Glucoamylase mutant M3 with improved secretion expression level as well as gene and application thereof

文档序号：373824 发布日期：2021-12-10 浏览：41次中文

阅读说明：本技术 分泌表达量提高的葡萄糖淀粉酶突变体m3及其基因和应用 (Glucoamylase mutant M3 with improved secretion expression level as well as gene and application thereof ) 是由罗会颖彤丽格秦星黄火清王亚茹杨浩萌王晓璐涂涛王苑苏小运柏映国于 2021-11-09 设计创作，主要内容包括：本发明涉及基因工程领域,具体涉及分泌表达量提高的葡萄糖淀粉酶突变体M3及其基因和应用。葡萄糖淀粉酶突变体GA2经定点突变后得到分泌表达量提高的突变体。本发明的葡萄糖淀粉酶突变体具有良好的酶学性质,可以应用于饲料、食品、医药、纺织等行业。(The invention relates to the field of genetic engineering, in particular to a glucoamylase mutant M3 with improved secretion expression level, and a gene and application thereof. After site-directed mutagenesis, the glucoamylase mutant GA2 can obtain a mutant with improved secretion expression. The glucoamylase mutant provided by the invention has good enzymatic properties, and can be applied to industries such as feed, food, medicine, textile and the like.)

1. The glucoamylase mutant M3 with improved secretion expression is characterized in that the amino acid sequence of the glucoamylase mutant M3 is shown as SEQ ID NO. 3.

2. A method for increasing the secretory expression level of glucoamylase, which comprises the following steps:

the method comprises the steps of mutating the 132 th amino acid of the amino acid sequence of the wild glucoamylase TlGA1931 from Ser to Cys, the 492 th amino acid from Tyr to Cys, the 548 th amino acid from Leu to Cys, the 562 th amino acid from Ala to Cys, the 108 th amino acid from Gln to Glu, the 599 th amino acid from Gln to Ala, the 600 th amino acid from Gly to Tyr and the 603 th amino acid from Val to Gln, wherein the amino acid sequence of the wild glucoamylase TlGA1931 is shown in SEQ ID NO 1.

3. A method for increasing the secretory expression level of glucoamylase, which comprises the following steps:

the method comprises the step of mutating the 599 th amino acid of a glucoamylase mutant GA2 from Gln to Ala, the 600 th amino acid from Gly to Tyr and the 603 th amino acid from Val to Gln, wherein the amino acid sequence of the glucoamylase mutant GA2 is shown as SEQ ID NO. 2.

4. A glucoamylase mutant gene encoding glucoamylase mutant M3 having increased secretion expression according to claim 1.

5. The glucoamylase mutant gene as set forth in claim 4, wherein the nucleotide sequence is as set forth in SEQ ID NO 5.

6. A recombinant vector comprising the glucoamylase mutant gene of claim 4.

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

8. The method for preparing the glucoamylase with the improved secretion expression level is characterized by comprising the following steps:

(1) transforming a host cell with a recombinant vector comprising the gene encoding glucoamylase mutant M3 with increased secretion expression according to claim 1 to obtain a recombinant strain;

(2) culturing the recombinant strain, and inducing and expressing the glucoamylase mutant;

(3) recovering and purifying the expressed glucoamylase.

9. The use of glucoamylase mutant M3 with increased secretion expression according to claim 1.

Technical Field

The invention relates to the field of genetic engineering, in particular to a glucoamylase mutant M3 with improved secretion expression level, and a gene and application thereof.

Background

Glucoamylase is one of glycoside hydrolases (exonucleases) that act on alpha-1, 4 glycosidic bonds and is known under the systematic name alpha-1, 4-glucan glucoside hydrolase (alpha-1, 4-glucan glucohydrolase, ec.3.2.1.3) or gamma-amylase (gamma-amylase), a saccharifying enzyme for short. Glucoamylase cleaves glucose molecules from the non-reducing sugar end with low substrate specificity, i.e., is capable of cleaving alpha-1, 4-glucosidic bonds and also has a slight hydrolytic capacity for alpha-1, 6-glucosidic and alpha-1, 3-glucosidic bonds. Glucoamylase consists of a catalytic domain, a linker domain, and a starch domain. According to the positional relationship of these 3 domains, fungal saccharifying enzymes belong to family 15 of glycoside hydrolases and are distinguished from family 13 α -amylases and family 14 β -amylases.

