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Thermostable amylase mutant and preparation method and application thereof

An amylase and mutant technology, which is applied in the field of thermostable amylase mutants and their preparation, can solve the problems of large blindness and difficulty in obtaining target strains, and achieves good thermostability, shortened transformation time, and wide application. Foreground effect

Active Publication Date: 2014-05-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The blindness of the screening is large, and it is not easy to obtain the target strain

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: Amylase thermostability improves mutation analysis and method

[0017] By analyzing the 3D spatial structure of amylase, the region A (catalytic region), region B and region C were determined. According to the amino acid sequence and structural correlation of amylase, a short peptide and amylase sequence were selected for fusion expression to improve its thermal stability.

[0018] According to the sequence of the short peptide and PT-linker, it was fully synthesized by chemical total synthesis and then cloned into the plasmid pUC57 to construct the recombinant plasmid pUC57-P. At the same time, according to the amylase sequence of Bacillus stearothermophilus, the amylase was fully synthesized by a chemical total synthesis method and then cloned into the plasmid pET-20b(+) to construct the recombinant plasmid pET-20b(+)-Amy. Primers were designed for the analyzed sequence, and the short peptide and PT-linker sequence in the recombinant plasmid pUC57-P wer...

Embodiment 2

[0019] Embodiment 2: Amylase activity assay method

[0020] Determination of amylase activity by DNS method:

[0021] 1) Preparation of DNS reagent: Weigh 6.5g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, transfer it into a 1L volumetric flask, add 262mL of 2mol / L sodium hydroxide solution, then add 185g of potassium sodium tartrate and 5g of phenol and 5g of anhydrous sodium sulfite, dilute to 1L, store in a brown bottle, and place in a refrigerator at 4°C until use.

[0022] 2) Preparation of glucose standard curve: glucose solutions with different concentrations of 0.1g / L-1.0g / L were prepared. Mix 2mL of different concentrations of glucose with 3mL of DNS solution, put them in a boiling water bath, and keep the water bath for 7min. Cool with cold water, dilute to 15mL, and measure the absorbance with A540. With the glucose concentration as the abscissa and the absorbance as the ordinate, a standard curve was made.

[0023] 3) Definition of en...

Embodiment 3

[0024] Embodiment 3: Determination and Analysis of Amylase Thermal Stability

[0025] The amylases before and after fusion were treated with 20% glycerol, pH 6.0, and 95°C, and samples were taken regularly, and the residual enzyme activity was measured by the method in Example 2. By comparison, it was found that the amylase after the fusion of short peptides was The half-life under the assay conditions was increased from 4h to 7h, and the half-life was increased by 1.7 times. The fusion amylase has good thermal stability.

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PUM

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Abstract

The invention discloses a thermostable amylase mutant and a preparation method and application thereof, belonging to the field of genetic engineering. Bacillus stearothermophilus amylase is taken as a female parent, the N-terminal area of the bacillus stearothermophilus amylase and oligopeptide are connected through PT-linker so as to obtain the mutant, and the amino acid sequence of the oligopeptide is shown as SEQIDNO.1. Under the transformation condition, the bacillus stearothermophilus amylase is improved to be 7h from 4h of a comparison example (before mutation) in half-life period at 95DEG C. The method remarkably improves the thermostability of the amylase and provides a foundation for industrial production of the amylase.

Description

technical field [0001] The invention relates to an amylase mutant and a preparation method thereof, in particular to a thermostable amylase mutant and a preparation method thereof. Background technique [0002] Alpha-amylase, the systematic name is alpha-1,4-glucose-4-glucohydrolase. The characteristic of α-amylase is that it cuts the α-1,4 glycosidic bond in the middle of the starch molecular chain, and cannot hydrolyze the α-1,6 glycosidic bond, nor can it hydrolyze the α-1,4 glycosidic bond close to the 1,6 branch. The hydrolysis products are soluble dextrin, oligosaccharides and a small amount of maltose and glucose. α-amylase has considerable industrial application value and is widely used in alcohol, organic acid, amino acid and textile industries. The thermostable amylase-producing strains are obtained mainly through screening, mutagenesis and enzyme molecular modification. The blindness of the screening is large, and it is not easy to obtain the target strain. Mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/70C12R1/07
CPCC07K2319/31C12N9/2417C12N15/70C12Y302/01001
Inventor 吴敬李祝
Owner JIANGNAN UNIV
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