CN113122525B - Formaldehyde conversion protein and application thereof - Google Patents

Abstract

The invention discloses a formaldehyde-converting protein mutant and application thereof, in particular relates to a plurality of formaldehyde-converting protein mutants with improved activity, and belongs to the field of biochemical engineering. Several mutants of formaldehyde-converting protein with improved activity are obtained by genetic engineering methods. The mutant can efficiently catalyze formaldehyde conversion.

Description

Formaldehyde conversion protein and application thereof

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a formaldehyde conversion protein variant and application thereof.

Background

One carbon compound is widely focused as a green energy substance with great application prospect, and can be used for synthesizing basic organic chemical raw materials, fuels and other high-added-value chemicals. Because of the characteristics of low cost and easy availability, the carbon compound becomes the compound which has the most development prospect for preparing high-value compounds by replacing petroleum, and has important scientific significance and development value in the fields of medicine, food and chemical industry. Meanwhile, the utilization and conversion of the carbon compound can also obviously reduce the negative influence on the ecological environment caused by people in the use process of fossil fuel and synthetic materials.

The formaldehyde can be converted from other carbon compounds, further is converted into a bioavailable intermediate substance, has the characteristics of wide sources, low price and the like, and has wide application prospects in industrial biotechnology. Although the use of a carbon compound by modifying a microorganism has been successful, a carbon compound such as formaldehyde, methanol, etc. does not provide the growth of the microorganism as the sole carbon source, and in addition, the yield of a high-value compound synthesized by metabolism of the microorganism using methanol is low and by-products are many.

Siegel et al reported in 2015 that a formaldehyde-converted protein (FLS) can catalyze formaldehyde condensation to produce DHA, but it has a high K _m Values. Formaldehyde has serious toxicity to thalli and high K _m FLS of the value is detrimental to the intracellular synthesis of DHA from formaldehyde and further the synthesis of high value compounds. Modification and application of FLS enzymes are now less studied. Obtaining high activity FLS is critical for formaldehyde conversion and utilization and offers more possibilities.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a formaldehyde conversion protein variant with improved formaldehyde conversion capability, and a coding gene and related application thereof.

In a first aspect, the invention claims formaldehyde-converting protein variants.

The formaldehyde conversion protein variant disclosed by the invention is obtained by performing point mutation on formaldehyde conversion protein, and the mutation site contains (or is) part or all of the following components: from the N-terminal, the 28 th, 90 th and 283 th bits are formed.

Preferably, the amino acid sequence of the formaldehyde-converting protein variant has more than 95% identity to a sequence comprising only the above-described mutation sites.

Further, the formaldehyde-converting protein variant is characterized in that: the formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites of the formaldehyde-converting protein: position 28 from the N-terminus.

Further, the formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites of the formaldehyde-converting protein: from the N end, the 28 th and 90 th positions are obtained.

Further, the formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites of the formaldehyde-converting protein: from the N end, the 28 th bit and the 283 th bit are arranged.

Still further, the formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites of the formaldehyde-converting protein: from the N end, the 28 th, 90 th and 283 th bits.

Further, the formaldehyde-converting protein variant may be any one of the following:

(A1) The formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites (or the following sites) of the formaldehyde-converting protein: position 28 from the N-terminus (corresponding mutant V1 or V2);

(A2) The formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites (or the following sites) of the formaldehyde-converting protein: from the N-terminus, at positions 28 and 90 (corresponding mutants V3, V4 or V5);

(A3) The formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites (or the following sites) of the formaldehyde-converting protein: positions 28 and 283 from the N-terminus (corresponding mutants V6, V7 or V8);

(A4) The formaldehyde-converting protein variant is a protein obtained by mutating amino acid residues of at least the following sites (or the following sites) of the formaldehyde-converting protein: position 28, 90 and 283 from the N-terminus (corresponding mutant V9);

wherein the amino acid sequence of the formaldehyde conversion protein is shown as SEQ ID No. 1.

In the formaldehyde-converting protein variant, the 28 th amino acid from the N end can be mutated to leucine or phenylalanine, the 90 th amino acid can be mutated to valine, leucine or alanine, and the 283 th amino acid can be mutated to histidine, serine or glutamine.