The glucoamylase is one of the most industrially used biological enzyme preparations, is widely applied to the industries of food, medicine, fermentation and the like, is one of the most China's biological enzyme products with the largest yield and the largest use amount, and has very high commercial value.

Chinese patent application CN202010131741.9 discloses a glucoamylase mutant GA2, a mutant strain derived fromTalaromyces leycettanusSaccharifying enzyme TlGA1931 obtained from JCM12802 strain is subjected to site-directed mutagenesis by Q108E/S132C/Y492C/S548C/A562C to obtain mutant GA2, after 10min treatment at 70 ℃, the relative residual enzyme activity of a wild type is 14 percent and is close to inactivation, the residual enzyme activity of glucoamylase mutant GA2 is 90 percent, after 2min treatment at 75 ℃, the relative residual enzyme activity of the wild type is 13 percent and is close to inactivation, and the residual enzyme activity of glucoamylase mutant GA2 is 95 percent. The optimum temperature of glucoamylase mutant GA2 was increased from 70 ℃ to 75 ℃.

The glucoamylase has 2 binding sites on Starch Binding Domain (SBD) which can simultaneously bind 2 molecules of substrate, wherein carbohydrate domains (CBM) are classified more and have wide sources, thus causing the diversity of sequence and structure. This makes the carbohydrate domain have different functions in promoting the combination of enzyme and substrate, the specific recognition of substrate, etc., and the characteristics of the carbohydrate domain are proved to have corresponding influences on the whole protein, such as stability, heat resistance and activity, but no report exists at present that the starch domain is closely related to the secretion amount of glucoamylase.

Disclosure of Invention

The present invention has been proposed and completed in order to further optimize the enzymatic properties of glucoamylase TlGA 1931.

The invention aims to provide a glucoamylase mutant with improved secretion expression.

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

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

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

It is still another object of the present invention to provide a method for preparing glucoamylase having increased secretion expression.

Still another object of the present invention is to provide the use of the glucoamylase mutant.

Chinese patent application CN202010131741.9 discloses a glucoamylase mutant GA2, a mutant strain derived fromTalaromyces leycettanusSaccharifying enzyme TlGA1931 obtained from JCM12802 strain is subjected to site-directed mutagenesis by Q108E/S132C/Y492C/S548C/A562C to obtain glucoamylase mutant GA2 with improved specific activity and thermostability, and glucoamylase mutant GA2 is subjected to further mutagenesis to obtain glucoamylase mutant with improved secretion expression, wherein the amino acid sequence of wild saccharifying enzyme TlGA1931 is shown as SEQ ID NO: 1.

The amino acid sequence of glucoamylase mutant GA2 is shown in SEQ ID NO. 2.

According to a specific embodiment of the invention: the glucoamylase mutant M3 was obtained by mutating the 599 th amino acid of the glucoamylase mutant GA2 from Gln to Ala, the 600 th amino acid from Gly to Tyr, and the 603 th amino acid from Val to Gln.

According to an embodiment of the invention, a glucoamylase is usedTlThe amino acid sequence of the mutant M3 of GA1931 is shown in SEQ ID NO 3.

The invention provides a method for coding the glucoamylaseTlThe gene of GA1931 mutant M3.

According to the specific embodiment of the invention, the gene sequence of the glucoamylase mutant GA2 is shown in SEQ ID NO: 4, respectively.

According to the specific embodiment of the invention, the sequence of the coding gene of glucoamylase mutant M3 is shown as SEQ ID NO. 5.

The method for improving the secretion expression level of the glucoamylase comprises the following steps:

The invention provides a recombinant vector containing the coding gene of the glucoamylase mutant M3.

