CN108611339A - Glucoamylase TlGa15 and its gene and application - Google Patents

Glucoamylase TlGa15 and its gene and application Download PDF

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CN108611339A
CN108611339A CN201810448537.2A CN201810448537A CN108611339A CN 108611339 A CN108611339 A CN 108611339A CN 201810448537 A CN201810448537 A CN 201810448537A CN 108611339 A CN108611339 A CN 108611339A
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glucoamylase
tlga15
thr
ala
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CN108611339B (en
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罗会颖
姚斌
郭玉杰
涂涛
王苑
黄火清
柏映国
苏小运
王亚茹
孟昆
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Institute of Animal Science of CAAS
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

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Abstract

The present invention relates to a kind of from the glucoamylase TlGa15 and its gene of fungi and application, and amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.The present invention provides a new glucose amylase gene, the glucoamylase of coding has good property, can make to be applied to the industry such as feed, food, medicine.Therefore, the present invention has produced the glucoamylase of the good properties of suitable commercial Application using genetic engineering means.

Description

Glucoamylase TlGa15 and its gene and application
Technical field
The present invention relates to agricultural biological technical fields, in particular it relates to which a kind of deriving from fungi glucoamylase Enzyme TlGa15 and its gene and application.
Background technology
Amylase is a kind of biocatalyst that purposes is extremely wide, can be applied to breadmaking industry, starch saccharification and Liquefaction, textile destarch, papermaking, detergent industry, chemistry, clinical medical analysis and pharmacy industry etc..Amylase family includes Alpha-amylase, beta amylase and glucoamylase.Alpha-amylase is restriction endonuclease, acts on α-Isosorbide-5-Nitrae sugar inside starch molecule Glycosidic bond generates dextrin and oligosaccharide.Beta amylase is excision enzyme, it sequentially cuts maltose from starch irreducibility.Glucose Amylase is a kind of excision enzyme acting on α-Isosorbide-5-Nitrae glycosidic bond, and glucose molecule is cut from non-reducing sugar end.Glucoamylase Zymolyte specificity is relatively low, can either cut off α-Isosorbide-5-Nitrae-glycosidic bond, and to α -1,6- glycosidic bonds and α -1,3- glycosidic bond also have slightly Hydrolysis ability.Glucoamylase is one of maximum biological enzyme formulation of industrial dosage, is widely used in food, doctor Medicine and fermentation etc. are industrial, are one of China's yield and the maximum biological enzyme product of dosage, have very high commercial value.
Glucoamylase (EC.3.2.1.3) is also known as carbohydrase, is that the important of starch sugar industry uses enzyme.Glucose Industrial production is mainly in two steps：(1) liquefaction of starch, high-temperatureα-amylase effect under by the starch of gelatinization resolve into oligosaccharides and Maltose reduces viscosity；(2) it is saccharified, 60-65 DEG C is cooled the temperature to after liquefaction, pH is adjusted to 4.0-4.5, is added suitable Glucoamylase, which is kept the temperature to concentration of glucose, reaches highest.Oligosaccharides and malt are mainly decomposed in the effect of glucoamylase Sugar generates glucose.
In sugaring reaction, higher reaction temperature is conducive to the progress entirely reacted.Higher reaction temperature is more advantageous to Saccharifying carries out, and is mainly reflected in the following aspects：(1) high temperature dissolves conducive to substrate, increases concentration of substrate；(2) it reduces Substrate viscosity is conducive to stirring；(3) risk of microbiological contamination is reduced；(4) increase reaction rate, reduce reaction time (5) reduction enzyme purification Cost；(6) extend the catalytic half-life of enzyme.
Currently, the carbohydrase industrially applied is mainly derived from aspergillus niger (Aspergillus niger), aspergillus awamori (Asperillus awamori) and Rhizopus oryzae (Rhizopus oryzae).The temperature of starch liquefacation is usually under the conditions of 95 DEG C It carries out, what the first step carried out under the high temperature conditions, entire temperature of reaction system is higher, therefore produces and carbohydrase is required also to have Preferable temperature stability, and common carbohydrase is typically only capable to stablize at 60 DEG C.Therefore, the selection and breeding of high temperature resistant carbohydrase are This field urgent problem to be solved.
Invention content
According to the bad problem of starch enzyme heat stability in the prior art, the object of the present invention is to provide a kind of acid, heat Stability improves, is suitable for the glucoamylase TlGa15 applied in the industries such as feed, food, medicine.
Another object of the present invention is to provide the gene of above-mentioned glucoamylase TlGa15.
Another object of the present invention is to provide the recombinant vector for including above-mentioned glucoamylase TlGa15.
Another object of the present invention is to provide the recombinant bacterial strain for including above-mentioned glucoamylase TlGa15 genes.
Another object of the present invention is to provide a kind of method preparing glucoamylase TlGa15.
Another object of the present invention is to provide the application of above-mentioned glucoamylase TlGa15.
