification by PCRgoxm10The gene fragment adopts a double-enzyme cutting method to obtain a vector pPIC9 nucleic acid fragment, and the two are connected by a recombinase to obtain a recombinant plasmid pPIC9-goxm10And transforming Pichia pastoris GS115 to obtain recombinant Pichia pastoris GS115(pPIC9-goxm10). The primers used for PCR were as follows:

GoxM10-F（SEQ ID NO: 20），GoxM10-R（SEQ ID NO: 21）。

wherein, GoxM10-F and GoxM10-R are used for amplifying the gene coding sequence of GoxM 10; the vector pPIC9 was obtained by inoculating the preserved strain to liquid LB medium overnight for culture, and extracting the plasmid.

After the amplification is finished, carrying out nucleic acid electrophoresis detection on the PCR product and the extracted plasmid,goxm10the sizes of the bands of the vector and the pPIC9 are 1746bp and 8088bp respectively, and the vector is usedEcoRI andNotand I, after enzyme digestion, respectively recovering and purifying the PCR product and the enzyme digestion product.

2. Construction of recombinant Strain GS115(pPIC 9)-goxm10)

To be recoveredgoxm10The recombinant plasmid was ligated with the pPIC9 gene fragment by recombinant enzyme, and the recombinant product was transformed into Escherichia coli JM109 competent cells, which were then plated on LB (100. mu.g/mL Amp) for selection. After sequencing is correct, use is made ofBglII restriction Endonuclease the recombinant plasmid pPIC9-goxm10Enzyme digestion is carried out, after enzyme digestion products are recovered, pichia pastoris GS115 competent cells are transformed by electric shock, and a recombinant expression strain GS115(pPIC 9) is obtained-goxm10)。

Example 2 preparation of recombinant mutant Strain GS115

1. Construction of recombinant plasmids with improved stability

Design of mutant primers based on the mutant, using the plasmid pPIC9-goxm10Introducing mutant amino acid by point mutation kit as template, and using PCR product containing mutant amino acidDpnI, digesting to remove a template, transforming the digested PCR product into escherichia coli JM109 competent cells, and carrying out sequencing verification on the escherichia coli JM109 competent cells to obtain escherichia coli pPIC9-goxm10-H113S，pPIC9-goxm10-D193N，pPIC9-goxm10-K362Q，pPIC9-goxm10-D497N，pPIC9-goxm10-R535N， pPIC9-goxm10-H113S/D497N，pPIC9-goxm10-D193N/D497N，pPIC9-goxm10-K362Q/D497N，pPIC9-goxm10D497N/R535N. The primers used are as follows:

Gox-H113S-F（SEQ ID NO：22），Gox-H113S-R（SEQ ID NO：23）；Gox-D193N-F（SEQ ID NO：24），Gox-D193N-R（SEQ ID NO：25）；Gox-K362Q-F（SEQ ID NO：26），Gox-K362Q-R（SEQ ID NO：27）；Gox-D497N-F（SEQ ID NO：28），Gox-D497N-R（SEQ ID NO：29）；

Gox-R535N-F（SEQ ID NO：30），Gox-R535N-R（SEQ ID NO：31）。

2. construction of acid-and Heat-Stable recombinant Strain GS115

By usingBglII, carrying out enzyme digestion on the mutation plasmid with correct sequencing, recovering the enzyme digestion product, and then carrying out electric shock transformation on the pichia pastoris GS115 competent cell to obtain a recombinant expression strain GS115(pPIC9-goxm10-H113S)，GS115(pPIC9-goxm10-D193N)，GS115(pPIC9-goxm10-K362Q)，GS115(pPIC9-goxm10-D497N)，GS115(pPIC9-goxm10-R535N)， GS115(pPIC9-goxm10-H113S/D497N)，GS115(pPIC9-goxm10-D193N/D497N)，GS115(pPIC9-goxm10-K362Q/D497N)，GS115(pPIC9-goxm10-D497N/R535N)。

Example 3 obtaining of glucose oxidase GoxM10 and high acid and Heat stability mutant enzyme proteins

1. Inducible expression of GoxM10 and mutants

Inoculating the obtained recombinant yeast expression strain into 50 mL YPD culture medium with the inoculation amount of 1% for seed liquid culture, culturing at 200 rpm and 30 ℃ for 48 h, then transferring into 400 mL BMGY culture medium with the inoculation amount of 1% for culturing at 200 rpm and 30 ℃ for 48 h, centrifugally collecting thalli, transferring into 200 mL BMMY culture medium, supplementing 1% methanol every 24 h for induction expression, and culturing for 72 h.