The invention also provides a recombinant strain containing the coding gene of the glucoamylase mutant M3, wherein the preferred strain is Pichia pastoris GS115, and the recombinant strain containing the glucoamylase mutant gene is recombinant gibberellin GS 115/M3.

According to an embodiment of the present invention, a method for preparing glucoamylase having increased secretory expression is as follows:

(1) transforming a host cell by using a recombinant vector containing a coding gene of the glucoamylase mutant M3 to obtain a recombinant strain;

(2) culturing the recombinant strain, and inducing expression of glucoamylase;

(3) recovering and purifying the expressed glucoamylase.

The invention has the beneficial effects that:

the invention mutates the mutant GA2 of the existing glucoamylase, the specific activity and the thermal stability of the glucoamylase mutant M3 are basically not lost, the soluble expression level of the glucoamylase mutant M3 in pichia pastoris is obviously improved, and the expression level of the mutant M3 is improved by 4.1 times compared with that of the mutant GA 2. Therefore, the invention also proves that the carbohydrate domain (CBM) is not only related to the activity and stability of the protein, but also has a large influence on the expression amount for the first time. The soluble expression level of the mutant M3 is greatly improved, better catalytic efficiency and thermal stability can be still maintained, the industrial application requirements can be completely met, the mutant M3 can be well applied to the industries of food, medicine, textile and feed, and the mutant M3 has wide application prospect.

Drawings

FIG. 1 shows SDS-PAGE analysis of expression of glucoamylase mutant GA2 and mutant M3;

FIG. 2 shows the temperature optimum curves for glucoamylase mutant GA2 and mutant M3;

FIG. 3 shows the specific activities of glucoamylase mutant GA2 and mutant M3;

FIG. 4 shows the transcriptional level measurements of glucoamylase mutant GA2 and mutant M3.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: pichia pastoris (Pichia pastorisGS115), Pichia pastoris expression vector pPIC9 and strain GS 115.

2. Enzymes and other biochemical reagents: ligase was purchased from Invitrogen, site-directed mutagenesis kit was purchased from allgold, and others were made-by-home reagents (all available from general Biochemical reagents).

3. Culture medium:

(1) coli culture medium LB (1% peptone, 0.5% yeast powder, 1% NaCl, pH7. O).

(2) BMGY medium; 1% yeast powder, 2% peptone, 1.34% YNB, 0.000049< Biotin, 1% glycerol (v/v).

(3) BMMY medium: glycerol was replaced by 0.5% methanol, and the balance was BMGY.

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

EXAMPLE 1 site-directed mutagenesis of glucoamylase

The plasmid pPIC9 containing the coding gene of glucoamylase mutant GA2TlGa1931-GA2 sequence as template, and glucoamylase mutantTlThe codon corresponding to the 599 th amino acid of the amino acid sequence of GA1931-GA2 is mutated from CAA to GCG, the codon corresponding to the 600 th amino acid is mutated from GGG to TAC, and the codon corresponding to the 603 th amino acid is mutated from GTG to CAG, so that the coding gene of the mutant M3 is obtained. Primers for each round of site-directed mutagenesis are shown in the table below. The mutation site of the present application is selected at the C-terminus of the CBM domain, i.e., at the C-terminus of the entire glucoamylase, and there is no report that the CBM domain is related to the expression level of the enzyme.

TABLE 1 primers required for site-directed mutagenesis

EXAMPLE 2 construction of glucoamylase engineered strains

(1) Construction of expression vector and expression in Yeast

Recombinant plasmid pPIC 9-containing glucoamylaseTlGa1931-GA2 is used as a template, and a site-directed mutagenesis reagent is used for amplifying mutants. After nucleic acid gel validation, 1 μ L DMT enzyme was added to the PCR product, mixed well and incubated at 37 ℃ for 1 h. 2-5. mu.L of DMT enzyme-digested PCR product was taken and transformed into competent cell DMT by heat shock. And (4) selecting positive transformants for DNA sequencing, wherein sequencing shows that the transformants with correct sequences are used for preparing a large amount of recombinant plasmids. Using restriction endonucleasesBglII, carrying out linear expression plasmid vector DNA, electrically shocking and transforming yeast GS115 competent cells, culturing for 2-3 days at 30 ℃, and selecting transformants growing on MD plates for further expression experiments, wherein the concrete operation refers to a Pichia pastoris expression operation manual. Glucoamylase positive clones were then screened for GS115/M3 by chromogenic reaction of MM plates.

