Acid and high temperature resistant alpha-amylase and genes and engineering bacteria of alpha-amylase and preparation method

A technology of amylase and acid resistance, which is applied in the field of bioengineering to achieve the effects of enhanced thermal stability, broad application prospects and simplified process

Active Publication Date: 2016-11-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Some domestic scholars have also carried out research on acid-resistant and high-temperature-resistant α-amylase, but the obtained α-amylase still cannot have both heat-resistant and acid-resistant properties, and can

Method used

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  • Acid and high temperature resistant alpha-amylase and genes and engineering bacteria of alpha-amylase and preparation method
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  • Acid and high temperature resistant alpha-amylase and genes and engineering bacteria of alpha-amylase and preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of Acid and High Temperature α-Amylase Gene

[0037] 1. Construction of BLA mutants

[0038] According to the BLA mutation sites reported in the literature, corresponding site-directed mutagenesis primers (Table 1) were designed. Using BLA as the starting sequence, five BLA mutants were constructed by whole-plasmid PCR, and these five mutants were expressed in the same expression vector. Express in the same host cell to obtain five mutant acid-resistant and high-temperature α-amylases;

[0039] 2. Determination and analysis of enzymatic properties of BLA mutants

[0040] According to the analysis of enzymatic properties, it is found that BLA-m9 and BLA-m6 have the best thermal stability at pH 4.5, and BLA-m9 has higher specific activity in the range of pH 4.0-5.0 and good acid resistance;

[0041] 3. Structural analysis of BLA mutants

[0042] The homology modeling of five mutants of BLA was carried out by SWISS-MODEL, and the models of BLA-4480, BLA-m6,...

Embodiment 2

[0049] Preparation of acid-resistant and high-temperature α-amylase engineered bacteria

[0050] 1. Construction of recombinant expression plasmids

[0051] Digest the target fragment on the mutant plasmid with Pst I and Hind III, enzyme digestion reaction system: 10 μL plasmid + 1 μL Pst I + 1 μL Hind III + 10 μL buffer + 15 μL ddH 2 O, reaction conditions: 37°C, 3h; purify and recover the digested fragment with a gel recovery kit;

[0052] The pNY326 vector plasmid ( figure 1 ) with Pst I and Hind III for double enzyme digestion and dephosphorylation treatment, enzyme digestion reaction system: 10 μL plasmid + 1 μL Pst I + 1 μL Hind III + 10 μL buffer + 15 μL ddH 2 O, reaction conditions: 37°C, 3h; pNY digested fragments were phosphorylated with alkaline phosphatase (CIAP), reaction system: 20 μL double digestion reaction solution + 2 μL CIAP + 3 μL buffer + 5 μL ddH 2 O, reaction conditions: 37°C, 3h; the phosphorylated pNY fragments were purified and recovered with a ge...

Embodiment 3

[0059] Preparation of acid-resistant and high-temperature α-amylase

[0060] Express and purify acid-resistant high-temperature α-amylase according to the following method: inoculate the acid-resistant high-temperature α-amylase recombinant strain in 20mL MTNm medium, 37°C, 180rpm constant temperature shaking culture for about 12h, until OD600 is 1.0 to obtain seed liquid; The seed solution was inoculated in a 5L fermenter containing 2L of TMNm medium with a 2% inoculum amount, cultured at a constant temperature of 30°C, with a rotation speed of 200rpm for 72 hours, and an air flow of 10vvm; the pH value was monitored in real time during the fermentation process, and acid and alkali were automatically replenished to maintain the culture The pH value of the liquid is about 6.86; after 72 hours of cultivation, the supernatant is centrifuged to obtain the crude enzyme liquid; the crude enzyme liquid is purified to obtain electrophoretic pure acid-resistant high-temperature α-amyla...

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Abstract

The invention discloses genes and engineering bacteria of acid and high temperature resistant alpha-amylase and a preparation method of the genes and engineering bacteria. Compared with a mutant reported by literature, under the condition that the pH ranges from 4.2 to 4.6, and the temperature is 95 DEG C, the thermostability of the acid and high temperature resistant alpha-amylase is enhanced, the expression quantity is 50 times that of existing acid and high temperature resistant alpha-amylase, the alpha-amylase is applied to the fields of chemical industry, food, medicine and the like, starch can be efficiently degraded under the conditions of strong acidity and high temperature, the process can be simplified, environmental pollution is reduced, and a wide application prospect is achieved. Meanwhile, according to genes and engineering bacteria of the acid and high temperature resistant alpha-amylase and the preparation method of the genes and engineering bacteria, rational design of applied combined construction prediction has important guiding significance in improving thermostability of an industrial enzyme.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to gene site-directed mutation and DNA recombination technology, in particular to an acid-resistant high-temperature α-amylase gene, an engineering bacterium and a preparation method thereof. Background technique [0002] α-amylase plays an extremely important role in industrial production, especially starch deep processing. Starch deep processing usually includes two steps of liquefaction and saccharification. When liquefying, mix the wet starch granules with the enzyme, heat them with steam at 105°C, and then hydrolyze them at 90°C for 1-1.5 hours. Before liquefying, adjust the pH value of starch slurry or corn steep liquor from natural pH4.5 to pH5.8- 6.2, lower it to pH 4.2-4.5 during the saccharification process. The use of α-amylase is limited by the optimum temperature and optimum reaction pH. Industrially used alpha-amylases are mainly derived from Bacillus, Thermotolerant Act...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/10C12N1/21C12N9/28C12N15/75C12N15/74C12R1/10C12R1/01
CPCC12N9/2417C12N15/102C12Y302/01001
Inventor 谢秋宏师瑞琳相宏宇刘玲
Owner JILIN UNIV
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