2. Purification of GoxM10 and mutants

Centrifuging the bacteria solution after induction expression at 12000 rpm for 10 min, collecting supernatant, concentrating with a 10 kDa membrane package, dialyzing with 10 mM disodium hydrogen phosphate-citric acid buffer solution (pH 6.5), purifying the crude enzyme solution after dialysis with an anion column, wherein the solution A is 10 mM disodium hydrogen phosphate-citric acid buffer solution (pH 6.5), the solution B is solution A, 1M NaCl is added, purifying protein, and collecting eluent.

3. Enzymatic characterization of GoxM10 and mutants

(1) Acid stability assay for GoxM10 and mutants

1 pH stability of GoxM10 and mutants was evaluated using two assays. The method comprises the following steps: the same concentrations of GoxM10 and mutant enzyme solutions were placed in accurately prepared 0.1M disodium hydrogen phosphate-citric acid buffers at pH 2.5, 2.75, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, followed by incubation at 37 ℃ for 1 h. And after the heat preservation is finished, measuring the residual enzyme activity under the standard condition, and recording the enzyme activity of the untreated sample as 100%. The second method comprises the following steps: the same concentration of GoxM10 and mutant enzyme solutions was placed in 0.1M disodium hydrogen phosphate-citric acid buffer at pH 2.5, followed by incubation at 37 ℃ for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. After the heat preservation is finished, the relative residual enzyme activity of the GoxM10 and the mutant enzyme solution thereof is measured under standard conditions. As shown in fig. 1, when treated at pH 2.5 for 30 min, the relative residual enzyme activities of mutants H113S, D193N, K362Q, D497N, R535N, H113S/D497N, D193N/D497N, K362Q/D497N, and D497N/R535N were 22%, 26%, 34%, 24%, 27%, 35%, 25%, and 28%, respectively, and were increased by about 5%, 24%, 62%, 14%, 29%, 67%, 19%, and 33%, respectively, as compared to GoxM10 (21%). As shown in FIG. 2, the relative residual enzyme activities of the mutants H113S, D193N, K362Q, D497N, R535N, H113S/D497N, D193N/D497N, K362Q/D497N and D497N/R535N are respectively 26%, 28%, 26%, 32%, 29%, 47%, 49%, 32% and 43% after treatment at pH 2.75 and 37 ℃ for 60 min, and are respectively improved by about 8%, 17%, 8%, 33%, 21%, 96%, 104%, 33% and 79% compared with GoxM10 (24%).

(4) pH optimum assay for GoxM10 and mutants

Firstly, substrate mixed liquor for GoxM enzyme activity determination with different pH gradients (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0) is respectively prepared, then, enzyme activity of GoxM10 and a mutant thereof is determined at 30 ℃, the enzyme activity under the condition of the optimal reaction pH is set as 100%, and then, relative enzyme activity under the other pH conditions is calculated, so that the optimal reaction pH of GoxM10 and the mutant thereof can be obtained. As shown in fig. 3, mutants D193N, D497N and D193N/D497N had an optimum pH of 5, which was decreased by one unit compared to GoxM10, mutants H113S/D497N, K362Q/D497N and D497N/R535N had an optimum reaction pH of 5.5, which was decreased by 0.5 unit compared to GoxM10, and mutants H113S, K362Q and R535N had an optimum reaction pH of 6.0 compared to GoxM 10.