EXAMPLE 3 preparation of recombinant glucoamylase

(1) Shake flask level bulk expression of glucoamylase in Pichia pastoris

Screening out transformants with higher enzyme activity, inoculating the transformants into a 1L triangular flask of 300 mL BMGY liquid medium, and carrying out shaking culture on a shaking table at 30 ℃ and 220 rpm for 48 h; centrifuging at 4500 rpm for 5min, removing supernatant, adding 200 mL BMMY liquid culture medium containing 0.5% methanol into thallus, and performing induction culture at 30 deg.C and 220 rpm for 48 h. During the induction culture period, the methanol solution is replenished once at intervals of 24 hours to compensate the loss of methanol, so that the concentration of the methanol is kept at about 0.5 percent; centrifuging at 12,000 Xg for 10min, collecting supernatant fermentation liquid, detecting enzyme activity and performing SDS-PAGE protein electrophoresis analysis.

(2) Purification of recombinant glucoamylase

The supernatant of the recombinant glucoamylase from the shake flask fermentation culture was collected, concentrated using a 10 kDa membrane pack while the medium was replaced with pH 6.310 mM disodium phosphate-citrate buffer, and then purified by anion exchange column and identified using SDS-PAGE. As shown in FIG. 1, the GA2 mutant showed no protein band, while the M3 mutant showed a clear band, when the amount of the same sample was 10. mu.l. The expression level of glucoamylase starch mutant M3 was 4-5 times higher than that of GA2 mutant by protein band thickness analysis. Because the specific activity of the mutant M3 is the same as that of the GA2 mutant, the measured M3 enzyme activity (namely expression quantity) is improved by 4.1 times compared with that of GA2, and the result is consistent with the analysis result of software.

Example 4 determination of enzymatic Properties of purified glucoamylase mutants

The glucoamylase of the invention was assayed for activity using the DNS method. The specific method comprises the following steps: under the conditions of the optimal pH and the optimal temperature of each mutant (the optimal pH is 4.5 and the optimal temperature is 75 ℃), wherein 1 mL of reaction system comprises L00 muL of appropriate diluted enzyme solution and 900 muL of substrate, the reaction is carried out for 30min, 1.5mL of DNS is added to stop the reaction, and the reaction is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm. Glucoamylase activity unit definition: under the conditions of corresponding optimal temperature and optimal pH, the enzyme amount required by the catalytic hydrolysis of the substrate to release 1 mu mol of reducing sugar per minute is one enzyme activity unit (U).

Determination of enzymatic activity and kinetic parameters of glucoamylase M3 mutants:

(1) determination of optimum temperature and thermostability of glucoamylase M3 mutant

The enzymatic activities of wild-type glucoamylase, GA2 mutant and M3 mutant were measured at 20, 30, 40, 50, 55, 60, 65, 70, 80, 85 and 90 ℃ at pH 4.5, respectively. As shown in FIG. 2, the optimum temperatures of the wild type glucoamylase, the GA2 mutant and the M3 mutant were 65 ℃ and 75 ℃ respectively.

