CN113755473B - Glucoamylase mutant M5 with improved secretion expression level as well as gene and application thereof - Google Patents
Glucoamylase mutant M5 with improved secretion expression level as well as gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a glucoamylase mutant M5 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.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a glucoamylase mutant M5 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 about 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 leycettanusGlucoamylase mutant GA2 with improved specific activity and thermostability is obtained after glucoamylase mutant GA2 is subjected to site-directed mutagenesis by Q108E/S132C/Y492C/S548C/A562C, and glucoamylase mutant GA 3 with improved secretion expression is obtained by further performing mutagenesis on glucoamylase mutant GA2, wherein glucoamylase mutant M5 is obtained by performing site-directed mutagenesis on amino acids 599, 600 and 603 of glucoamylase mutant GA2, glucoamylase mutant M8 is obtained by performing site-directed mutagenesis on amino acids 589, 607, 608, 609 and 613 of glucoamylase mutant GA2, and the combination of the sites is mutated to obtain glucoamylase mutant M8, and the amino acid sequence of wild-type glucoamylase TlGA1931 is shown in SEQ ID NO 1.
The amino acid sequence of glucoamylase mutant GA2 is shown in SEQ ID NO. 2.
According to the specific embodiment of the invention, the glucoamylase mutant M3 is 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. The glucoamylase mutant M5 was obtained by mutating the amino acid at position 589 of the glucoamylase mutant GA2 from Ser to Asp, the amino acid at position 607 from Thr to Ile, the amino acid at position 608 from Val to Leu, the amino acid at position 609 from Asn to Asp, and the amino acid at position 613 from Arg to Gln. The method comprises the steps of mutating the 589 th amino acid of a glucoamylase mutant GA2 from Ser to Asp, mutating the 599 th amino acid from Gln to Ala, mutating the 600 th amino acid from Gly to Tyr, mutating the 603 th amino acid from Val to Gln, mutating the 607 th amino acid from Thr to Ile, mutating the 608 th amino acid from Val to Leu, mutating the 609 th amino acid from Asn to Asp, and mutating the 613 th amino acid from Arg to Gln, thus obtaining the glucoamylase mutant M8.
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.
According to an embodiment of the invention, a glucoamylase is usedTlThe amino acid sequence of the mutant M5 of GA1931 is shown in SEQ ID NO. 4.
According to an embodiment of the invention, a glucoamylase is usedTlThe amino acid sequence of the mutant M8 of GA1931 is shown in SEQ ID NO: 5.
The invention provides a method for coding the glucoamylaseTlThe gene of GA1931 mutant M5.
According to the specific embodiment of the invention, the gene sequence of the glucoamylase mutant GA2 is shown in SEQ ID NO: and 6.
According to the specific embodiment of the invention, the sequence of the coding gene of glucoamylase mutant M3 is shown as SEQ ID NO. 7.
According to the specific embodiment of the invention, the sequence of the coding gene of glucoamylase mutant M5 is shown as SEQ ID NO. 8.
According to the specific embodiment of the invention, the sequence of the coding gene of glucoamylase mutant M8 is shown as SEQ ID NO. 9.
The method for improving the secretion expression level of the glucoamylase comprises the following steps:
the method comprises the steps of mutating the amino acid at the position 589 of a glucoamylase mutant GA2 from Ser to Asp, mutating the amino acid at the position 607 from Thr to Ile, mutating the amino acid at the position 608 from Val to Leu, mutating the amino acid at the position 609 from Asn to Asp, and mutating the amino acid at the position 613 from Arg to Gln, wherein the amino acid sequence of the glucoamylase mutant GA2 is shown as SEQ ID NO. 2.
The invention provides a recombinant vector containing the coding gene of the glucoamylase mutant M5.
The invention also provides a recombinant strain containing the coding gene of the glucoamylase mutant M5, wherein the preferred strain is Pichia pastoris GS115, and the recombinant strain containing the glucoamylase mutant gene is recombinant gibberellin GS 115/M5.