For amino acid substitutions, the following nomenclature is used: original amino acid (wild type), position (i.e. position in SEQ ID No. 1), substituted amino acid. Accordingly, substitution of leucine for the original isoleucine at position 28 of SEQ ID No.1 is designated "I28L".

In a specific embodiment of the present invention, the formaldehyde-converting protein variant is specifically any one of the following:

(a1) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L (corresponding mutant V1, the amino acid sequence of which is shown as SEQ ID No. 2);

(a2) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-T90V (corresponding mutant V3, the amino acid sequence of which is shown as SEQ ID No. 4);

(a3) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-T90L (corresponding mutant V4, the amino acid sequence of which is shown as SEQ ID No. 5);

(a4) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-T90A (corresponding mutant V5, the amino acid sequence of which is shown as SEQ ID No. 6);

(a5) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-N283H (corresponding mutant V6, amino acid sequence shown as SEQ ID No. 7);

(a6) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-N283S (corresponding mutant V7, amino acid sequence shown in SEQ ID No. 8);

(a7) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-N283Q (corresponding mutant V8, amino acid sequence shown in SEQ ID No. 9);

(a8) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L-N283H-T90L (corresponding mutant V9, amino acid sequence shown as SEQ ID No. 10);

(a9) The formaldehyde conversion protein variant is a protein obtained by mutating amino acid residues of the following sites of the formaldehyde conversion protein shown in SEQ ID No. 1: I28L (corresponding mutant V2, the amino acid sequence of which is shown as SEQ ID No. 3);

in a second aspect, the invention claims a formaldehyde-converting protein variant related biomaterial that is any one of the following:

(I) A nucleic acid molecule encoding the formaldehyde-converting protein variant of any one of claims 1-5;

(II) expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.

Wherein, the nucleic acid molecule encoding the formaldehyde conversion protein is a DNA molecule shown in SEQ ID No. 11.

Further, the nucleic acid molecule encoding the formaldehyde-converting protein variant is specifically any one of the following:

(B1) A DNA molecule shown in SEQ ID No.12 (corresponding mutant V1);

(B2) A DNA molecule shown in SEQ ID No.14 (corresponding mutant V3);

(B3) A DNA molecule shown in SEQ ID No.15 (corresponding mutant V4);

(B4) A DNA molecule shown in SEQ ID No.16 (corresponding mutant V5);

(B5) A DNA molecule shown in SEQ ID No.17 (corresponding mutant V6);

(B6) A DNA molecule shown in SEQ ID No.18 (corresponding mutant V7);

(B7) A DNA molecule shown in SEQ ID No.19 (corresponding mutant V8);

(B8) A DNA molecule shown in SEQ ID No.20 (corresponding mutant V9);

(B9) The DNA molecule shown in SEQ ID No.13 (corresponding mutant V2).

In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning the "nucleic acid molecule encoding the formaldehyde-converting protein variant" into the region of the multiple cloning site of pET21 a.

The recombinant bacterium may be E.coli containing the nucleic acid molecule.

In a third aspect, the invention claims the use of a formaldehyde conversion variant or biomaterial as hereinbefore described in any of the following:

(C1) Producing 1, 3-dihydroxyacetone or hydroxyacetaldehyde;

(C2) Improving the yield of 1, 3-dihydroxyacetone or hydroxy acetaldehyde;

(C3) Preparing a downstream product thereof by producing 1, 3-dihydroxyacetone;

(C4) Preparation of saccharides, organic acids and amino acids

In a fourth aspect, the invention claims a process for producing 1, 3-dihydroxyacetone or hydroxyacetaldehyde and/or for producing a downstream product of 1, 3-dihydroxyacetone or hydroxyacetaldehyde. The method can comprise the following steps: expressing any one of the formaldehyde conversion protein variants in a recipient bacterium to obtain a recombinant bacterium; fermenting and culturing the recombinant bacteria to obtain 1, 3-dihydroxyacetone or hydroxyaldehyde or downstream products from fermentation broth;

further, expression of the formaldehyde-converting protein variant in the recipient bacterium is achieved by introducing into the recipient bacterium the "nucleic acid molecule encoding the formaldehyde-converting protein variant".