The stability of the wild type glucoamylase, the GA2 mutant and the M3 mutant at 70 ℃ is determined, the wild type glucoamylase, the GA2 mutant and the M3 mutant are respectively treated at 70 ℃ for 0, 2, 5, 10, 20, 30 and 60min in a 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution (pH 6.3) buffer solution system, and then relative residual enzyme activities are determined at the corresponding optimal temperatures, after the GA2 mutant and the M3 are treated at 70 ℃ for 10min, the residual enzyme activities of the wild type glucoamylase are about 12-15%, and the residual enzyme activities of the GA2 mutant and the M3 mutant are about 85-90%. After 1h of treatment, the wild type only has 5-8% of residual enzyme activity, and the GA2 mutant and the M3 mutant still have 60-70% of residual enzyme activity, so that the heat stability is obviously improved compared with the wild type.

(2) Determination of the enzymatic Activity of the glucoamylase M3 mutant

The purified glucoamylase M3 mutant of the invention was enzymatically reacted at pH 4.5 and 75 ℃ to determine its enzymatic activity. As shown in FIG. 3, the specific activity of glucoamylase M3 was 1093.8U/mg, which was substantially stable and increased by 1.2 times compared with the specific activity 1054.0U/mg of glucose starch mutant GA 2.

(3) Determination of the kinetic parameters of the wild-type glucoamylase, the GA2 and the M3 mutant were determined at the optimum pH and temperature, respectively:

preparing soluble starch with the concentration of 1-10 mg/mL by using a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5, and gelatinizing the soluble starch to be used as a reaction substrate. Then reacting for 15 min under the conditions of pH 4.5, 65 ℃ and 75 ℃ respectively and measuring the enzyme activity under different substrate concentrations. Purified wild-type glucoamylase using soluble starch as substrateK _mThe value was 0.77 mg/mL,V _maxthe value was 719.2. mu. mol/(min. mg). ProjectionVariant M3 was tested at 75 ℃ and pH 4.5,K _mthe value was 0.72 mg/mL,V _maxthe value was 1195.2. mu. mol/(min. mg),K _mthe value did not change, but the catalytic efficiency (kcat @)KM) increased from 982.3 mL/s/mg to 1759.8 mL/s/mg, the catalytic efficiency of mutant M3 was slightly lower than that of GA2 but improved over the wild type.

(4) Determination of transcriptional levels of glucoamylase GA2 and M3 mutants

The GA2 and M3 mutants are provided with three parallel biological repeats, each biological repeat is further subjected to three technical repeats, the three types of ARG4, gap and re-act are selected as internal reference genes, and the ARG4 is finally determined to be the used internal reference gene. RT-PCR experiments were performed and the calculation of the transcript level changes was performed using 2^-ΔΔCtAnd (4) calculating. As shown in fig. 4, the transcription level of mutant M3 was 1.9 times higher than that of mutant GA2, which was significant.

Sequence listing

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

<120> glucoamylase mutant M3 with improved secretion expression level and gene and application thereof

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Leu Val Ile Ala Ala Pro His Pro Thr Glu Leu Leu Pro Arg Ala Ser

20 25 30

Gly Ser Leu Asp Ser Trp Leu Ser Thr Glu Val Pro Tyr Ala Leu Asp

35 40 45

Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala Lys Ala Gln Gly Ala

50 55 60

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

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Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ile Lys Cys Leu Ile

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Asp Glu Phe Ile Ser Thr Gly Asp Ala Asn Leu Gln Ser Val Ile Gln

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Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser

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Gly Gly Leu Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn

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Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro

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Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala

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

180 185 190

Asn Asp Leu Ser Tyr Val Ser Gln Tyr Trp Asn Gln Ser Thr Phe Asp

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Leu Trp Glu Glu Ile Asp Ser Ser Ser Phe Phe Thr Thr Ala Val Gln

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

225 230 235 240

Thr Cys Ser Asn Cys Asp Ser Gln Ala Pro Leu Val Leu Cys Phe Leu

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Gln Ser Tyr Trp Thr Gly Ser Tyr Ile Leu Ser Asn Thr Gly Gly Gly

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Arg Ser Gly Lys Asp Ala Asn Ser Leu Leu Gly Ser Ile His Thr Phe

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Asp Pro Ala Ala Ala Gly Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser

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Ala Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser

305 310 315 320

Ile Tyr Ser Ile Asn Ser Gly Ile Pro Gln Gly Gln Ala Val Ala Val

325 330 335

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

340 345 350

Cys Thr Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp

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Asn Arg Ile Gly Ser Leu Thr Ile Thr Asp Val Ser Leu Ala Phe Phe

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Gln Asp Leu Tyr Pro Ser Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser

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Ser Thr Tyr Gln Ser Ile Val Ala Ala Val Lys Thr Tyr Ala Asp Gly

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Tyr Met Ser Ile Val Gln Lys Tyr Thr Pro Ser Asn Gly Ala Leu Ala

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Glu Gln Phe Ser Arg Asn Asp Gly Ser Pro Leu Ser Ala Val Asp Leu

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Thr Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ala Ala Arg Arg Asn Phe

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Ser Val Pro Ala Tyr Ser Trp Gly Glu Ala Ser Ala Asn Thr Val Pro

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Ser Ser Cys Ser Ala Ser Ser Ala Ser Gly Pro Tyr Ala Thr Ala Thr

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Asn Thr Asn Trp Pro Ala Pro Thr Cys Thr Ser Pro Pro Ala Asn Val

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Ala Val Arg Phe Asn Glu Met Val Thr Thr Asn Phe Gly Glu Asn Val

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Phe Val Val Gly Ser Ile Ala Ala Leu Gly Ser Trp Ser Pro Ser Ser

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Ala Ile Pro Leu Ser Ala Ala Glu Tyr Asn Ser Gln Thr Pro Leu Trp

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Arg Ser Tyr Thr Val Pro Gln Gly Cys Gly Val Thr Thr Ala Thr Val

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Leu Val Ile Ala Ala Pro His Pro Thr Glu Leu Leu Pro Arg Ala Ser

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Gly Ser Leu Asp Ser Trp Leu Ser Thr Glu Val Pro Tyr Ala Leu Asp

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Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala Lys Ala Gln Gly Ala

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

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Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser

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Gly Gly Leu Cys Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn

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Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro

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

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Asn Asp Leu Ser Tyr Val Ser Gln Tyr Trp Asn Gln Ser Thr Phe Asp

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Leu Trp Glu Glu Ile Asp Ser Ser Ser Phe Phe Thr Thr Ala Val Gln

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

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Thr Cys Ser Asn Cys Asp Ser Gln Ala Pro Leu Val Leu Cys Phe Leu

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Gln Ser Tyr Trp Thr Gly Ser Tyr Ile Leu Ser Asn Thr Gly Gly Gly

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Arg Ser Gly Lys Asp Ala Asn Ser Leu Leu Gly Ser Ile His Thr Phe

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Asp Pro Ala Ala Ala Gly Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser

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Ala Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser

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Ile Tyr Ser Ile Asn Ser Gly Ile Pro Gln Gly Gln Ala Val Ala Val

325 330 335

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

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Cys Thr Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp

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Asn Arg Ile Gly Ser Leu Thr Ile Thr Asp Val Ser Leu Ala Phe Phe

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Gln Asp Leu Tyr Pro Ser Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser

385 390 395 400

Ser Thr Tyr Gln Ser Ile Val Ala Ala Val Lys Thr Tyr Ala Asp Gly

405 410 415

Tyr Met Ser Ile Val Gln Lys Tyr Thr Pro Ser Asn Gly Ala Leu Ala

420 425 430

Glu Gln Phe Ser Arg Asn Asp Gly Ser Pro Leu Ser Ala Val Asp Leu

435 440 445

Thr Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ala Ala Arg Arg Asn Phe

450 455 460

Ser Val Pro Ala Tyr Ser Trp Gly Glu Ala Ser Ala Asn Thr Val Pro

465 470 475 480

Ser Ser Cys Ser Ala Ser Ser Ala Ser Gly Pro Cys Ala Thr Ala Thr

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Asn Thr Asn Trp Pro Ala Pro Thr Cys Thr Ser Pro Pro Ala Asn Val

