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Thermostable amylase mutant and a preparation method thereof

A technology of amylase and mutants, applied in the field of alkaline amylase mutants and their preparation, can solve problems such as poor stability, achieve the effects of improved thermal stability, shortened transformation time, and broad application prospects

Inactive Publication Date: 2013-11-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to meet the requirements of continuous production in the printing and dyeing industry, amylases need to be used at high temperatures, but most amylases are currently less stable at high temperatures

Method used

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  • Thermostable amylase mutant and a preparation method thereof
  • Thermostable amylase mutant and a preparation method thereof
  • Thermostable amylase mutant and a preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Analysis and method of amylase thermostability site-directed mutation

[0019] The amylase derived from Bacillus alkalophilus (SEQ ID NO.1) was simulated by Swiss-model software to obtain the spatial structure of the amylase, and the software Disulfide by Design was used TM Predict possible disulfide bonds in the amylase structure. On this basis, the Swiss-model was used to simulate the spatial structure of the mutated enzyme, and the Discovery Studio software was used to analyze whether a disulfide bond was formed, and the amino acid sites capable of forming a disulfide bond were obtained. Finally, the Discovery Studio software was used to analyze the effect of the mutation of the amino acid site capable of forming disulfide bonds on the formation of hydrogen bonds and salt bridges inside the enzyme molecule. At the same time, considering the distance between the mutation site and the active center, it was finally determined that the amylase catalytic domain...

Embodiment 2

[0024] Example 2: Analysis and method of amylase thermostability site-directed mutation

[0025] Determination of alkaline amylase activity by DNS method:

[0026] 1) Preparation of DNS reagent: Weigh 3.25g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, transfer it into a 500mL volumetric flask, add 162.5mL of 2mol / L sodium hydroxide solution, then add 22.5g of glycerol, shake Mix well, dilute to 500mL, store in a brown bottle and place in a refrigerator at 4°C until use.

[0027] 2) Preparation of glucose standard curve: Prepare glucose solutions with different concentrations from 0.2g / L to 1.0g / L. Take 1mL of different concentrations of glucose and mix it with the same volume of DNS solution, put it in a boiling water bath, and keep the water bath for 10min. Cool with cold water, dilute to 10mL, A 540 Measure the absorbance. Take the concentration of glucose as the abscissa and the absorbance as the ordinate to make a standard curve.

[0028] 3...

Embodiment 3

[0033] Example 3: Determination and Analysis of Thermostability of Amylase at 60°C

[0034] Through the measurement, it was found that the half-life of the single pair of disulfide bond mutants P25C-G416C, G106C-Q110C and H426C-M470C at 60°C (Table 2) all increased, among which G106C-Q110C had the most significant effect, and the half-life increased to 3.3 times the original . Combined mutations were carried out on this basis to obtain four mutants: P25C-G416C / G106C-Q110C, P25C-G416C / H426C-M470C, G106C-Q110C / H426C-M470C and P25C-G416C / G106C-Q110C / H426C-M470C. It was found that their half-lives at 60°C (Table 3) were all improved, among which the hexamutant P25C-G416C / G106C-Q110C / H426C-M470C, which can form three pairs of disulfide bonds, had the most significant effect, and the half-life was increased to the original 7 times. The amylase has strong thermal stability under alkaline conditions.

[0035] Table 2 Thermostability of single pair disulfide bond recombinase at 60°C...

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Abstract

The invention discloses a thermostable amylase mutant and a preparation method thereof, which belongs to the field of genetic engineering. According to invention, bacillus alcalophilus JN21 (CCTCC NO:M2011231) amylase is used as female parent; molecular biological technique is adopted to conduct site-directed mutagenesis for bacillus alcalophilus amylase sequence; a pair or multiple pairs of disulfide linkages are introduced in amylase catalytic structural domain to obtain amylase mutant with higher thermal stability; under the modification condition, the half-life period of bacillus alcalophilus amylase at 60 DEG C is improved to 22.3 min from 3.2 min of a comparison example (before mutation); by utilizing the strategy, the thermostability of amylase can be obviously improved to provide basis for industrialized production, and the strategy has significant guiding significance for the modification of properties of other enzymes.

Description

technical field [0001] The invention relates to a thermostable amylase mutant and a preparation method thereof, in particular to an alkaline amylase mutant with relatively high thermostability and a preparation method thereof. Background technique [0002] Amylase can hydrolyze the α-1,4-glucosidic bonds inside starch molecules, and the hydrolyzed products are dextrin, maltooligosaccharides, maltose and glucose, which are widely used in food, textile, medicine and feed industries. The potential of alkaline amylase to hydrolyze starch under strongly alkaline conditions makes it useful in industrial fields such as starch processing, textile desizing, and detergent addition for automatic washing machines. Adding alkaline amylase can effectively remove starchy dirt on tableware and clothing, improve the quality of textile printing and dyeing, has good practical application effects and broad market demand, and related research has therefore received extensive attention. [0003]...

Claims

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Application Information

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IPC IPC(8): C12N9/28C12N15/56C12N15/10C12N15/70C12N1/21C12R1/07
Inventor 陈坚堵国成刘龙李江华杨海泉邓壮梅
Owner JIANGNAN UNIV
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