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Glucoamylase mutant m5 with increased secreted expression and its gene and application

A technique for secreting and expressing glucoamylase, which is applied in the field of genetic engineering to achieve the effect of increasing the level of soluble expression, meeting the needs of industrial applications, and improving catalytic efficiency

Active Publication Date: 2022-02-25
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes the carbohydrate domain play different roles in promoting enzyme-substrate binding, substrate-specific recognition, etc., and its own characteristics have also been confirmed to have a corresponding impact on the entire protein, such as stability and heat resistance and activity , but it has not been reported that the starch domain is closely related to the secretion of glucoamylase

Method used

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  • Glucoamylase mutant m5 with increased secreted expression and its gene and application
  • Glucoamylase mutant m5 with increased secreted expression and its gene and application
  • Glucoamylase mutant m5 with increased secreted expression and its gene and application

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Experimental program
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Effect test

Embodiment 1

[0046] The site-directed mutation of embodiment 1 glucoamylase

[0047] With the plasmid pPIC9- containing the gene encoding the glucoamylase mutant GA2 Tl The Ga1931-GA2 sequence was used as a template, and the codon corresponding to the 599th amino acid of the amino acid sequence of the glucoamylase mutant GA2 was mutated from CAA to GCG, the codon corresponding to the 600th amino acid was mutated from GGG to TAC, and the 603rd amino acid was mutated from GGG to TAC. The codon of amino acid position was mutated from GTG to CAG to obtain mutant M3. The codon corresponding to the 589th amino acid of the amino acid sequence of the glucoamylase mutant GA2 is mutated from AGT to GAC, the codon corresponding to the 607th amino acid is mutated from ACG to ATT, and the codon corresponding to the 608th amino acid The codon corresponding to the 609th amino acid was mutated from AAT to GAC from GTG to CTT, and the codon corresponding to the 613th amino acid was mutated from AGG to CAG...

Embodiment 2

[0051] Embodiment 2 Construction of glucoamylase engineering strain

[0052] (1) Construction of expression vector and expression in yeast

[0053] Recombination of plasmid pPIC9- with glucoamylase Tl Ga1931-GA2 was used as a template, and mutants were amplified using site-directed mutagenesis reagents. After verification by nucleic acid gel, add 1 μL of DMT enzyme to the PCR product, mix well and incubate at 37°C for 1 hour. Take 2-5 μL of the PCR product digested with DMT enzyme and transform it into DMT competent cells by heat shock. Positive transformants were picked for DNA sequencing, and the transformants with the correct sequence were used to prepare a large number of recombinant plasmids. restriction endonuclease Bgl II Linearize the expression plasmid vector DNA, transform yeast GS115 competent cells by electric shock, culture at 30°C for 2-3 days, pick the transformants grown on the MD plate for further expression experiments, please refer to Pichia pastoris e...

Embodiment 3

[0054] Embodiment 3 Preparation of recombinant glucoamylase

[0055] (1) Massive expression of glucoamylase at shake flask level in Pichia pastoris

[0056] The transformant with higher enzyme activity was screened out, inoculated in a 1 L Erlenmeyer flask with 300 mL of BMGY liquid medium, 30°C, 220 rpm shaking culture for 48 h; centrifuged at 4500 rpm for 5 min, discarded the supernatant, and poured The cells were added to 200 mL of BMMY liquid medium containing 0.5% methanol, and cultured at 30°C, 220 rpm for 48 h. During the induction culture period, methanol solution was added once every 24 h to compensate for the loss of methanol, so that the methanol concentration was kept at about 0.5%; centrifuged at 12,000×g for 10 min, the supernatant fermentation liquid was collected, and the enzyme activity was detected for SDS-PAGE protein electrophoresis analyze.

[0057] (2) Purification of recombinant glucoamylase

[0058] Collect the recombinant glucoamylase supernatant fr...

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Abstract

The invention relates to the field of genetic engineering, in particular to the glucoamylase mutant M5 with increased secreted expression and its gene and application. Glucoamylase mutant GA2 was subjected to site-directed mutation to obtain a mutant with increased secretion expression. The glucoamylase mutant of the present invention has good enzymatic properties and can be applied to feed, food, medicine, textile and other industries.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the glucoamylase mutant M5 with increased secreted expression and its gene and application. Background technique [0002] Glucoamylase is a kind of glycoside hydrolase (glycoside hydrolase), which acts on the exonuclease of α-1,4 glycosidic bond, and its systematic name is α-1,4-glucan glucoside hydrolase (α-1 , 4-glucanglucohydrolase, EC.3.2.1.3) or γ-amylase (γ-amylase), referred to as glucoamylase. Glucoamylase cleaves glucose molecules from non-reducing sugar ends, and has low substrate specificity, that is, it can cut α-1,4-glucosidic bonds, and it is suitable for α-1,6-glycosidic bonds and α-1,3-glucosidic bonds. Glycosidic linkages are also slightly hydrolyzable. Glucoamylase consists of a catalytic domain, a linker domain and a starch domain. According to the positional relationship of these three structural domains, fungal glucoamylase belongs to the 15th family of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/34C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2428C12N15/815C12Y302/01003
Inventor 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张杰张红莲于会民姚斌
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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