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Ala Val Arg Phe Asn Glu Met Val Thr Thr Asn Phe Gly Glu Asn Val

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Phe Val Val Gly Ser Ile Ala Ala Leu Gly Ser Trp Ser Pro Ser Ser

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Ala Ile Pro Cys Ser Ala Ala Glu Tyr Asn Ser Gln Thr Pro Leu Trp

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Tyr Cys Ile Val Thr Leu Pro Ala Gly Thr Ser Phe Gln Tyr Lys Tyr

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

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Arg Ser Tyr Thr Val Pro Gln Gly Cys Gly Val Thr Thr Ala Thr Val

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Asn Asp Ser Trp Arg

610

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<213> Artificial Sequence (Artificial Sequence)

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Met Gln Tyr Leu Leu Lys Thr Thr Leu Gly Ala Leu Ser Val Ala Gln

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Leu Val Ile Ala Ala Pro His Pro Thr Glu Leu Leu Pro Arg Ala Ser

20 25 30

Gly Ser Leu Asp Ser Trp Leu Ser Thr Glu Val Pro Tyr Ala Leu Asp

35 40 45

Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala Lys Ala Gln Gly Ala

50 55 60

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

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Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ile Lys Cys Leu Ile

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Asp Glu Phe Ile Ser Thr Gly Asp Ala Asn Leu Glu Ser Val Ile Gln

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Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser

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Gly Gly Leu Cys Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn

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Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro

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Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala

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

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Asn Asp Leu Ser Tyr Val Ser Gln Tyr Trp Asn Gln Ser Thr Phe Asp

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Leu Trp Glu Glu Ile Asp Ser Ser Ser Phe Phe Thr Thr Ala Val Gln

210 215 220

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

225 230 235 240

Thr Cys Ser Asn Cys Asp Ser Gln Ala Pro Leu Val Leu Cys Phe Leu

245 250 255

Gln Ser Tyr Trp Thr Gly Ser Tyr Ile Leu Ser Asn Thr Gly Gly Gly

260 265 270

Arg Ser Gly Lys Asp Ala Asn Ser Leu Leu Gly Ser Ile His Thr Phe

275 280 285

Asp Pro Ala Ala Ala Gly Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser

290 295 300

Ala Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser

305 310 315 320

Ile Tyr Ser Ile Asn Ser Gly Ile Pro Gln Gly Gln Ala Val Ala Val

325 330 335

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

340 345 350

Cys Thr Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp

355 360 365

Asn Arg Ile Gly Ser Leu Thr Ile Thr Asp Val Ser Leu Ala Phe Phe

370 375 380

Gln Asp Leu Tyr Pro Ser Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser

385 390 395 400

Ser Thr Tyr Gln Ser Ile Val Ala Ala Val Lys Thr Tyr Ala Asp Gly

405 410 415

Tyr Met Ser Ile Val Gln Lys Tyr Thr Pro Ser Asn Gly Ala Leu Ala

420 425 430

Glu Gln Phe Ser Arg Asn Asp Gly Ser Pro Leu Ser Ala Val Asp Leu

435 440 445

Thr Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ala Ala Arg Arg Asn Phe

450 455 460

Ser Val Pro Ala Tyr Ser Trp Gly Glu Ala Ser Ala Asn Thr Val Pro

465 470 475 480

Ser Ser Cys Ser Ala Ser Ser Ala Ser Gly Pro Cys Ala Thr Ala Thr

485 490 495

Asn Thr Asn Trp Pro Ala Pro Thr Cys Thr Ser Pro Pro Ala Asn Val

500 505 510

Ala Val Arg Phe Asn Glu Met Val Thr Thr Asn Phe Gly Glu Asn Val

515 520 525

Phe Val Val Gly Ser Ile Ala Ala Leu Gly Ser Trp Ser Pro Ser Ser

530 535 540

Ala Ile Pro Cys Ser Ala Ala Glu Tyr Asn Ser Gln Thr Pro Leu Trp

545 550 555 560

Tyr Cys Ile Val Thr Leu Pro Ala Gly Thr Ser Phe Gln Tyr Lys Tyr

565 570 575

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

580 585 590

Arg Ser Tyr Thr Val Pro Ala Tyr Cys Gly Gln Thr Thr Ala Thr Val

595 600 605

Asn Asp Ser Trp Arg

610

<210> 4

<211> 1842

<212> DNA

<213> fungus (Talaromyces leycettanus JCM12802)