Advantageous effects

The invention obtains the mutant of the enzyme by carrying out molecular transformation on formaldehyde conversion protein (FLS), thereby greatly improving the efficiency of producing 1, 3-dihydroxyacetone by formaldehyde condensation.

Drawings

Fig. 1: DHA standard curve

Terminology and definitions

In the present invention, amino acids are represented by single letter or three letter codes, having the following meanings: a: ala (alanine); i: ile (isoleucine); g: gly (glycine); n: asn (asparagine); w: trp (tryptophan); l: leu (leucine); t: thr (threonine); k: lys (lysine); r: arg (arginine); h: his (histidine); f: phe (phenylalanine); v: val (valine); s: ser (serine); q: gln (glutamine).

In the present invention, "homology" has the meaning conventional in the art, referring to "identity" between two nucleic acid or amino acid sequences, the percentage of which represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences to be compared obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length.

The term "mutation" with respect to an amino acid position or residue means that the amino acid at a particular position has been replaced with another amino acid.

The expression "XaY" is used herein to denote a mutation or substitution of an amino acid, wherein a denotes the position of the amino acid in SEQ ID No.1, X denotes the wild-type amino acid species at the a-position in SEQ ID No.1, and Y denotes the amino acid species after the mutation at the a-position in SEQ ID No. 1. For example, "I28L" means that isoleucine I at position 28 corresponding to SEQ ID No.1 is replaced with leucine L as aligned with SEQ ID No. 1.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The experimental methods used in the following examples are conventional methods unless otherwise specified.

EXAMPLE 1 construction of expression vector for Formaldehyde converting protein

Formaldehyde converting protein (FLS) is an amino acid mutant of benzaldehyde lyase (benzaldehyde lyase) derived from Pseudomonas fluorescens (Pseudomonas fluorescens biovar I), and comprises mutation into A28I-A394G-G419N-A480W-L90T-W89K-R188H, the nucleotide sequence of which is SEQ ID No.11, and the formaldehyde converting protein shown in SEQ ID No.1 is encoded. The recombinant plasmid pET21a-fls of the mutant is used as a template, a primer (an upstream primer: 5'-GTTTAACTTTAAGAAGGAGATATACATATGGC-3' downstream primer: 5'-GCTTTGTTAGCAGCCGGATC-3') is designed, and error-prone PCR is carried out to randomly mutate a target gene.

After the PCR is completed, the target gene is recovered by gel, and the mutated FLS gene is connected to a vector pET21 a. The vector linear fragment was transformed into E.coli BL21 Gold (DE 3) and incubated in an incubator at 37℃until a monoclonal was grown.

EXAMPLE 2 screening of mutants of Formaldehyde converting protein

The obtained clones of the mutants were picked up and put into 96-well plates containing LB medium, cultured overnight in a shaker at 37℃at 800rpm, and transferred into 96-well plates, cultured for a certain period of time in a shaker at 37℃at 800rpm, and then cultured at 20 ℃.

After the completion of the culture, the cells were collected by centrifugation. The cells were resuspended in pH7.4 buffer and reacted at 30℃for 3 hours with the addition of formaldehyde solution (pH 7.4) and 1mM TPP. After completion of the reaction, 60. Mu.L of Buffer1 (0.6 mg/mL galactose oxidase, 32U/mL horseradish peroxidase, pH 7.4) was added, followed by 50. Mu.L of Buffer2 (8 mM ABTS, pH 7.4) and detection was performed at 410nm for 20min.

Compared with the original FLS, the obtained strain is a beneficial mutant strain with higher catalytic efficiency, and then the corresponding mutant amino acid is found through gene sequencing. Through screening of three site-directed saturated mutant libraries, seven mutants with remarkably improved enzyme activity and stable activity are finally obtained. The names and corresponding mutant amino acids of the seven mutants are shown in Table 1 through gene sequencing.