Specific implementation mode according to the present invention, the amino acid sequence such as SEQ ID NO.1 of glucoamylase TlGa15 It is shown：
636 amino acid of the enzyme overall length, 20 amino acid of N-terminal are signal peptide sequence " MTARLASALC ALAFGQAVVA ".
Specific implementation mode according to the present invention, ripe glucoamylase TlGa15 amino acid sequences such as SEQ ID Shown in NO.2：
Specific implementation mode according to the present invention encodes the gene order such as SEQ ID NO.3 of above-mentioned glucoamylase It is shown：
Specific implementation mode according to the present invention, glucose amylase gene TlGa15 structural gene overall length 2186bp, contains There are 4 intrones, cDNA long 1911bp, cDNA sequence is as shown in SEQ ID NO.4：
Maturation protein theoretical molecular weight is 65.8kDa, which belongs to the 15th family of glycosyl hydrolase, a kind of new glucose Amylase.
The cDNA sequence after signal peptide is removed as shown in SEQ ID NO.5
The present invention also provides the recombinant vector for including above-mentioned glucoamylase TlGa15 genes, preferably pPIC9- TlGa15.The glucose amylase gene of the present invention is inserted between suitable restriction enzyme cleavage sites of the expression vector, it is made Nucleotide sequence is operable to be linked to the expression control sequence.As the most preferred embodiment of the present invention, preferably To be inserted into glucose amylase gene between EcoR I and Not the I restriction enzyme sites on plasmid pPIC9, make the core Nucleotide sequence is located at the downstream of AOXl promoters and is regulated and controled by it, obtains expression of recombinant yeast plasmid pPIC9-TlGa15.
The present invention also provides the recombinant bacterial strains for including above-mentioned glucoamylase TlGa15 genes, preferably recombinant bacterial strain GS115/TlGa15。
Specific implementation mode according to the present invention, the method for preparing glucoamylase TlGa15 include the following steps：
(1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
(2) recombinant bacterial strain, the expression of induction recombination glucoamylase TlGa15 are cultivated；
(3) it and recycles and purifies expressed glucoamylase TlGa15.
Specific implementation mode according to the present invention, the host cell be Pichia pastoris (Pichia pastoris) cell, Brewer's yeast (Saccharomyces cerevisiae) cell or Hansenula polymorpha (Hansenula polymorpha) are thin Born of the same parents.
Specific implementation mode according to the present invention, by expression of recombinant yeast plasmid conversion Pichia pastoris (Pichic Pastoris) GS115 obtains recombinant bacterial strain GS115/TlGa15.
The glucoamylase TlGa15 optimal pHs of the present invention are 4.5, in 5.5 ranges of pH 4.0-pH, can be tieed up Hold its 70% or more enzyme activity；75 DEG C of optimum temperature, at 70 DEG C still with 80% or more enzyme activity, at 65 DEG C 60min is managed, remaining enzyme activity handles 10min 80% or more at 70 DEG C, can still keep 70% enzyme activity, has good Good stability.
The glucoamylase TlGa15 of the present invention can be using genetic engineering means come industrialization production.The Portugal of the present invention Grape saccharogenic amylase TlGa15 genes can be applied to feed, food, medicine and other fields.It may be implemented according to the technique and scheme of the present invention Utilize the glucoamylase TlGa15 of the excellent suitable commercial Application of genetic engineering means nature of production.
Description of the drawings
The optimum pH of the recombination glucoamylase TlGa15 of Fig. 1 display present invention.
The pH stability of the glucoamylase TlGa15 of Fig. 2 display present invention.
The optimal reactive temperature of the glucoamylase TlGa15 of Fig. 3 display present invention.
The thermal stability of the glucoamylase TlGa15 of Fig. 4 display present invention.
Specific implementation mode
Test material and reagent
1, bacterial strain and carrier：Pichia pastoris (Pichia pastoris GS115), yeast expression vector pPIC9 and Bacterial strain GS115 is purchased from biochemical reagents company.
2, enzyme and other biochemical reagents：Restriction endonuclease, ligase and other biochemical reagents are purchased from Reagent Company.
3, culture medium：
(1) culture medium：30g/L wheat bran, 30g/L maize cob meals, 30g/L dregs of beans, 5g/L barleys, 5g/L (NH4)SO4, 1g/L KH2PO4, 0.5g/L MgSO4·7H2O, 0.01g/L FeSO4·7H2O, 0.2g/L CaCl2In 1L go from In sub- water, sterilization treatment 20min under the conditions of 121 DEG C, 15 pounds
(2) Escherichia coli culture medium LB (126 peptones, 0.5% yeast extract, 126NaCl, pH7.0).
(3) BMGY culture mediums；1% yeast extract, 2% peptone, 1.34%YNB, 0.000049<Biotin, 1% is sweet Oily (v/v).
(4) BMMY culture mediums：Divided by 0.5% methanol replace glycerine, remaining composition is identical as BMGY, pH4.0.
