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Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method

An amylase and acid-resistant technology, applied in the field of bioengineering, can solve the problems that α-amylase cannot adapt to high temperature resistance, acid resistance and high activity, so as to reduce consumption, improve raw material utilization rate and product quality, The effect of obvious social benefits

Active Publication Date: 2012-11-21
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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Problems solved by technology

[0006] At present, it is difficult for α-amylase to take into account the characteristics of high temperature resistance, acid resistance and high activity, so it cannot meet the requirements of my country's sugar, brewing, organic acid and other deep processing industries that use starch as raw material.

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  • Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method
  • Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method
  • Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method

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[0033] The technical contents of the present invention will be further described below in conjunction with the examples; the following examples are illustrative, not restrictive, and the protection scope of the present invention cannot be limited with the following examples.

[0034] The present invention uses site-directed mutagenesis technology to perform directional transformation on the high-temperature-resistant α-amylase gene of Bacillus licheniformis to obtain an acid-resistant high-temperature α-amylase gene, specifically using overlapping PCR technology to perform site-directed mutation on the high-temperature-resistant α-amylase gene, No. 1058 The base C→T, the 1199th base A→G, the acid-resistant high-temperature α-amylase gene was obtained, and then cloned into the Escherichia coli-Bacillus shuttle expressing the secreted expression vector pBSA43, and transforming six kinds of extracellular proteases. Bacillus subtilis WB600 realized the secretion and expression of a...

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Abstract

The invention relates to an acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method. A technical proposal of the method consists of: utilizing a recombinant DNA technology for site-directed mutagenesis of a high temperature resistant alpha-amylase gene of Bacillus licheniformis, thus obtaining an acid resistant mutant gene of the high temperature resistant alpha-amylase, i.e. an acid and high temperature resistant alpha-amylase gene, and making use of a Bacillus subtilis expression system for secreting expression of the acid and high temperature resistant alpha-amylase. The acid and high temperature resistant alpha-amylase provided in the invention solves the problem of limited application of current high temperature resistant alpha-amylase due to its poor acid resistance and absence of high temperature resistance and acid resistance at the same time, satisfies some requirements for conducting a starch raw material liquefaction process under an acid and high temperature environment, and can improve product yield and quality as well as simplify the process.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to gene site-directed mutation and recombinant DNA technology, in particular to an acid-resistant high-temperature α-amylase, its gene, engineering bacteria and a preparation method. Background technique [0002] α-amylase (α-1,4-glucan-4-glucanohydrolase EC 3.2.1.1) can use starch as a substrate to hydrolyze α-1,4 glucosidic bonds from the inside of starch to generate maltose, maltotriose, and dextrin Wait for reducing sugar. Under the action of α-amylase, the starch is separated and degraded rapidly, and the viscosity decreases, that is, the liquefaction is completed. α-amylase can be produced by microbial fermentation, and can also be extracted from plants and animals. At present, most of the industrial production is large-scale production of α-amylase by microbial fermentation. Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus, Aspergillus oryzae, Aspergillus ...

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Application Information

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IPC IPC(8): C12N15/56C12N15/10C12N1/21C12N9/28C12R1/125C12R1/10
Inventor 刘逸寒路福平樊帅徐艳静胡博王春霞王建玲
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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