1. A glucose oxidase mutant with improved thermostability is characterized in that the glucose oxidase mutant is prepared by reacting an amino acid sequence shown as SEQ ID NO: 1 at least one amino acid of the 4 th, 70 th, 86 th, 96 th, 274 th and 508 th positions of the glucose oxidase.

2. The glucose oxidase mutant with improved thermostability according to claim 1,

the mutation of the 4 th amino acid is I4A, I4P or I4Y;

the mutation of the 70 th amino acid is D70Q or D70K;

the amino acid 86 is mutated to A86F or A86Q or deleted;

the mutation of the 96 th amino acid is S96A, S96N or S96F;

the mutation of the 274 th amino acid is G274S, G274Q or G274H;

the mutation of amino acid 508 is P508A, P508H or P508C.

3. The glucose oxidase mutant with improved thermostability according to claim 1,

the substitutions are I4A, D70K, S96A, G274Q, P508H and deletion of amino acid 86; or

The mutations are I4P, D70K, A86F, S96A, G274S, P508A; or

The mutations are I4A, D70D, A86Q, S96F, G274Q, P508H; or

The mutations are I4Y, D70Q, A86Q, S96A, G274H and P508C; or

The mutations are I4Y, D70Q, A86Q, S96N, G274Q, P508A; or

The mutations are I4P, D70K, S96A, G274S, P508C and deletion of amino acid 86.

4. A gene encoding a glucose oxidase mutant with improved thermostability according to claim 1.

5. A recombinant vector comprising the gene of claim 4.

6. The recombinant vector according to claim 5, wherein the recombinant vector is a Pichia pastoris recombinant expression vector or an Aspergillus niger recombinant expression vector.

7. A recombinant strain comprising the gene of claim 4.

8. The recombinant strain according to claim 8, wherein the recombinant strain is a recombinant yeast cell or a filamentous fungal cell, more preferably pichia or aspergillus niger.

9. A method of preparing a glucose oxidase enzyme having improved thermal stability, comprising the steps of:

constructing a recombinant expression vector encoding a gene encoding the glucose oxidase mutant with improved thermostability according to claim 1;

introducing the obtained recombinant expression vector into a host cell;

inducing the host cell to express glucose oxidase.

10. Use of a glucose oxidase mutant with improved thermostability according to claim 1.

Background

Glucose oxidase (EC 1.1.3.4, GOD) as a highly specific oxidoreductase is capable of catalyzing the conversion of beta-D-glucose to gluconic acid and producing hydrogen peroxide (H)₂O₂) Plays an extremely important role in the fields of medical detection, food, biofuel cells, livestock breeding and the like. The glucose oxidase as a novel green additive has a unique action mode,unlike conventional enzyme preparations, it does not improve the effect on animal productivity by improving the digestive utilization of nutrients and reducing antinutritional factors. This may be done by acting on the one hand on gluconic acid produced by the action with glucose in the feed and on the other hand consuming O during the action with glucose₂So that the digestive tract is easy to form an anaerobic environment and the proliferation of anaerobic beneficial bacteria is promoted. Taken together, glucose oxidase may act as an acidifier and a probiotic by acting in the gut.

GOD is widely distributed in animals, plants and microorganisms, and is a main source for producing GOD due to the rapid growth and the wide source of the microorganisms, and main production strains are Aspergillus niger and Penicillium. The glucose oxidase has poor thermal stability, when the temperature exceeds 65 ℃, the enzyme activity of the glucose oxidase is rapidly reduced, and the temperature in the feed granulation process is above 75 ℃, so that the poor thermal stability of the glucose oxidase becomes a key factor which hinders the wide application of the glucose oxidase. Therefore, the ability to provide a thermostable glucose oxidase has important application value and will bring great market competitiveness. How to more effectively improve the heat resistance of the glucose oxidase is also the direction of future research. With the continuous and deep research, the application prospect of the glucose oxidase can be wider.

CN 103981159A discloses a glucose oxidase mutant and application thereof, wherein the heat resistance of the mutant is improved by changing Asn at position 172 into Arg and changing Cys at position 525 into Asn.