The clone of 1 glucoamylase enzyme coding gene TlGa15 of embodiment
It extracts Talaromyces leycettanus JCM12802 genomic DNAs and designs cloning primer, with Talaromyces leycettanus JCM12802 total DNAs are that template carries out PCR amplification.PCR response parameters are：95℃ 5min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2min, 35 cycles, 72 DEG C of 10min obtain about 2000bp segments.
The acquisition of 2 glucoamylase cDNA of embodiment
The total serum IgE for extracting Talaromyces leycettanus JCM12802, utilizes Oligo (dT)20And reverse transcriptase Obtain a chain of cDNA, then design amplification open reading frame primer 12802GH15-3F and 12802GH15-3R (see Table 1), the single-stranded cDNA is expanded, the cDNA sequence of glucoamylase is obtained.
Primer needed for 1 gene cloning this experiment of table
It finds that the gene has after comparing by genome sequence to glucoamylase and cDNA sequence to include containing 4 Son, cDNA long 1911bp encode 636 amino acid and a terminator codon, and 20 amino acid of N-terminal are its signal peptide sequence, The gene for the coding glucoamylase that separation clone obtains from Talaromyces leycettanus JCM12802 is new Gene.
The structure of 3 glucoamylase engineered strain of embodiment
(1) structure of expression vector and the expression in yeast
Using the cDNA that correct glucoamylase enzyme coding gene TlGa15 is sequenced as template, designs and synthesized with EcoR The primer 12802GH15-3-F and 12802GH15-3-R (being shown in Table 1) of I and Not I restriction enzyme sites, to TlGa15 at The white code area of soft-boiled eggs is expanded, and utilizes EcoR I and Not I digestion PCR products, and connection enters expression vector pPIC9 The sequence of (Invitrogen, San Diego), glucoamylase TlGa15 maturation proteins are inserted into the letter of above-mentioned expression vector The downstream of number peptide sequence, forms correct reading frame with signal peptide, is built into Yeast expression carrier pPIC9-TlGa15, converts Competent escherichia coli cell Trans1.Positive transformant carries out DNA sequencing, and sequencing shows that the correct transformant of sequence is used for greatly Measure Prepare restructuring plasmid.Linearisation expression plasmid carrier DNA, electroporated yeast GS115 are carried out with restriction enzyme Dra I Competent cell, 30 DEG C are cultivated 2-3 days, and the transformant that picking is grown on MD tablets carries out further expression experiment.
The expression vector of the cDNA of the signal peptide sequence containing TlGa15 is built in the same way, and is converted.
(2) screening of glucoamylase high activity transformant
With sterilized toothpick from picking single bacterium colony on the MD plates with transformant, first put on MD tablets according to number, MD tablets are placed in 30 DEG C of incubators and are cultivated 1~2 day, until bacterium colony is grown.It is inoculated with by number from picking transformant on MD tablets In the centrifuge tube equipped with 3mL BMGY culture mediums, 30 DEG C, 220rpm shaking table cultures 48h；By the bacterium solution 3 of shaking table culture 48h, 000 × g centrifuge 15min, remove supernatant, the BMMY culture mediums that 1mL contains 0.5% methanol added in centrifuge tube, 30 DEG C, 220rpm Fiber differentiations；After Fiber differentiation 48h, 3,000 × g centrifuges 5min, takes supernatant for Enzyme assay, therefrom filters out The highest transformant of glucose-amylase activity.
Embodiment 4 recombinates the preparation of glucoamylase
(1) great expression of glucose amylase gene TlGa15 shaking flask levels in Pichia pastoris
The higher transformant of enzyme activity is filtered out, is inoculated in the 1L triangular flasks of 400mL BMGY fluid nutrient mediums, 30 DEG C, 220rpm shaking table shaken cultivations 48h；4500rpm centrifuges 5min, softly abandons supernatant, then 200mL is added to thalline and contains 0.5% first The BMMY fluid nutrient mediums of alcohol, 30 DEG C, 220rpm Fiber differentiations 48h.During Fiber differentiation, it is molten that a methanol is added at interval for 24 hours Liquid makes methanol concentration be maintained at 0.5% or so to compensate the loss of methanol；(3) 12,000 × g centrifuge 10min, collect supernatant hair Zymotic fluid detects enzymatic activity and carries out SDS-PAGE protein electrophoresis analyses.
(2) purifying of glucoamylase is recombinated
The recombination glucoamylase supernatant for collecting shaking flask expression, is concentrated, while using less salt by 10kDa film packets Buffer exchange culture medium therein, is then further concentrated with 10kDa super filter tubes.Concentration can be diluted to the weight of certain multiple Group TlGa15, is purified by ion-exchange chromatography.Specifically, take TlGa15 concentrates 2.0mL through using 20mM in advance HiTrap Q Sepharose XL anion columns equilibrated Tris-HCl (pH 7.5), then use 0-1mol/L NaCl into Row linear gradient elution, to the measurement of the eluent detection enzymatic activity and progress albumen concentration of Fraction collection.