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 M5 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 M5 are basically not lost, the soluble expression level of the glucoamylase mutant M5 in pichia pastoris is obviously improved, and the expression level of the mutant M5 is improved by 4.2 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 M5 is greatly improved, better catalytic efficiency and thermal stability can be still maintained, the industrial application requirements can be completely met, the mutant M5 can be well applied to the industries of food, medicine, textile and feed, and the mutant M5 has wide application prospect.
Drawings
FIG. 1 shows SDS-PAGE analysis of expression of glucoamylase mutant GA2 and mutants M3, M5, M8;
FIG. 2 shows the optimal temperature curves for glucoamylase mutant GA2 and mutants M3, M5, M8;
FIG. 3 shows the specific activities of glucoamylase mutant GA2 and mutants M3, M5, M8;
FIG. 4 shows the transcriptional level measurements of glucoamylase mutant GA2 and mutants M3, M5, M8.
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 GA2TlThe Ga1931-GA2 sequence is used as a template, a codon corresponding to 599 th amino acid of an amino acid sequence of a glucoamylase mutant GA2 is mutated from CAA to GCG, a codon corresponding to 600 th amino acid is mutated from GGG to TAC, and a codon corresponding to 603 th amino acid is mutated from GTG to CAG, so that the mutant M3 is obtained. The mutant M5 is obtained by mutating a codon corresponding to the 589 th amino acid of an amino acid sequence of a glucoamylase mutant GA2 from AGT to GAC, a codon corresponding to the 607 th amino acid from ACG to ATT, a codon corresponding to the 608 th amino acid from GTG to CTT, a codon corresponding to the 609 th amino acid from AAT to GAC, and a codon corresponding to the 613 th amino acid from AGG to CAG. The method comprises the steps of mutating the 589 th amino acid of a glucoamylase mutant GA2 from Ser to Asp, mutating the 599 th amino acid from Gln to Ala, mutating the 600 th amino acid from Gly to Tyr, mutating the 603 th amino acid from Val to Gln, mutating the 607 th amino acid from Thr to Ile, mutating the 608 th amino acid from Val to Leu, mutating the 609 th amino acid from Asn to Asp, and mutating the 613 th amino acid from Arg to Gln, thus obtaining the mutant M8.
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. Then glucoamylase positive clones were screened by chromogenic reaction of MM plates for GS115/M3, GS115/M5, GS 115/M8.
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, when the amount of the same sample was 10. mu.l, protein bands were not observed in the mutant GA2, but in the mutants M3, M5 and M8, protein bands were evident. The expression level of glucoamylase starch mutants M3, M5 and M8 is improved by 4-5 times compared with that of mutant GA2 by protein band thickness analysis through software. Due to the fact that the specific activities of the mutants M3, M5 and M8 and the mutant GA2 are at the same level, the enzyme activities (namely expression quantities) of the mutants M3, M5 and M8 are measured to be improved by 4.1 times, 4.2 times and 4.0 times compared with the mutant GA2, and the results are consistent with the results of software analysis.
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 (pH 4.5) and the optimal temperature of each mutant, 1 mL of a 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 mutants M3, M5, M8:
(1) determination of the optimum temperature and thermostability of the glucoamylase M3, M5 and M8 mutants
The enzymatic activities of wild type glucoamylase, mutant GA2, and mutants M3, M5 and M8 were measured at 20, 30, 40, 50, 55, 60, 65, 70, 80, 85 and 90 ℃ at pH4.5, respectively. As shown in FIG. 2, the optimum temperatures for glucoamylase wild type, mutant GA2, and mutants M3, M5, and M8 were 65 deg.C, 75 deg.C, and 70 deg.C, respectively.