TABLE 1 mutant names and corresponding mutant amino acids

EXAMPLE 3 expression of Formaldehyde-converting protein and mutants thereof in E.coli

The inoculating needle picks up positive bacteria of the original FLS and mutant, inoculates in LB medium, cultures for 12h at 37 ℃, then inoculates in 100mL LB medium with 1% (V/V) inoculum size, cultures at 37 ℃ and carries on IPTG induction expression. Collecting the culture bacterial liquid into a centrifuge tube, centrifuging, discarding the supernatant, re-suspending and cleaning the bacterial cells with a buffer solution, and preserving the bacterial cells at a low temperature.

EXAMPLE 4DHA standard curve

90. Mu.L of DHA solutions of different concentrations, 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, 5mM and 8mM, respectively, were added to the reaction system, 60. Mu.L of Buffer1 (0.6 mg/mL galactose oxidase, 32U/mL horseradish peroxidase, pH 7.4) was added, and then 50. Mu.L of Buffer2 (8 mM ABTS, pH 7.4) was added, followed by detection under light absorption at 410nm for 20min. A DHA standard curve (y=0.0058x+0.0004) was obtained

EXAMPLE 5 Formaldehyde-converting protein and assay for mutant Activity thereof

The cells of example 3 were collected by centrifugation at 4℃and the supernatant was collected by centrifugation after re-suspension and ultrasonic disruption.

The cell resuspension was mixed with formaldehyde solution and 1mM TPP, reacted at 30℃for 3 hours, after the completion of the reaction, 60. Mu.L of tool enzyme Buffer1 (0.6 mg/mL galactose oxidase, 32U/mL horseradish peroxidase, pH 7.4) was added, and then 50. Mu.L of Buffer2 (8 mM ABTS, pH 7.4) was added, followed by detection under light absorption at 410nm for 20 minutes. The wild-type FLS and mutant whole-cell enzyme activities were calculated, and the percentage of mutant enzyme activities relative to the wild-type is shown in the following table (100% of the wild-type whole-cell enzyme activity).

TABLE 2 Whole cells of formaldehyde transformed variants were active at 5mM formaldehyde concentration

EXAMPLE 6 detection of formaldehyde to DHA by chromogenic method

The cells of example 3 were collected by centrifugation at 4℃and Hepes buffer (100 mM Hepes-NaCl, pH 7.4) was resuspended, and the supernatant was collected by centrifugation.

The cell resuspension was mixed with formaldehyde solution, reacted at 30℃for 3 hours, after the completion of the reaction, 60. Mu.L of Toolse Buffer1 (0.6 mg/mL galactose oxidase, 32U/mL horseradish peroxidase, pH 7.4) was added, and then 50. Mu.L of Buffer2 (8 mM ABTS, pH 7.4) was added, followed by detection under light absorption at 410nm for 20 minutes. The amounts of wild-type FLS and mutant DHA were calculated from the DHA standard curve in example 4 (y=0.0058x+0.0004), as shown in the table below.

TABLE 3 amount of DHA produced by whole cells of Formaldehyde converting protein variants at 5mM formaldehyde concentration

EXAMPLE 7 purification of Formaldehyde converting proteins and mutants thereof

The cells collected in example 3 were resuspended in pH7.4 buffer, sonicated in an ice bath, and the supernatant collected by centrifugation using Ni ²⁺ And (3) performing affinity chromatography purification on the expressed mutant by using a chromatographic column, eluting by using imidazole, desalting by using a desalting column, and storing the purified protein in a buffer solution for later use.

Example 8 determination of kinetic parameters of Formaldehyde converting proteins and their mutant pure enzymes

Adding pure enzyme with the same concentration into a reaction system, taking substrates with different concentrations to detect absorbance values, and determining the initial rate of each reaction. The specific detection method comprises the following steps:

1. the purified mutant was diluted to a concentration of 3mg/mL with pH7.4 buffer.

2. Formaldehyde solutions with different concentrations (pH 7.4buffer containing formaldehyde with different concentrations and 1mM TPP) are added as substrates (the final concentration is 2-225mM respectively), 3mg/mL of pure enzyme is added into the reaction system, and the reaction is carried out at 30 ℃. The reaction samples were taken over time and 90. Mu.L of Toolse Buffer1 (0.6 mg/mL galactose oxidase, 32U/mL horseradish peroxidase, pH 7.4) was added, followed by 50. Mu.L of Toolse Buffer2 (8 mM ABTS, pH 7.4) and detected at 410nm for 20min.