Embodiment 5 recombinates the analysis of glucoamylase some properties
Activity analysis is carried out to the glucoamylase of the present invention using DNS methods.The specific method is as follows：In pH 4.5,75 Under the conditions of DEG C, the reaction system of 1mL includes l00 μ L dilution enzyme solutions appropriate, and 900 μ L substrates react 10min, and 1.5mL is added DNS terminates reaction, boiling water boiling 5min.540nm measures OD values after cooling.Glucose-amylase activity unit definition：In 75 DEG C of pH Under the conditions of 4.5, it is an enzyme-activity unit that interior catalyzing hydrolysis substrate per minute, which releases the enzyme amount needed for 1 μm of ol reduced sugar,.
(1) optimal pH of glucoamylase TlGa15 and pH stability
The glucoamylase TlGa15 that purified embodiment 3 is expressed carries out enzymatic reaction to measure at different pH Its optimal pH.Buffer solution used is pH 1.0-3.0 glycine-HCI buffer solutions, the one phosphoric acid hydrogen two of citric acid of pH 3.0-8.0 Sodium buffer solution and pH 8.0-10.0 are Tris-HCl buffer solutions.
The glucoamylase TlGa15 of purifying different pH buffer system as shown in Figure 1, the pH that is measured at 75 DEG C is suitable Property the result shows that：The optimal pH of TlGa15 is 4.5, in 5.5 ranges of pH 4.0-pH, the enzyme be able to maintain that its 70% or more Enzyme activity.
Enzyme solution is handled into 60min in the buffer solution of different pH value at 37 DEG C, then measures the residual activity of enzyme to study The pH stability of enzyme.As shown in Fig. 2, analysis result shows to be able to maintain that 85% or more enzyme activity between pH 3.0-pH 7.0, Illustrate that the enzyme has excellent pH stability.
(2) glucoamylase TlGa15 reacts optimum temperature and thermal stability
As shown in figure 3, the glucoamylase of purifying under the conditions of 4.5 pH, measures under different temperatures (40-90 DEG C) Enzymatic activity, the experimental results showed that display, the optimal reactive temperature of the enzyme is 75 DEG C, still has 80% or more at 65-75 DEG C Enzyme activity.
As shown in figure 4, temperature tolerance is measured as glucoamylase handles different time at different temperatures, then 75 Enzyme assay is carried out at DEG C.Thermal stability experiment shows：The glucoamylase handles 60min, remaining enzyme activity at 65 DEG C 80% or more, even if the enzyme handles 10min at 70 DEG C, 70% enzyme activity can be still kept, this shows that the enzyme has Preferable stability.
Sequence table
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>Glucoamylase TlGa15 and its gene and application
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<213>Blue shape bacterium JCM12802 (Talaromyces leycettanus JCM12802)
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Met Thr Ala Arg Leu Ala Ser Ala Leu Cys Ala Leu Ala Phe Gly Gln
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Ala Val Val Ala Ala Pro Gln Pro Val Leu Pro Arg Ala Thr Thr Ser
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Leu Asp Ser Trp Leu Ala Ser Glu Thr Ser Val Ala Leu Thr Gly Ile
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Leu Asn Asn Ile Gly Ser Ser Gly Ala Tyr Ala Gln Thr Ala Ser Pro
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Gly Ile Val Ile Ala Ser Pro Ser Thr Ser Asn Pro Asp Tyr Tyr Tyr
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Thr Trp Thr Arg Asp Ala Ala Leu Thr Met Lys Val Leu Ile Asp Leu
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Phe Lys Asn Gly Asn Ser Ser Leu Leu Thr Val Ile Glu Gln Tyr Ile
100 105 110
Asp Ala Gln Ala Tyr Leu Gln Thr Val Ser Asn Pro Ser Gly Ser Leu
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Ser Ser Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala
130 135 140
Phe Thr Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg
145 150 155 160
Ala Thr Ala Leu Ile Ala Phe Gly Gln Trp Leu Ile Asp Asn Gly Tyr
165 170 175
Ser Thr Tyr Ala Ser Ser Ile Val Trp Pro Ile Val Arg Asn Asp Leu
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Ser Tyr Leu Ala Gln Tyr Trp Asn Gln Thr Gly Tyr Glu Leu Trp Glu
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Glu Val Ser Gly Ser Ser Phe Phe Thr Leu Ala Val Gln His Arg Ala
210 215 220
Leu Val Glu Gly Ser Ser Phe Ala Ser Gln Leu Gly Thr Ser Cys Pro
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Tyr Cys Asp Ser Gln Ala Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe
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Trp Thr Gly Glu Tyr Ile Leu Ala Asn Phe Gly Ser Ser Arg Ser Gly
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Lys Asp Ala Asn Thr Leu Leu Gly Ser Ile His Thr Phe Asp Pro Ala
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Ala Gly Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser Ser Arg Ala Leu
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Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser Ile Tyr Thr Ile
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Asn Ser Gly Ile Ser Glu Gly Thr Ala Val Ala Val Gly Arg Tyr Pro
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Glu Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe Ile Cys Thr Leu Ala
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Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Asn Arg Ile Gly
355 360 365
Ser Ile Thr Ile Thr Ser Ile Ser Leu Ala Phe Phe Gln Asp Leu Tyr
370 375 380
Pro Ser Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Thr Val Phe Ser
385 390 395 400
Ser Ile Val Ser Ala Ala Lys Thr Tyr Ala Asp Gly Tyr Met Ser Ile
405 410 415
Val Glu Thr His Ala Met Thr Asn Gly Ser Leu Ser Glu Gln Phe Ser
420 425 430
Lys Ser Asp Gly Thr Glu Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr
435 440 445
Ala Ala Leu Leu Thr Ala His Leu Arg Arg Asn Ser Ile Val Pro Ala
450 455 460
Ser Trp Gly Glu Thr Thr Ala Asn Ser Val Pro Ser Val Cys Ser Ala
465 470 475 480
Thr Ser Ala Thr Gly Thr Tyr Ser Thr Ala Thr Asn Thr Ala Trp Pro
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Ala Thr Leu Thr Ser Gly Ser Gly Ser Ala Thr Thr Thr Thr Ser Ser
500 505 510
Ile Thr Ser Gly Thr Ser Thr Pro Ser Thr Thr Thr Ser Ser Thr Ala
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Cys Ser Thr Pro Thr Ser Val Ala Val Thr Phe Asp Leu Ile Ala Thr
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Thr Tyr Tyr Gly Glu Asn Ile Lys Ile Ala Gly Ser Ile Ser Glu Leu
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Gly Asn Trp Asp Thr Asn Asn Ala Val Pro Leu Ser Ala Ser Gln Tyr
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Thr Ser Ser Asn Pro Leu Trp Tyr Val Thr Ile Asp Leu Pro Ala Gly
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Glu Ser Phe Glu Tyr Lys Tyr Ile Arg Ile Glu Ser Asp Gly Ser Ile
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Val Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Val Cys
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Gly Glu Thr Ala Val Thr Glu Asn Asp Thr Trp Arg
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<211> 616
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<213>Blue shape bacterium JCM12802 (Talaromyces leycettanus JCM12802)
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Ala Pro Gln Pro Val Leu Pro Arg Ala Thr Thr Ser Leu Asp Ser Trp
1 5 10 15
Leu Ala Ser Glu Thr Ser Val Ala Leu Thr Gly Ile Leu Asn Asn Ile
20 25 30
Gly Ser Ser Gly Ala Tyr Ala Gln Thr Ala Ser Pro Gly Ile Val Ile
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Asp Ala Ala Leu Thr Met Lys Val Leu Ile Asp Leu Phe Lys Asn Gly
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Asn Ser Ser Leu Leu Thr Val Ile Glu Gln Tyr Ile Asp Ala Gln Ala