The stability of wild type, mutant GA2, mutant M3, M5 and M8 of glucoamylase at 70 ℃ is determined, the wild type, mutant GA2, mutant M3, M5 and M8 of the glucoamylase are respectively treated at 70 ℃ for 0, 2, 5, 10, 20, 30 and 60min in a buffer system of 0.1mol/L citric acid-disodium hydrogen phosphate buffer (pH 6.3), and then relative residual enzyme activities are determined at corresponding optimal temperatures, after the wild type, mutant GA2, mutant M3, M5 and M8 of the glucoamylase are treated at 70 ℃ for 10min, the residual enzyme activity of the wild type of the glucoamylase is about 12-15%, the residual enzyme activities of the mutant GA2 and mutant M3 are basically the same and about 85-90%, and the residual enzyme activities of the mutant M5 and M8 are respectively 92% and 22%. After 1h of treatment, the wild type only has 5-8% of residual enzyme activity, while the residual enzyme activity of the mutant GA2 and the mutant M3 is 60-70%, the mutants M5 and M8 have 62% and 9% of residual enzyme activity, the heat stability of the mutant M5 is obviously improved compared with the wild type, and although the mutant M8 has partially same mutation sites as the mutant M5 and the mutant M3, the heat stability of the mutant M8 is not improved compared with the wild type.
(2) Determination of the enzymatic Activity of the glucoamylase mutants M3, M5, M8
The purified glucoamylase M3 and M5 mutants of the invention were enzymatically reacted at pH4.5 and 75 ℃ and the M8 mutant was enzymatically reacted at pH4.5 and 70 ℃ to determine the enzymatic activity. As shown in FIG. 3, the specific activity of glucoamylase M3 is 1093.8U/mg, that of M5 is 1112.9U/mg, that of M8 is 1004.3U/mg, and that of M3 and M5 is slightly higher than that of GA2 and that of M8 is slightly lower than that of GA2, compared with 1054.0U/mg, which is the specific activity of glucose starch mutant GA 2.
(3) Determination of kinetic parameters of wild-type glucoamylase, mutant GA2 and mutants M3, M5 and M8 at 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 at pH4.5, 65 deg.C, 75 deg.C and 70 deg.C respectively15 min 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). M3 and M5 mutants were added at 75 ℃ and pH4.5,K mthe values are 0.72 mg/mL and 0.38 mg/mL respectively,V maxvalues are 1195.2. mu. mol/(min. mg), 1226.8. mu. mol/(min. mg), respectively; m8 mutant was cultured at 70 ℃ and pH4.5,K mthe value was 0.31 mg/mL,V maxthe value was 1004.4. mu. mol/(min. mg), the M3 mutant was more wild-typeK mThe values are not changed, and the M5 and M8 mutants are compared with the wild typeK mThe value decreases significantly and the substrate affinity increases. Catalytic efficiency (kcat;) of mutant M3KM) 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 mutant GA2 but improved compared with the wild type; catalytic efficiency (kcat;) of mutants M5 and M8Km) is respectively increased from 982.3 mL/s/mg to 3378.2mL/s/mg and 3412.9mL/s/mg, which are respectively increased by 3.4 times and 3.5 times compared with the wild type.
