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Higher-heat-stability amylase mutant, and preparation method and application thereof

A technology of thermal stability and amylase, applied in the field of enzyme engineering, can solve problems such as poor stability, and achieve the effects of improved thermal stability, efficient degradation and broad application prospects

Active Publication Date: 2015-01-07
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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  • Higher-heat-stability amylase mutant, and preparation method and application thereof
  • Higher-heat-stability amylase mutant, and preparation method and application thereof
  • Higher-heat-stability amylase mutant, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Determination of Amylase Mutation Sites and Obtaining of Mutants

[0022] The amylase (SEQ ID NO.1) derived from Bacillus alkalophilus was simulated by Swiss-model software, based on the amylase spatial structure model obtained, and the solvent availability of all amino acids in the enzyme protein molecule was calculated using Macrodox software. Contact area. On the one hand, select lysine, asparagine, glutamine, and serine located on the surface of the protein (solvent accessible area is greater than 100 Å), and replace them with arginine to enhance the electrostatic interaction on the protein surface, and analyze the corresponding positions at the same time. The effect of amino acid mutation at the point on the formation of hydrogen bonds and salt bridges inside the enzyme molecule was determined to carry out amino acid substitutions at the following positions: Lys 98Arg, Asn 166Arg, Lys 192Arg, Ser 258Arg, Asn 275Arg and Gln 317Arg. On the other hand, sele...

Embodiment 2

[0028] Example 2 Amylase thermostability site-directed mutation analysis and method

[0029] Determination of alkaline amylase activity by DNS method:

[0030] 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, and then add 22.5g of glycerol, Shake well, dilute to 500mL, store in a brown bottle and place in a refrigerator at 4°C until use.

[0031] 2) Preparation of glucose standard curve: glucose solutions with different concentrations of 0.2g / L-1.0g / L were prepared. 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.

[0032] 3) A...

Embodiment 3

[0036] The thermostability determination analysis of embodiment 3 amylase at 60 ℃

[0037]It was found by measurement that the half-lives of the single mutants S66V, K98R, N166R, K192R, S258R, N275R, Q317R, Q349V and S438V were all increased at 60°C (Table 2), among which Q317R had the most significant effect, and the half-life was increased to 1.8 times the original . On this basis, compound mutations were carried out, and six single mutants that enhanced electrostatic interaction were compounded to obtain a six-mutant K98R / N166R / K192R / S258R / N275R / Q317R, and three single mutants that enhanced hydrophobic interaction were compounded A triple mutant S66V / Q349V / S438V was obtained. Finally, a nine-mutant S66V / K98R / N166R / K192R / S258R / N275R / Q317R / Q349V / S438V was obtained by introducing all nine amino acid substitutions into the catalytic domain of amylase. It was found by measurement that their half-lives at 60° C. (Table 3) were all improved, and the effect of the mutant with 9 a...

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Abstract

The invention discloses a higher-heat-stability amylase mutant, and a preparation method and application thereof, belonging to the field of enzyme engineering. A molecular biology technique is utilized to perform multiple site-directed mutagenesis to obtain the higher-heat-stability amylase mutant. The half life of the obtained amylase at 60 DEG C is enhanced to 23.9 minutes from 3.2 minutes of the control sample (before mutation). The scheme can obviously enhance the heat stability of the amylase, and provides basis for industrial production. The scheme has important instruction meanings for property modification of other enzymes.

Description

[0001] This application is a divisional application with the application number: 201310422992.2, the application date: September 17, 2013, and the application name: an amylase mutant with improved thermal stability, its preparation method and application. technical field [0002] The invention relates to an amylase mutant with improved thermostability and a preparation method thereof, belonging to the field of enzyme engineering. Background technique [0003] α-amylase (EC3.2.1.1) can hydrolyze the α-1,4-glucosidic bond inside the starch molecule, and the hydrolyzed products are dextrin, maltooligosaccharide, maltose and glucose. It is widely used in industries such as food, textile, medicine and feed Wide range of applications. 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. Alkaline amylase desizi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/70
CPCC12N9/2417C12N15/70C12Y302/01001
Inventor 陈坚刘龙邓壮梅堵国成杨海泉
Owner JIANGNAN UNIV
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