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Tyr Leu Gln Thr Val Ser Asn Pro Ser Gly Ser Leu Ser Ser Gly Gly
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Leu Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Phe Thr Gly Ala
115 120 125
Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Leu
130 135 140
Ile Ala Phe Gly Gln Trp Leu Ile Asp Asn Gly Tyr Ser Thr Tyr Ala
145 150 155 160
Ser Ser Ile Val Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr Leu Ala
165 170 175
Gln Tyr Trp Asn Gln Thr Gly Tyr Glu Leu Trp Glu Glu Val Ser Gly
180 185 190
Ser Ser Phe Phe Thr Leu Ala Val Gln His Arg Ala Leu Val Glu Gly
195 200 205
Ser Ser Phe Ala Ser Gln Leu Gly Thr Ser Cys Pro Tyr Cys Asp Ser
210 215 220
Gln Ala Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe Trp Thr Gly Glu
225 230 235 240
Tyr Ile Leu Ala Asn Phe Gly Ser Ser Arg Ser Gly Lys Asp Ala Asn
245 250 255
Thr Leu Leu Gly Ser Ile His Thr Phe Asp Pro Ala Ala Gly Cys Asp
260 265 270
Asp Thr Thr Phe Gln Pro Cys Ser Ser Arg Ala Leu Ala Asn His Lys
275 280 285
Val Val Thr Asp Ser Phe Arg Ser Ile Tyr Thr Ile Asn Ser Gly Ile
290 295 300
Ser Glu Gly Thr Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Ser Tyr
305 310 315 320
Tyr Asn Gly Asn Pro Trp Phe Ile Cys Thr Leu Ala Ala Ala Glu Gln
325 330 335
Leu Tyr Asp Ala Ile Tyr Gln Trp Asn Arg Ile Gly Ser Ile Thr Ile
340 345 350
Thr Ser Ile Ser Leu Ala Phe Phe Gln Asp Leu Tyr Pro Ser Ala Ala
355 360 365
Thr Gly Thr Tyr Ser Ser Ser Ser Thr Val Phe Ser Ser Ile Val Ser
370 375 380
Ala Ala Lys Thr Tyr Ala Asp Gly Tyr Met Ser Ile Val Glu Thr His
385 390 395 400
Ala Met Thr Asn Gly Ser Leu Ser Glu Gln Phe Ser Lys Ser Asp Gly
405 410 415
Thr Glu Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu
420 425 430
Thr Ala His Leu Arg Arg Asn Ser Ile Val Pro Ala Ser Trp Gly Glu
435 440 445
Thr Thr Ala Asn Ser Val Pro Ser Val Cys Ser Ala Thr Ser Ala Thr
450 455 460
Gly Thr Tyr Ser Thr Ala Thr Asn Thr Ala Trp Pro Ala Thr Leu Thr
465 470 475 480
Ser Gly Ser Gly Ser Ala Thr Thr Thr Thr Ser Ser Ile Thr Ser Gly
485 490 495
Thr Ser Thr Pro Ser Thr Thr Thr Ser Ser Thr Ala Cys Ser Thr Pro
500 505 510
Thr Ser Val Ala Val Thr Phe Asp Leu Ile Ala Thr Thr Tyr Tyr Gly
515 520 525
Glu Asn Ile Lys Ile Ala Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp
530 535 540
Thr Asn Asn Ala Val Pro Leu Ser Ala Ser Gln Tyr Thr Ser Ser Asn
545 550 555 560
Pro Leu Trp Tyr Val Thr Ile Asp Leu Pro Ala Gly Glu Ser Phe Glu
565 570 575
Tyr Lys Tyr Ile Arg Ile Glu Ser Asp Gly Ser Ile Val Trp Glu Ser
580 585 590
Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Val Cys Gly Glu Thr Ala
595 600 605
Val Thr Glu Asn Asp Thr Trp Arg
610 615
<210> 3
<211> 2186
<212> DNA
<213>Blue shape bacterium JCM12802 (Talaromyces leycettanus JCM12802)
<400> 3
atgactgcac gtcttgcctc cgcgctgtgt gcgctcgctt ttggccaggc agtcgtcgct 60
gcaccacagc ctgtgcttcc cagagcaaca accagcttgg actcgtggct cgcttcggaa 120
acttctgtcg cgcttactgg catcttgaac aatattggct cgagcggtgc ctatgctcag 180
actgcgagcc cgggcattgt cattgctagt cctagtacta gcaatccaga ctgtacgtga 240
catcctttat cagtttgttg acgacatcga ggatttgaat gagaaagaaa gcagagttgg 300
ttaatgttgt acgaacatag actactatac ctggacccgt gatgctgccc tgaccatgaa 360
ggtcctcatc gacctcttca agaacggcaa ctccagtctg ctcaccgtga tcgagcagta 420
catcgacgcg caggcgtatc tccagacagt ctcgaacccc tctggctctc tttccagcgg 480
tggcctgggc gaacccaagt tcaatgtcga cgagaccgcc ttcactggcg cctggggacg 540
gcctcagcgt gacggtcctg cgctgcgagc tactgctctg attgcttttg gccagtggtt 600
gattgtacgt cgctaggaga taccgcaccg aactgaccgt ggactactaa cacactcgta 660
ggataatggc tactcaacct atgcttcgag cattgtctgg cccatcgtgc ggaacgatct 720
ctcgtacctc gcccagtact ggaatcagac cggttatggt gagttgacaa cgatctggat 780
tcatcctggt ccccagagcc tatgttaatg gtcgtcaacc acagaactct gggaggaggt 840
tagtggctcc tccttcttca cccttgctgt ccaacatcgt gccctcgttg aaggcagctc 900