<400> 4

atgcagtacc ttcttaaaac taccctcggc gctctgagcg ttgctcagct tgtcatcgcg 60

gcaccacatc ccacggaact tctccctcgg gcatcagggt ccctggattc atggctttcc 120

accgaagttc cttacgctct cgatggtgta ttgaacaaca tcggacccaa tggtgcaaag 180

gcccaggggg ccagctccgg cattgtggtt gcaagcccca gcacaagtaa tcctgactac 240

ttctactctt ggactcggga cgctgcgctc accatcaaat gcctgatcga tgagttcatc 300

tcgactgggg atgcgaacct ggagtcggtg attcagaact atatcagctc ccaggccttc 360

ttgcaaacag tgtccaaccc ctctggcggc ctgtgtactg gaggtctcgg cgagcccaag 420

tttgaggtca atgaggcggc atttactggt gcttggggcc ggccacaaag agatgggccg 480

gccttgagag cgactgccat gatcaattac gccaactggc ttattgcaaa tggacaggct 540

tcactcgcca attcgatcgt ctggccgatc gtccagaatg atctctccta cgtcagccag 600

tactggaatc agagtacctt tgacctttgg gaggaaatcg acagctcctc cttcttcacg 660

acggctgtgc agcaccgtgc tcttgttgag ggctctgctc tggcaaaaaa gcttggccat 720

acctgctcaa actgcgactc tcaagcaccg cttgtcttgt gtttcctgca atcctactgg 780

accggttcct atattctttc caacaccgga ggcggacgtt ccggaaagga cgccaactcc 840

ctacttggaa gtattcatac ttttgaccca gcagcggcgg gatgcgacga caccactttc 900

cagccttgct ctgcccgagc cctagcgaac cacaaggtcg tcaccgactc gttccgttca 960

atctactcaa tcaactcggg catcccacag ggccaagcag tcgccgtggg tcgctaccct 1020

gaagatgtat atcagggcgg aaacgcatgg tatctctgca ccctcgctgc tgcagagcag 1080

ctgtacgacg cactctatca gtggaacagg atcggatctc tcacgatcac ggacgtcagc 1140

ttggcattct tccaggatct ctacccatcg gcggcaacag gcacttattc ctcatcctcg 1200

tcgacctacc aatccatcgt tgccgctgtc aagacgtacg cggacggata catgagcatt 1260

gttcaaaaat acaccccttc caacggcgcc ctcgccgagc agttctcccg caacgatggc 1320

tcccccctct cagccgtcga cctaacctgg tcctacgcct ccctgctcac tgccgccgcg 1380

cgcagaaatt tctccgtccc cgcctactcc tggggcgaag ccagcgccaa caccgtccca 1440

tcgtcttgct cggcctcgtc tgcctcaggc ccctgtgcca ccgcgaccaa cacgaactgg 1500

cccgcaccca catgcacctc gccaccggca aacgtggccg tccgattcaa cgagatggtc 1560

actaccaact ttggagagaa cgtctttgtc gtgggctcga tcgccgcgtt gggatcttgg 1620

agtcctagtt ccgctatccc gtgtagcgcg gccgaataca actcacagac gccgttgtgg 1680

tattgtatcg tgacgttgcc ggcgggcacg agcttccagt ataagtatat caagaaagag 1740

ccggatggca gtgtggtctg ggagagtgat ccgaacaggt cctatacggt gcctcaaggg 1800

tgtggcgtga cgactgcgac ggtgaatgat agttggaggt ag 1842

<210> 5

<211> 1842

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5