Most of the current enzyme reactions need to be carried out under mild conditions to maintain the normal activity of the enzyme, and in practical application adverse conditions (such as high heat, high acid, high salt and the like), the enzyme has poor tolerance and is easy to inactivate, so that the reaction efficiency is reduced, and the popularization and the application of the enzyme are greatly limited. Therefore, it is also a difficult point in the current research to perform stress-resistant modification on the enzyme molecule to improve the stability and catalytic activity of the enzyme molecule. The glucose oxidase mutant provided by the invention has important value for widening the practical application of glucose oxidase.

Disclosure of Invention

The invention aims to provide a glucose oxidase mutant.

It is still another object of the present invention to provide genes encoding glucose oxidase mutants.

It is still another object of the present invention to provide a recombinant vector comprising the gene encoding the glucose oxidase mutant as described above.

It is still another object of the present invention to provide a recombinant strain comprising the gene encoding the above-mentioned glucose oxidase mutant.

The glucose oxidase with improved thermal stability provided by the invention is based on the amino acid sequence shown as SEQ ID NO: 1, wherein at least one amino acid selected from the group consisting of 4 th, 70 th, 86 th, 96 th, 274 th and 508 th amino acids of the glucose oxidase is mutated.

According to a particular embodiment of the invention, the mutations are a combination of one or more of I4A, I4P, I4Y, D70Q, D70K, a86F, a86 (amino acid 86 deleted), a86Q, S96A, S96N, S96F, G274S, G274Q, G274H, P508A, P508H or P508C.

In a preferred embodiment of the invention, the substitution is I4A, D70K, a86, S96A, G274Q, P508H.

In a preferred embodiment of the invention, the substitutions are I4P, D70K, a86F, S96A, G274S, P508A.

In a preferred embodiment of the invention, the substitutions are I4A, D70D, a86Q, S96F, G274Q, P508H.

In a preferred embodiment of the invention, the substitutions are I4Y, D70Q, a86Q, S96A, G274H, P508C.

In a preferred embodiment of the invention, the substitutions are I4Y, D70Q, a86Q, S96N, G274Q, P508A.

In a preferred embodiment of the invention, the substitution is I4P, D70K, a86, S96A, G274S, P508C.

The invention also provides a gene for coding the mutant.

The invention also provides a recombinant expression vector containing the gene for coding the glucose oxidase mutant, and preferably, the recombinant expression vector is a pichia pastoris recombinant expression vector or an aspergillus niger recombinant expression vector.

The present invention also provides recombinant strains comprising a gene encoding a mutant of the above-described glucose oxidase, which in some embodiments of the invention is a fungal cell, preferably a yeast cell or a filamentous fungal cell, more preferably pichia or aspergillus niger.

The invention further provides application of the glucose oxidase mutant in the fields of food, chemical industry, medicine, agriculture or feed.

Compared with wild glucose oxidase, the glucose oxidase mutant provided by the invention has obviously improved thermal stability, and is beneficial to application of the glucose oxidase in industrial production. The invention carries out molecular modification on the glucose oxidase from Aspergillus niger, improves the thermal stability of the glucose oxidase by a site-directed mutagenesis technology and an error-prone PCR method, and lays a foundation for the industrial application of the glucose oxidase.

Drawings

FIG. 1 shows the determination of the enzymatic activity of glucose oxidase at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ respectively under the condition of pH5.5;

FIG. 2 shows the determination of the enzyme activity of glucose oxidase at 37 ℃ under the conditions of pH3, pH3.5, pH4, pH4.5, pH5, pH5.5, pH6, pH6.5, pH7 and pH7.5, respectively.

Detailed Description

The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.

The procedures and methods for vector construction of the present invention are those conventionally used in the field of genetic engineering.

Defining:

"Gene" refers to a segment of DNA involved in the production of a polypeptide, including regions preceding and following the coding region, and intervening sequences (introns) between individual coding segments (exons).

"amino acid substitution" refers to: 1 or more amino acid residues are substituted with other chemically similar amino acid residues. For example: a hydrophobic residue is substituted with another hydrophobic residue and a polar residue is substituted with another polar residue having the same charge. Functionally similar amino acids capable of such substitution are well known in the art and are classified by amino acid. Specific examples of the nonpolar (hydrophobic) amino acid include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.

In the present specification, "identity" with respect to a base sequence or an amino acid sequence means: the degree of identity of the bases or amino acid residues constituting each sequence between the compared sequences. Any numerical value of "identity" shown in the present specification may be any numerical value calculated by using a homology search program known to those skilled in the art.