ctttgccagc cagctcggca cctcctgtcc ctactgcgat tcgcaagcgc ctcagatcct 960
ctgcttcctg caaagctttt ggaccggcga atatatcctt gccaattttg gcagcagccg 1020
ctccggcaag gatgccaaca ccctcctcgg gagcatccac acgtttgacc cggcagcggg 1080
atgcgatgac acgaccttcc agccttgctc ttctcgtgct ctcgccaatc ataaggtcgt 1140
cacggactct ttccggtcga tctacacgat caattcgggg atcagtgaag gcacggctgt 1200
ggctgtggga cgctatcctg aggattctta ctataacggc aacccttggt tcatctgcac 1260
gctggctgcc gctgaacagc tgtatgatgc gatctatcag tggaatcgca ttggatcgat 1320
cacgatcacg agtatctcgt tggcgttctt ccaagatctg tacccttccg cagcgactgg 1380
gacgtattcg tcttccagta cggtgttctc gtccattgtc agtgccgcca agacctatgc 1440
agatggatat atgagcattg tggtatgctc ctttttttct cctcttggaa ttgtaatccc 1500
tggatggtta tctgacacgt ctaaaggaaa cccatgctat gaccaacggc agcctgtctg 1560
agcaattctc caagtcagat ggcactgagc tctctgctcg cgatctcacc tggtcgtacg 1620
ccgccttgct gacagcccat ctgcgtcgca actctatcgt ccctgcttca tggggtgaga 1680
ctaccgccaa cagcgttcca tctgtctgct ctgcaacctc tgctaccggc acatacagca 1740
ctgcaaccaa taccgcctgg cccgctactc tgaccagcgg gtccggctct gcaaccacca 1800
ccacttcttc gatcacgagc gggacttcaa ctcccagtac gacaacatca tcgaccgcat 1860
gcagcactcc cacgtctgtt gccgtgacgt ttgatttgat cgcgacgacg tactacggcg 1920
agaacatcaa gatcgccggc tcgatatccg agctcggaaa ctgggacaca aataacgctg 1980
ttcctttgag cgcgagccag tacactagca gtaaccctct gtggtatgtg acgatcgact 2040
tgcctgcggg tgaatcgttt gagtacaagt atatccggat cgagagcgat ggtagcatcg 2100
tgtgggaaag cgatcctaat cgatcctata cggtgcccgc ggtctgtggt gagacggcgg 2160
tgacggagaa cgatacctgg agatag 2186
<210> 4
<211> 1911
<212> DNA
<213>Blue shape bacterium JCM12802 (Talaromyces leycettanus JCM12802)
<400> 4
atgactgcac gtcttgcctc cgcgctgtgt gcgctcgctt ttggccaggc agtcgtcgct 60
gcaccacagc ctgtgcttcc cagagcaaca accagcttgg actcgtggct cgcttcggaa 120
acttctgtcg cgcttactgg catcttgaac aatattggct cgagcggtgc ctatgctcag 180
actgcgagcc cgggcattgt cattgctagt cctagtacta gcaatccaga ctactactat 240
acctggaccc gtgatgctgc cctgaccatg aaggtcctca tcgacctctt caagaacggc 300
aactccagtc tgctcaccgt gatcgagcag tacatcgacg cgcaggcgta tctccagaca 360
gtctcgaacc cctctggctc tctttccagc ggtggcctgg gcgaacccaa gttcaatgtc 420
gacgagaccg ccttcactgg cgcctgggga cggcctcagc gtgacggtcc tgcgctgcga 480
gctactgctc tgattgcttt tggccagtgg ttgattgata atggctactc aacctatgct 540
tcgagcattg tctggcccat cgtgcggaac gatctctcgt acctcgccca gtactggaat 600
cagaccggtt atgaactctg ggaggaggtt agtggctcct ccttcttcac ccttgctgtc 660
caacatcgtg ccctcgttga aggcagctcc tttgccagcc agctcggcac ctcctgtccc 720
tactgcgatt cgcaagcgcc tcagatcctc tgcttcctgc aaagcttttg gaccggcgaa 780
tatatccttg ccaattttgg cagcagccgc tccggcaagg atgccaacac cctcctcggg 840
agcatccaca cgtttgaccc ggcagcggga tgcgatgaca cgaccttcca gccttgctct 900
tctcgtgctc tcgccaatca taaggtcgtc acggactctt tccggtcgat ctacacgatc 960
aattcgggga tcagtgaagg cacggctgtg gctgtgggac gctatcctga ggattcttac 1020
tataacggca acccttggtt catctgcacg ctggctgccg ctgaacagct gtatgatgcg 1080
atctatcagt ggaatcgcat tggatcgatc acgatcacga gtatctcgtt ggcgttcttc 1140
caagatctgt acccttccgc agcgactggg acgtattcgt cttccagtac ggtgttctcg 1200
tccattgtca gtgccgccaa gacctatgca gatggatata tgagcattgt ggaaacccat 1260
gctatgacca acggcagcct gtctgagcaa ttctccaagt cagatggcac tgagctctct 1320
gctcgcgatc tcacctggtc gtacgccgcc ttgctgacag cccatctgcg tcgcaactct 1380
atcgtccctg cttcatgggg tgagactacc gccaacagcg ttccatctgt ctgctctgca 1440
acctctgcta ccggcacata cagcactgca accaataccg cctggcccgc tactctgacc 1500
agcgggtccg gctctgcaac caccaccact tcttcgatca cgagcgggac ttcaactccc 1560
agtacgacaa catcatcgac cgcatgcagc actcccacgt ctgttgccgt gacgtttgat 1620
ttgatcgcga cgacgtacta cggcgagaac atcaagatcg ccggctcgat atccgagctc 1680
ggaaactggg acacaaataa cgctgttcct ttgagcgcga gccagtacac tagcagtaac 1740
cctctgtggt atgtgacgat cgacttgcct gcgggtgaat cgtttgagta caagtatatc 1800
cggatcgaga gcgatggtag catcgtgtgg gaaagcgatc ctaatcgatc ctatacggtg 1860