The above examples are only for explaining the technical solutions of the present application, and do not limit the scope of protection of the present application.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> mutant D497N with improved acid stability of glucose oxidase GoxM10, and derivative mutant and application thereof

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 2

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

Ser Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 3

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccatcta aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 4

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asn Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 5

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaaaaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 6

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Gln Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 7

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagcaagctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 8

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asn Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 9

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctaa cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 10

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 11

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctaacgttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 12

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

Ser Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asn Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 13

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccatcta aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctaa cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 14

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asn Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asn Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 15

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaaaaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctaa cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 16

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Gln Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asn Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 17

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagcaagctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctaa cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 18

<211> 581

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Val Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Lys Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ala Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

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

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

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

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Ser Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

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

260 265 270

Asn Phe His Asn Val Thr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

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

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asn Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

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

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 19

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60

atcattgctg gtggtggatt gactggtttg gttgtcgctg ccagattgac tgagaaccca 120

gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180

gaagacttga acgcttacgg taaaatcttc ggatcttccg ttgaccacgc ttacgagact 240

gtctgccttg ccactaacaa tagaaccgct ttgattagag ctggtaacgg tttgggtggt 300

tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360

gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420

gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcaacgcc 480

tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540

tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600

gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660

caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720

caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780

agagctgttg gtgtcgagtt cggaactcac aagggtaact ttcacaatgt taccgctaaa 840

cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900

atcggaatga agtccatttt ggaaccactt ggtattaaga ccgtcgttga cttgcctgtt 960

ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020

gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080

gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140

agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200

attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260

ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320

ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380

ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440

tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500

tgggttgaat acattccata ccacttcaga cctaactacc atggtgtcgg aacctgttct 1560

atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctaacgttta cggtgtccag 1620

ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680

gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740

caataa 1746

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggtattgagg cttccttgtt gactgaccca 30

<210> 21

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttgcatggag gcgtagtcag ccaaaacagc 30

<210> 22

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tggaccagac catctaaggc tcaagttgac tc 32

<210> 23

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aacttgagcc ttagatggtc tggtccaagt ac 32

<210> 24

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tccgctgttg aaaacgatgg tgtcccaact aa 32

<210> 25

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgggacacca tcgttttcaa cagcggacat ca 32

<210> 26

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gactacactg agcaagctca cgaattgctt aa 32

<210> 27

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

caattcgtga gcttgctcag tgtagtcacc ga 32

<210> 28

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

caattcgtga gcttgctcag tgtagtcacc ga 32

<210> 29

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccaagcatcc aagttagcat cgtaggccaa gt 32

<210> 30

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gacaacgccg ctaacgttta cggtgtccag gg 32

<210> 31

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gacaccgtaa acgttagcgg cgttgtcaac g 31

Claims

1. The glucose oxidase GoxM10 mutant with improved acid stability is characterized in that the mutant has an amino acid sequence obtained by carrying out H113S or D193N or K362Q or D497N or R535N mutation on glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO: 1.

2. The glucose oxidase GoxM10 mutant with improved acid stability according to claim 1, wherein the mutant has an amino acid sequence obtained by subjecting glucose oxidase GoxM10 with an amino acid sequence shown as SEQ ID NO. 1 to H113S/D497N, D193N/D497N, K362Q/D497N or D497N/R535N mutation.

3. A glucose oxidase gene encoding the enzyme GoxM10 mutant with improved acid stability according to claim 1 or 2.

4. The glucose oxidase gene of claim 3, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO 3 or 5 or 7 or 9 or 11 or 13 or 15 or 17 or 19.

5. A recombinant vector comprising the glucose oxidase gene of claim 3.

6. A recombinant strain comprising the glucose oxidase gene of claim 3.

7. A method of making a glucose oxidase having improved acid stability, comprising the steps of:

constructing a recombinant vector comprising a gene encoding the glucose oxidase GoxM10 mutant with improved acid stability according to claim 1 or 2;

introducing the recombinant vector into a host cell;

inducing expression and separating glucose oxidase.

8. A method for improving the acid stability of glucose oxidase is characterized in that the method comprises the step of carrying out H113S or D193N or K362Q or D497N or R535N mutation on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO: 1.

9. The method of improving the acid stability of glucose oxidase according to claim 8, comprising:

carrying out H113S/D497N mutation on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO: 1; or

Carrying out D193N/D497N mutation on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO: 1; or

A step of mutating a glucose oxidase GoxM10 having an amino acid sequence shown in SEQ ID NO:1 into K362Q/D497N; or

Carrying out D497N/R535N mutation on glucose oxidase GoxM10 with the amino acid sequence shown as SEQ ID NO: 1.

10. Use of the glucose oxidase GoxM10 mutant with improved acid stability as claimed in claim 1 or 2 as feed additive and food additive.