An "expression vector" as used herein refers to a DNA construct containing a DNA coding sequence operably linked to one or more suitable control sequences capable of effecting the expression of the coding sequence in a host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may integrate into the genome itself. Plasmids are the most commonly used form of expression vector. However, the present invention is intended to include other forms of expression vectors which serve equivalent functions and which are or will be known in the art.

"promoter" refers to a regulatory sequence involved in binding RNA polymerase to initiate transcription of a gene. The promoter may be an inducible promoter or a constitutive promoter. A non-limiting example of an inducible promoter useful in the present invention is a glucoamylase promoter, which is an inducible promoter.

The term "host cell" refers to a cell or cell line into which a recombinant expression vector for polypeptide production can be transfected to express a polypeptide.

Fungal cells can be transformed in a manner known per se by processes involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall.

According to the invention, a vector comprising a polynucleotide sequence encoding a catalase is introduced into a host cell. The polypeptide comprising catalase activity is expressed from an expression vector comprising a polynucleotide encoding the polypeptide and comprising a regulatory sequence operably linked to a catalase coding sequence.

The term "recombinant strain" when used in reference to a cell, nucleic acid, protein or vector, refers to a cell, nucleic acid, protein or vector modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or the cell is derived from a cell so modified.

The term "selectable marker" or "selectable marker" refers to a gene capable of being expressed in a host cell, allowing for easy selection of those hosts containing the introduced nucleic acid or vector.

The modified amino acid sequence of the protein of the present invention may preferably be an amino acid sequence having 1 or more (preferably 1 or several or 1, 2, 3 or 4) conservative substitutions of the amino acids.

As used herein, the term "transformed" refers to a cell having a non-native nucleic acid sequence integrated into its genome or maintained as an episomal plasmid over multiple generations. Introduction of a vector comprising a polynucleotide sequence encoding a catalase polypeptide into an A.niger host cell can be carried out using any of a variety of techniques commonly known in the art, e.g., transformation, electroporation, nuclear microinjection, transduction, transfection, incubation with calcium phosphate DNA precipitates, high velocity bombardment with DNA-coated particles, or protoplast fusion.

Experimental materials and reagents:

1. bacterial strains and vectors

Coli strain Top10, Pichia pastoris X33, vector pPICZ α A, vector pGAPz α A, antibiotic Zeocin were purchased from Invitrogen.

2. Enzyme and kit

PCR enzyme, plasmid extraction kit and gel purification kit were purchased from Shanghai Producer, while restriction enzyme was purchased from NEB.

3. Culture medium

The E.coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCL, pH 7.0). LB-Amp is LB medium plus 100. mu.g/ml ampicillin. LB-Zeocin is LB culture medium added with 25 mug/ml Zeocin. The yeast medium was YPD (1% yeast extract, 2% peptone, 2% glucose). The yeast selection medium was YPDZ (YPD + 100. mu.g/ml Zeocin). Yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v/v)) and BMMY (the remainder was identical to BMGY except that glycerol was replaced with 0.5% methanol). Recombinant yeast fermentation basic salt culture medium: 5% of diammonium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 1.5% of magnesium sulfate heptahydrate, 1.95% of potassium sulfate, 0.1% of calcium sulfate and 0.03% of defoaming agent. 4.35ml PTM1 per liter after the high pressure. PTM1 (trace salt solution): copper sulfate 0.6% and potassium iodide 0.018%. 0.3 percent of manganese sulfate monohydrate, 0.02 percent of sodium molybdate dihydrate, 0.002 percent of boric acid, 0.05 percent of flowing cobalt chloride, 2 percent of zinc chloride, 6.5 percent of ferric sulfate heptahydrate, 0.5 percent of concentrated sulfuric acid and 0.02 percent of biotin

4. Chemical reagents:

glucose oxidase standards, o-dianisidine hydrochloride, and horseradish peroxide were purchased from Sigma, glucose from OXIOD, and other reagents from guangzhou chemical reagent factory.

5. Glucose oxidase determination method

The activity of the glucose oxidase is measured by o-dianisidine spectrophotometry. Under the action of glucose oxidase, glucose and oxygen react to generate gluconic acid and hydrogen peroxide, and the hydrogen peroxide and colorless reduced o-dianisidine generate water and red oxidized o-dianisidine under the action of peroxidase. And (3) measuring the light absorption value of the reaction liquid at 540nm, and calculating the enzyme activity of the glucose oxidase according to a standard curve.