cccgcggtct gtggtgagac ggcggtgacg gagaacgata cctggagata g 1911
<210> 5
<211> 1851
<212> DNA
<213>Blue shape bacterium JCM12802 (Talaromyces leycettanus JCM12802)
<400> 5
gcaccacagc ctgtgcttcc cagagcaaca accagcttgg actcgtggct cgcttcggaa 60
acttctgtcg cgcttactgg catcttgaac aatattggct cgagcggtgc ctatgctcag 120
actgcgagcc cgggcattgt cattgctagt cctagtacta gcaatccaga ctactactat 180
acctggaccc gtgatgctgc cctgaccatg aaggtcctca tcgacctctt caagaacggc 240
aactccagtc tgctcaccgt gatcgagcag tacatcgacg cgcaggcgta tctccagaca 300
gtctcgaacc cctctggctc tctttccagc ggtggcctgg gcgaacccaa gttcaatgtc 360
gacgagaccg ccttcactgg cgcctgggga cggcctcagc gtgacggtcc tgcgctgcga 420
gctactgctc tgattgcttt tggccagtgg ttgattgata atggctactc aacctatgct 480
tcgagcattg tctggcccat cgtgcggaac gatctctcgt acctcgccca gtactggaat 540
cagaccggtt atgaactctg ggaggaggtt agtggctcct ccttcttcac ccttgctgtc 600
caacatcgtg ccctcgttga aggcagctcc tttgccagcc agctcggcac ctcctgtccc 660
tactgcgatt cgcaagcgcc tcagatcctc tgcttcctgc aaagcttttg gaccggcgaa 720
tatatccttg ccaattttgg cagcagccgc tccggcaagg atgccaacac cctcctcggg 780
agcatccaca cgtttgaccc ggcagcggga tgcgatgaca cgaccttcca gccttgctct 840
tctcgtgctc tcgccaatca taaggtcgtc acggactctt tccggtcgat ctacacgatc 900
aattcgggga tcagtgaagg cacggctgtg gctgtgggac gctatcctga ggattcttac 960
tataacggca acccttggtt catctgcacg ctggctgccg ctgaacagct gtatgatgcg 1020
atctatcagt ggaatcgcat tggatcgatc acgatcacga gtatctcgtt ggcgttcttc 1080
caagatctgt acccttccgc agcgactggg acgtattcgt cttccagtac ggtgttctcg 1140
tccattgtca gtgccgccaa gacctatgca gatggatata tgagcattgt ggaaacccat 1200
gctatgacca acggcagcct gtctgagcaa ttctccaagt cagatggcac tgagctctct 1260
gctcgcgatc tcacctggtc gtacgccgcc ttgctgacag cccatctgcg tcgcaactct 1320
atcgtccctg cttcatgggg tgagactacc gccaacagcg ttccatctgt ctgctctgca 1380
acctctgcta ccggcacata cagcactgca accaataccg cctggcccgc tactctgacc 1440
agcgggtccg gctctgcaac caccaccact tcttcgatca cgagcgggac ttcaactccc 1500
agtacgacaa catcatcgac cgcatgcagc actcccacgt ctgttgccgt gacgtttgat 1560
ttgatcgcga cgacgtacta cggcgagaac atcaagatcg ccggctcgat atccgagctc 1620
ggaaactggg acacaaataa cgctgttcct ttgagcgcga gccagtacac tagcagtaac 1680
cctctgtggt atgtgacgat cgacttgcct gcgggtgaat cgtttgagta caagtatatc 1740
cggatcgaga gcgatggtag catcgtgtgg gaaagcgatc ctaatcgatc ctatacggtg 1800
cccgcggtct gtggtgagac ggcggtgacg gagaacgata cctggagata g 1851

Claims (10)

1. a kind of glucoamylase TlGa15, which is characterized in that its amino acid sequence such as SEQ ID NO.1 or SEQ ID Shown in NO.2.
2. a kind of glucoamylase TlGa15 genes, which is characterized in that encode glucoamylase described in claim 1 TlGa15。
3. glucoamylase TlGa15 genes according to claim 2, which is characterized in that the glucoamylase The nucleotide sequence of TlGa15 genes is as shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
4. including the recombinant expression carrier of glucoamylase TlGa15 genes described in claim 2.
5. including the recombinant expression carrier pPIC9-TlGa15 of glucoamylase TlGa15 genes described in claim 2.
6. including the recombinant bacterial strain of glucoamylase TlGa15 genes described in claim 2.
7. including the recombinant bacterial strain GS115/TlGa15 of glucoamylase TlGa15 genes described in claim 2.
8. a kind of method preparing glucoamylase TlGa15 described in claim 1, which is characterized in that including following step Suddenly：
(1) host cell is converted with the recombinant expression carrier comprising coding glucoamylase TlGa15 genes；
(2) host cell, the TlGa15 expression of induced glucose amylase are cultivated；
(3) it isolates and purifies and obtains glucoamylase TlGa15.
9. the purposes of glucoamylase TlGa15 described in claim 1.
10. applications of the glucoamylase TlGa15 described in claim 1 in feed, field of food and medicine.
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