3. Calculating the initial speed of the enzymatic reaction under different substrate concentrations according to the detection result, and calculating K _m Value sum V _max Further calculate k _cat The values and calculation results are shown in Table 3.

TABLE 4 kinetic parameters of Formaldehyde conversion protein variants

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, all changes and modifications that may be made without departing from the spirit of the invention are intended to be within the scope of the invention as claimed.

Sequence listing

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Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

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Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

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

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

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Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

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Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

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Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

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

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Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

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Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

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Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

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Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

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Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

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Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

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Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

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Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

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Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

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Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

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Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

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Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

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Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

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Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

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His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

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

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

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Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

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Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

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

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

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Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

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Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

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

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

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Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

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Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

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Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

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

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Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

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Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

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Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

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Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

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Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

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Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

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Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

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Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

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Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

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

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Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

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Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

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Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

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Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

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Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

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Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

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Ala Gly Val Glu His Leu Phe Gly Leu His Gly Phe His Ile Asp Thr

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Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

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His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

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

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

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Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

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Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

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

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

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Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

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Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

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Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

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

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

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Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

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Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

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Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

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Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

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Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

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Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

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Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

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Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

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Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

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Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

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Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

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Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 5

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 5

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 6

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 6

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Asn Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 7

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 7

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu His Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 8

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 8

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu Ser Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 9

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 9

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

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

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 10

<211> 575

<212> PRT

<213> Artificial sequence ()

<400> 10

Met Ala Met Ile Thr Gly Gly Glu Leu Val Val Arg Thr Leu Ile Lys

1 5 10 15

Ala Gly Val Glu His Leu Phe Gly Leu His Gly Leu His Ile Asp Thr

20 25 30

Ile Phe Gln Ala Cys Leu Asp His Asp Val Pro Ile Ile Asp Thr Arg

35 40 45

His Glu Ala Ala Ala Gly His Ala Ala Glu Gly Tyr Ala Arg Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

Phe Leu Thr Gly Ser Gly Ala Leu Arg Asp Asp Glu Thr Asn Thr Leu

100 105 110

Gln Ala Gly Ile Asp Gln Val Ala Met Ala Ala Pro Ile Thr Lys Trp

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Trp Asp Ile Leu Met Asn Gln Ile Asp Glu Asp Ser Val Ile

165 170 175

Ile Pro Asp Leu Val Leu Ser Ala His Gly Ala His Pro Asp Pro Ala

180 185 190

Asp Leu Asp Gln Ala Leu Ala Leu Leu Arg Lys Ala Glu Arg Pro Val

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Ser Met Leu Ser Gly Leu Pro Asp Ala Met Arg Gly Gly Leu