Example 1 Aspergillus niger Glucose Oxidase (GOD) Gene Synthesis

A Glucose Oxidase (GOD) gene of Aspergillus niger is optimized for codon preference of Pichia pastoris, is synthesized by Jinweizhi biotechnology, Suzhou, and is artificially synthesized (a nucleic acid sequence is shown as SEQ ID No.1, and an amino acid sequence is shown as SEQ ID No. 2). EcoRI enzyme cutting sites and XbaI enzyme cutting sites are respectively introduced into the 5 'end and the 3' end of a glucose oxidase optimization Gene (GOX) and connected to a Puc57-amp vector, the GOX-Puc57 is inoculated to an LBA culture medium and cultured for 24 hours, plasmids are extracted, enzyme cutting is carried out by EcoRI and XbaI, a target gene fragment is recovered by cutting gel, a product is purified and recovered and is connected to an expression vector pPICz alpha A, and the expression vector pPICz alpha A-GOX is obtained.

Example 2 error-prone PCR mutagenesis

Mutations were randomly introduced using the GeneMorph II random mutation PCR kit (Stratagene) using pPICz. alpha.A-GOX as described above as a template.

PCR amplification was performed with the primers in table 1:

TABLE 1

The primers used were:
		GOX-F	5’-tctaatggtattgaggcttccttg-3’
GOX-R	5’-ttactgcatagaagcgtagtcagc-3’

the reaction sequence is shown in table 2:

TABLE 2

And detecting the PCR amplification result by agarose electrophoresis, and purifying and recovering the PCR product. Decomposing the original plasmid by using restriction endonuclease DpnI, transferring the decomposed product into escherichia coli Top10 by using a heat shock method, verifying a recombinant transformant by using a bacterial liquid PCR, extracting a plasmid of the transformant which is verified to be correct, and sequencing to determine a corresponding mutant. Correctly sequenced mutants were linearized with PmeI and transformed into pichia pastoris X33.

Example 3 high throughput screening of Heat-elevated glucose oxidase mutant strains

The recombinant yeast transformants obtained in example 2 were picked up one by one with a toothpick into 24-well plates, 1mL of BMGY-containing medium was added to each well, cultured at 30 ℃ and 220rpm for about 24 hours, and the supernatant was centrifuged. And respectively adding 1.6mL of BMMY culture medium for induction culture. And after 24h of culture, centrifuging to obtain supernatant, respectively taking out 200 mu L of the supernatant to a 96-pore plate, and performing glucose oxidase enzyme activity determination and heat treatment residual enzyme activity determination. Through multiple screening comparison. Finally, 17 effective mutation sites which are obtained by screening the heat-resistant mutation sites capable of obviously improving GOD of glucose oxidase through high-throughput screening are I4A, I4P, I4Y, D70Q, D70K, A86F, A86, A86Q, S96A, S96N, S96F, G274S, G274Q, G274H, P508A, P508H and P508C. The relative specific activities of these 17 mutants are shown in table 3.

TABLE 3 comparison of Heat resistance of original and mutant glucose oxidases

As can be seen from the data in table 1, the glucose oxidase mutants of the present invention, I4A, I4P, I4Y, D70Q, D70K, a86F, a86, a86Q, S96A, S96N, S96F, G274S, G274Q, G274H, P508A, P508H and P508C, have improved thermostability compared to the wild-type glucose oxidase.

Example 4 site-directed mutagenesis

The effective mutations I4A, I4P, I4Y, D70Q, D70K, a86F, a86 (deletion of amino acid 86), a86Q, S96A, S96N, S96F, G274S, G274Q, G274H, P508A, P508H and P508C with improved enzyme activity in example 2 were subjected to combined mutation. The resulting recombinant yeast transformants were picked up one by one with toothpicks into 24-well plates, 1mL of BMGY-containing medium was added to each well, cultured at 30 ℃ and 220rpm for about 24 hours, and centrifuged to remove the supernatant. Then respectively adding 1.6mLBMMY culture medium to carry out induction culture. And after 24h of culture, centrifuging to obtain supernatant, respectively taking out 200 mu L of the supernatant to a 96-pore plate, and performing glucose oxidase enzyme activity determination and heat treatment residual enzyme activity determination. Through multiple screening comparison. The 6 combined mutations with obviously improved enzyme activity obtained by experiments are respectively named as GOX-MUT-1, GOX-MUT-2, GOX-MUT-3, GOX-MUT-4, GOX-MUT-5 and GOX-MUT-6.

