Glucoamylase mutants GA1, GA2 and GA4 with improved specific activity and thermal stability, and genes and applications of glucoamylase mutants

A glucoamylase and thermal stability technology, applied in the field of genetic engineering, can solve the problems of consumption of starch substrate, low starch utilization rate and ethanol yield, etc., and achieves improved specific activity and thermal stability, good thermal stability, The effect of high catalytic efficiency

Pending Publication Date: 2020-06-26
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are also fermentative production methods that use mixed culture saccharification and fermentation strains to produce ethanol at home and abroad, but the disadvantage of this process is that the growth of the strain itself needs to consume most of the starch substrate, and the actual starch utilization rate and ethanol yield are still quite low

Method used

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  • Glucoamylase mutants GA1, GA2 and GA4 with improved specific activity and thermal stability, and genes and applications of glucoamylase mutants
  • Glucoamylase mutants GA1, GA2 and GA4 with improved specific activity and thermal stability, and genes and applications of glucoamylase mutants
  • Glucoamylase mutants GA1, GA2 and GA4 with improved specific activity and thermal stability, and genes and applications of glucoamylase mutants

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Experimental program
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Effect test

Embodiment 1

[0054] The site-directed mutation of embodiment 1 glucoamylase

[0055] Using the plasmid pPIC9-Tlga1931 sequence of glucoamylase TlGA1931 as a template, the 132nd base of the amino acid sequence of glucoamylase TlGA1931 was mutated from Ser to Cys, the 492nd base was mutated from Tyr to Cys, and the 548th base The glucoamylase mutant GA1 is obtained by mutating Leu to Cys, and the 562nd base is mutated from Ala to Cys, and the 108th base of the amino acid sequence of the mutant GA1 of glucoamylase TlGA1931 is mutated from Gln to Glu to obtain glucose starch For the enzyme mutant GA2, the 475th base of the amino acid sequence of the mutant GA2 of the glucoamylase T1GA1931 was mutated from Ser to Ala to obtain the glucoamylase mutant GA4. The primers for each round of site-directed mutagenesis are listed in the table below:

[0056] Table 1 Primers required for this experiment

[0057]

Embodiment 2

[0058] The construction of embodiment 2 glucoamylase engineering strains

[0059] (1) Construction of expression vector and expression in yeast

[0060] Using the glucoamylase recombinant plasmid pPIC9-Tlga1931 as a template, the 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. Linearize expression plasmid vector DNA with restriction endonuclease Bgl II, transform yeast GS115 competent cells by electroporation, culture at 30°C for 2-3 days, pick transformants grown on MD plates for further expression experiments, specific operations Please refer to the Pichia expr...

Embodiment 3

[0061] The preparation of embodiment 3 recombinant glucoamylases

[0062] (1) Massive expression of glucoamylase at shake flask level in Pichia pastoris

[0063] Screen the transformant with higher enzyme activity, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY liquid medium, culture it on a shaker at 30°C and 220rpm for 48h; centrifuge at 4500rpm for 5min, discard the supernatant, and then add 200mL containing 0.5% Methanol BMMY liquid medium, 30°C, 220rpm induction culture for 48h. During the induction culture period, methanol solution was added once every 24 hours 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 minutes, the supernatant fermentation liquid was collected, and the enzyme activity was detected and analyzed by SDS-PAGE protein electrophoresis.

[0064] (2) Purification of recombinant glucoamylase

[0065] Collect the recombinant glucoamylase supernatant from the shake f...

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Abstract

The invention discloses glucoamylase mutants GA1, GA2 and GA4 with improved specific activity and thermal stability, and genes and applications of the glucoamylase mutants, relates to the field of genetic engineering, and particularly relates to the glucoamylase mutants with improved thermal stability, and the genes and applications of the glucoamylase mutants. In the amino acid sequence of glucoamylase TlGA1931, the amino acid at the position 132 is mutated from Ser to Cys, the amino acid at the position 492 is mutated from Tyr to Cys, the amino acid at the position 548 is mutated from Leu toCys, and the amino acid at position 562 is mutated from Ala to Cys. The mutant provided by the invention has excellent properties of high catalytic efficiency and good thermal stability, can completely meet industrial application requirements, can be well applied to food, medicine, textile and feed industries, and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to glucoamylase mutants GA1, GA2, GA4 with improved specific activity and thermal stability, genes and applications thereof. Background technique [0002] Amylase is a biocatalyst with a wide range of uses, which can be used in bread making industry, starch saccharification and liquefaction, textile desizing, paper making, cleaning agent industry, chemistry, clinical medicine analysis and pharmaceutical industry, etc. The amylase family includes alpha-amylases, beta-amylases and glucoamylases. α-amylase is an endonuclease that acts on the α-1,4 glycosidic bonds inside the starch molecule to generate dextrins and oligosaccharides. β-Amylase is an exonuclease that non-reductively and sequentially cleaves maltose from starch. Glucoamylase is an exonuclease acting on α-1,4 glycosidic bonds, and its systematic name is α-1,4-glucan glucohydrolase (α-1,4-glucan glucohydrolase, EC. 3.2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/34C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2428C12N15/815C12Y302/01003
Inventor 王兴吉王克芬张杰佟新伟刘文龙
Owner SHANDONG LONGKETE ENZYME PREPARATION
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