245 250 255

Val Gln Asn Leu Tyr Ser Phe Ala Lys Ala Asp Ala Ala Pro Asp Leu

260 265 270

Val Leu Met Leu Gly Ala Arg Phe Gly Leu His Thr Gly His Gly Ser

275 280 285

Gly Gln Leu Ile Pro His Ser Ala Gln Val Ile Gln Val Asp Pro Asp

290 295 300

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

305 310 315 320

Asp Val Gly Gly Thr Ile Glu Ala Leu Ala Gln Ala Thr Ala Gln Asp

325 330 335

Ala Ala Trp Pro Asp Arg Gly Asp Trp Cys Ala Lys Val Thr Asp Leu

340 345 350

Ala Gln Glu Arg Tyr Ala Ser Ile Ala Ala Lys Ser Ser Ser Glu His

355 360 365

Ala Leu His Pro Phe His Ala Ser Gln Val Ile Ala Lys His Val Asp

370 375 380

Ala Gly Val Thr Val Val Ala Asp Gly Gly Leu Thr Tyr Leu Trp Leu

385 390 395 400

Ser Glu Val Met Ser Arg Val Lys Pro Gly Gly Phe Leu Cys His Gly

405 410 415

Tyr Leu Asn Ser Met Gly Val Gly Phe Gly Thr Ala Leu Gly Ala Gln

420 425 430

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

435 440 445

Gly Ser Val Gly Tyr Ser Ile Gly Glu Phe Asp Thr Leu Val Arg Lys

450 455 460

Gln Leu Pro Leu Ile Val Ile Ile Met Asn Asn Gln Ser Trp Gly Trp

465 470 475 480

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

485 490 495

Thr Arg Leu Glu Asn Gly Ser Tyr His Gly Val Ala Ala Ala Phe Gly

500 505 510

Ala Asp Gly Tyr His Val Asp Ser Val Glu Ser Phe Ser Ala Ala Leu

515 520 525

Ala Gln Ala Leu Ala His Asn Arg Pro Ala Cys Ile Asn Val Ala Val

530 535 540

Ala Leu Asp Pro Ile Pro Pro Glu Glu Leu Ile Leu Ile Gly Met Asp

545 550 555 560

Pro Phe Ala Gly Ser Thr Glu Asn Leu Tyr Phe Gln Ser Gly Ala

565 570 575

<210> 11

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 11

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cattcatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 12

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 12

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 13

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 13

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg ctttcatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 14

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 14

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcgggtg gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 15

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 15

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggctg gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 16

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 16

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcgggcg gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 17

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 17

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgcata ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 18

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 18

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgtcga ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 19

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 19

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgcaga ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

<210> 20

<211> 1725

<212> DNA

<213> Artificial sequence ()

<400> 20

atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60

catctgtttg gcctgcatgg cctacatatt gacaccattt ttcaggcttg cctggaccac 120

gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180

gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240

gccgttacgc cgatcgcgaa cgctcggctg gatcgcactc cggttctgtt cctgaccggt 300

tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360

atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420

ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480

ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540

gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600

ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660

aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720

ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780

tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840

ggtctgcata ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900

gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960

gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020

gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080

gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140

aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200

tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260

atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320

cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380

ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440

accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500

aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560

gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620

aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680

ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcg 1725

Claims (5)

1. A formaldehyde-converting protein variant, characterized by: the amino acid sequence of the mutant is shown as SEQ ID No.2-SEQ ID No. 10.

2. A formaldehyde-converted protein variant related biomaterial characterized by: is any one of the following:

(I) A nucleic acid molecule encoding the formaldehyde-converting protein variant of claim 1;

(II) an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of (I), said transgenic cell line being of a non-animal or plant variety.

3. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule encoding the formaldehyde-converting protein variant is any one of the following:

(B1) A DNA molecule shown in SEQ ID No. 12;

(B2) A DNA molecule shown in SEQ ID No. 14;

(B3) A DNA molecule shown in SEQ ID No. 15;

(B4) A DNA molecule shown in SEQ ID No. 16;

(B5) A DNA molecule shown in SEQ ID No. 17;

(B6) A DNA molecule shown in SEQ ID No. 18;

(B7) A DNA molecule shown in SEQ ID No. 19;

(B8) A DNA molecule shown in SEQ ID No. 20;

(B9) A DNA molecule shown in SEQ ID No. 13; or (b)

The recombinant bacterium is escherichia coli containing the nucleic acid molecule.

4. Use of a formaldehyde conversion variant or biomaterial according to any one of claims 1 to 3 in any one of the following:

(C1) Producing 1, 3-dihydroxyacetone or hydroxyacetaldehyde;

(C2) Improving the yield of 1, 3-dihydroxyacetone or hydroxyacetaldehyde;

(C3) The downstream products are prepared by producing 1, 3-dihydroxyacetone or hydroxyacetaldehyde.

5. A process for producing 1, 3-dihydroxyacetone or hydroxyacetaldehyde and/or for producing a downstream product of 1, 3-dihydroxyacetone or hydroxyacetaldehyde, characterized by: the method comprises the following steps: expressing the formaldehyde conversion protein variant of claim 1 in a recipient bacterium to obtain a recombinant bacterium; fermenting and culturing the recombinant bacteria to obtain 1, 3-dihydroxyacetone or hydroxyaldehyde or downstream products from fermentation broth; further, expression of the formaldehyde-converting protein variant in the recipient bacterium is achieved by introducing into the recipient bacterium the nucleic acid molecule encoding the formaldehyde-converting protein variant of claim 3.

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