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Thermophilic acidic raw starch alpha-amylase mutant capable of improving cornstarch degradation capability as well as preparation method and application thereof

A technology of corn starch and raw starch, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems that the degradation capacity of corn starch cannot be satisfied, and achieve the effects of improving degradation capacity, saving energy, and simplifying the process

Active Publication Date: 2018-11-16
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem that the corn starch degradation ability of raw starch α-amylase GTamy cannot meet the requirements of the starch liquefaction process, the present invention provides a thermophilic acidic raw starch α-amylase mutant GTamy-S498 with improved corn starch degradation ability , whose amino acid sequence is shown in SEQ ID NO: 1

Method used

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  • Thermophilic acidic raw starch alpha-amylase mutant capable of improving cornstarch degradation capability as well as preparation method and application thereof
  • Thermophilic acidic raw starch alpha-amylase mutant capable of improving cornstarch degradation capability as well as preparation method and application thereof
  • Thermophilic acidic raw starch alpha-amylase mutant capable of improving cornstarch degradation capability as well as preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0031] Example 1: Preliminary screening of GTamy circular rearrangement mutants

[0032] (1) Synthesis of gene gtamy

[0033]According to the NCBI accession number JQ409473.1 of the thermophilic acidic starch α-amylase GTamy derived from the thermophilic bacterium Geobacillus thermoleovorans, its gene sequence was obtained by searching, as shown in SEQ ID NO: 4, and submitted to Shanghai Boyi Biotechnology Co., Ltd. Whole gene synthesis of alpha-amylase GTamy was performed.

[0034] (2) Construction of expression vector pSTOP1622-gtamyh

[0035] PCR primers P1 and P2 were designed according to the gene sequence of GTamy (Table 1), and the synthetic gene gtamy was used as a template and P1 and P2 were used as primers for PCR amplification. The PCR amplification conditions are: 98°C for 5 min; 98°C for 20 sec, 60°C for 40 sec, 74°C for 2 min, 30 cycles; 74°C for 10 min. The amplified product was digested with Spe I and Sac I, and connected to the vector pSTOP1622 to construct...

Embodiment 2

[0057] Example 2: Verification of enzymatic properties of GTamy circular rearrangement mutant GTamy-S498

[0058] Determination of optimum reaction temperature of recombinant α-amylase: Determination of enzyme activity of recombinant α-amylase on corn starch refers to Example 1, referring to Example 1 to mix enzyme solution and substrate, and react the mixture at 30-100° C. for 30 minutes , Measure the specific activity of the enzyme under different temperature conditions, and plot the specific activity of the enzyme against the temperature to determine the optimum reaction temperature. The optimal reaction temperature determination results of recombinant α-amylase GTamy and GTamy-S498 are as follows: figure 2 shown.

[0059] Determination of optimum reaction pH of recombinant α-amylase: the enzyme solution was mixed with 1% (W / V) cornstarch solutions of different pH, and the enzyme activity was determined at 80°C. The enzyme specific activity was plotted against the pH to ...

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Abstract

The invention discloses a thermophilic acidic raw starch alpha-amylase mutant capable of improving the cornstarch degradation capability as well as a preparation method and application thereof and belongs to the field of genetic engineering and enzyme engineering. According to the thermophilic acidic raw starch alpha-amylase mutant, an SBD structure domain of raw starch alpha-amylase GTamy is subjected to cyclic rearrangement mutation; then SBD in the GTamy is replaced with an SBD cyclic rearrangement mutant, so as to construct a GTamy cyclic rearrangement mutant. The enzyme activity of the GTamy cyclic rearrangement mutant to the cornstarch is compared to screen a raw starch alpha-amylase mutant GTamy-S498 with the cornstarch degradation capability which is remarkably improved. The enzymeactivity of the raw starch alpha-amylase mutant GTamy-S498 provided by the invention on the cornstarch is improved to 114.77 U / mg from 21.08 U / mg of a control group (before mutation) and is improvedby 5.44 times. The cornstarch degradation capability of the raw starch alpha-amylase mutant GTamy-S498 is remarkably improved and the enzymatic property meets the requirements of a starch liquefactiontechnology and is more suitable for the starch liquefaction technology.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and in particular relates to a thermophilic acidic raw starch α-amylase mutant with improved cornstarch degradation ability, a preparation method and application thereof. Background technique [0002] Natural raw starch is a complex and dense granule, which often needs to be treated with strong acid, strong alkali, high temperature or enzymatic treatment to destroy the structure of raw starch granules before application. Enzymatic degradation of raw starch can simplify the pretreatment process of raw starch in the modern fermentation industry, which is more environmentally friendly and saves energy consumption. Therefore, the research on raw starch degrading enzymes has always been highly concerned. Raw amylase refers to enzymes that can exhibit strong hydrolysis activity on raw starch granules that have not been cooked and gelatinized. Some enzymes in α-amylase, β-amyl...

Claims

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Application Information

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IPC IPC(8): C12N9/28C12N15/56C12N15/75C12N1/21C12R1/125
CPCC12N9/2417C12Y302/01001
Inventor 曾静袁林郭建军
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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