(4) Determination of transcriptional levels of glucoamylase GA2 and M3, M5 and M8 mutants
The GA2 and M3, M5 and M8 mutants are provided with three parallel biological repeats, each biological repeat is subjected to three technical repeats, the three types of ARG4, gap and re-act are selected as reference genes, and the ARG4 is finally determined to be the used 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 levels of mutants M3, M5, and M8 were 1.9 times, 2.4 times, and 5.8 times of mutant GA2, respectively, and the transcription levels of M5 and M8 were significantly increased.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> glucoamylase mutant M5 with improved secretion expression level and gene and application thereof
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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
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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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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
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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
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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
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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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Tyr Ala 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
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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
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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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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
130 135 140
Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro
145 150 155 160
Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala
165 170 175
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
195 200 205
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 Gln Gly Cys Gly Val Thr Thr Ala Thr Val
595 600 605
Asn Asp Ser Trp Arg
610
<210> 3
<211> 613
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Gln Tyr Leu Leu Lys Thr Thr Leu Gly Ala Leu Ser Val Ala Gln
1 5 10 15
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
65 70 75 80
Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ile Lys Cys Leu Ile
85 90 95
Asp Glu Phe Ile Ser Thr Gly Asp Ala Asn Leu Glu Ser Val Ile Gln
100 105 110
Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser
115 120 125
Gly Gly Leu Cys Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn
130 135 140
Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro
145 150 155 160
Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala
165 170 175
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
195 200 205
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> 613
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Gln Tyr Leu Leu Lys Thr Thr Leu Gly Ala Leu Ser Val Ala Gln
1 5 10 15
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
65 70 75 80
Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ile Lys Cys Leu Ile
85 90 95
Asp Glu Phe Ile Ser Thr Gly Asp Ala Asn Leu Glu Ser Val Ile Gln
100 105 110
Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser
115 120 125
Gly Gly Leu Cys Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn
130 135 140
Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro
145 150 155 160
Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala
165 170 175
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
195 200 205
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 Asp Asp Pro Asn
580 585 590
Arg Ser Tyr Thr Val Pro Gln Gly Cys Gly Val Thr Thr Ala Ile Leu
595 600 605
Asp Asp Ser Trp Gln
610
<210> 5
<211> 613
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Gln Tyr Leu Leu Lys Thr Thr Leu Gly Ala Leu Ser Val Ala Gln
1 5 10 15
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
65 70 75 80
Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ile Lys Cys Leu Ile
85 90 95
Asp Glu Phe Ile Ser Thr Gly Asp Ala Asn Leu Glu Ser Val Ile Gln
100 105 110
Asn Tyr Ile Ser Ser Gln Ala Phe Leu Gln Thr Val Ser Asn Pro Ser
115 120 125
Gly Gly Leu Cys Thr Gly Gly Leu Gly Glu Pro Lys Phe Glu Val Asn
130 135 140
Glu Ala Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro
145 150 155 160
Ala Leu Arg Ala Thr Ala Met Ile Asn Tyr Ala Asn Trp Leu Ile Ala
165 170 175
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
195 200 205
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 Asp Asp Pro Asn
580 585 590
Arg Ser Tyr Thr Val Pro Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu
595 600 605
Asp Asp Ser Trp Gln
610
<210> 6
<211> 1842
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
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> 7
<211> 1842
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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 gcctgcgtac 1800
tgtggccaga cgactgcgac ggtgaatgat agttggaggt ag 1842
<210> 8
<211> 1842
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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 ggaggatgat ccgaacaggt cctatacggt gcctcaaggg 1800
tgtggcgtga cgactgcgat tcttgacgat agttggcagt ag 1842
<210> 9
<211> 1842
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
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 ggaggacgac ccgaaccggt cgtacacggt cccagcgtac 1800
tgtggcgtga cgactgcgat tcttgacgat agttggcagt ag 1842
Claims (8)
1. The glucoamylase mutant M5 with improved secretion expression is characterized in that the amino acid sequence of the glucoamylase mutant is shown as SEQ ID NO. 4.
2. A method for increasing the secretory expression level of glucoamylase, which comprises the following steps:
the method comprises the steps of mutating the amino acid at the position 589 of a glucoamylase mutant GA2 from Ser to Asp, mutating the amino acid at the position 607 from Thr to Ile, mutating the amino acid at the position 608 from Val to Leu, mutating the amino acid at the position 609 from Asn to Asp, and mutating the amino acid at the position 613 from Arg to Gln, wherein the amino acid sequence of the glucoamylase mutant GA2 is shown as SEQ ID NO. 2.
3. A glucoamylase mutant gene encoding glucoamylase mutant M5 having increased secretion expression according to claim 1.
4. The glucoamylase mutant gene as set forth in claim 3 having the nucleotide sequence as set forth in SEQ ID NO 8.
5. A recombinant vector comprising the glucoamylase mutant gene of claim 3.
6. A recombinant strain comprising the glucoamylase mutant gene of claim 3.
7. 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 M5 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.
8. Use of the glucoamylase mutant M5 with increased secretion expression according to claim 1 for hydrolyzing alpha-1, 4 glucosidic bonds.
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