Wherein the content of the first and second substances,

GOX-MUT-1 contains mutation sites as follows: I4A, D70K, a86 (deletion of amino acid 86), S96A, G274Q, P508H; GOX-MUT-2 contains the following mutation sites: I4P, D70K, a86F, S96A, G274S, P508A;

GOX-MUT-3 contains the mutation sites as follows: I4A, D70D, a86Q, S96F, G274Q, P508H; GOX-MUT-4 contains mutation sites of: I4Y, D70Q, a86Q, S96A, G274H, P508C; GOX-MUT-5 contains the following mutation sites: I4Y, D70Q, a86Q, S96N, G274Q, P508A; GOX-MUT-6 contains the following mutation sites: I4P, D70K, a86, S96A, G274S, P508C.

TABLE 4 relative specific Activity of original and combinatorial mutants of glucose oxidase

Numbering	Relative specific activity (%)
		Original GOX control	100
GOX-MUT-1	86
		GOX-MUT-2	77
GOX-MUT-3	95
		GOX-MUT-4	39
GOX-MUT-5	56
		GOX-MUT-6	89

The specific activity of the enzyme is the number of enzyme activity units of unit weight (mg) of protein under a specific condition, and the relative specific activity is the ratio of the specific activities of the original strain and the mutant, as shown in table 4, the specific activities of 6 mutants are all higher than that of the original strain, and the enzyme activities are also all improved.

TABLE 5 comparison of Heat resistance of original glucose oxidase and combination mutant glucose oxidase

Numbering	Residual enzyme activity (%) in water bath at 75 ℃ for 3 minutes
		Original GOX control	2.6％
GOX-MUT-1	31.2％
		GOX-MUT-2	41.5％
GOX-MUT-3	27.3％
		GOX-MUT-4	39.8％
GOX-MUT-5	33.7％
		GOX-MUT-6	23.6％

6 mutants which can improve the heat resistance of the glucose oxidase are screened by multipoint combined mutation. Compared with the retention rate of 2.6 percent of the original glucose oxidase, the residual enzyme activity of the mutated glucose oxidase after being subjected to water bath for 3 minutes at 75 ℃ is improved to 23.6 to 41.5 percent.

Example 5 optimal reaction temperatures for original glucose oxidase and mutants GOX-MUT-1, GOX-MUT-2, GOX-MUT-3, GOX-MUT-4, GOX-MUT-5 and GOX-MUT-6

The results of measuring the enzyme activity of glucose oxidase at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃ respectively under the condition of pH5.5 are shown in FIG. 1. As can be seen from FIG. 1, the optimum reaction temperature range of glucose oxidase is 25 ℃ to 55 ℃. The activity of the reaction enzyme of MUT-1 and MUT-2 is higher than that of the original glucose oxidase at the temperature of 60 ℃ and 70 ℃.

Example 6 relative enzyme activities under different pH conditions of original glucose oxidase and mutants GOX-MUT-1, GOX-MUT-2, GOX-MUT-3, GOX-MUT-4, GOX-MUT-5 and GOX-MUT-6.

The enzyme activity of glucose oxidase was measured at 37 ℃ under the conditions of pH3, pH3.5, pH4, pH4.5, pH5, pH5.5, pH6, pH6.5, pH7 and pH7.5, respectively, and the results are shown in FIG. 2. As can be seen from FIG. 2, while improving the heat resistance of glucose oxidase, the relative enzyme activities of GOX-MUT-1, GOX-MUT-2, GOX-MUT-3, GOX-MUT-4, GOX-MUT-5 and GOX-MUT-6 under different pH conditions were substantially the same as those of the original glucose oxidase, and the relative enzyme activities were higher than those of the original glucose oxidase under the conditions of pH6.0 and pH 6.5.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Sequence listing

<110> Guangdong overflow Multi-interest Biotech Ltd

<120> glucose oxidase mutant with improved thermal stability, and coding gene and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 583

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

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

355 360 365

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

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580