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Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof

A technology of catalytic efficiency and amylase, which is applied in the field of amylase mutants with high catalytic efficiency and its preparation, can solve the problems of high blindness and difficulty in obtaining target strains, and achieve shortened transformation time, high catalytic efficiency, and broad application prospects Effect

Inactive Publication Date: 2014-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof
  • Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof
  • Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Amylase high catalytic efficiency improves mutation analysis and method

[0017] By analyzing the 3D spatial structure of amylase ( figure 2 ) to analyze and determine area A (catalytic area), area B, and area C. At the same time, analysis of the catalytic region (catalytic region) found 3 active sites (Asp238, Glu268, Asp330). According to the amino acid sequence and structural correlation of amylase, a short peptide and alkaline amylase sequence were selected for fusion expression to improve its catalytic efficiency.

[0018] According to the sequence of the short peptide, it was fully synthesized by chemical total synthesis and then cloned into the plasmid pET-22b(+) to construct the recombinant plasmid pET-22b(+)-P. Then, according to the amylase sequence of Bacillus alcalophilus, it was fully synthesized by chemical total synthesis. Design primers for the analyzed sequence, clone the fully synthesized amylase sequence into the recombinant plasmid ...

Embodiment 2

[0019] Embodiment 2: Determination of amylase specific enzyme activity before and after fusion

[0020] Determination of Alkaline Amylase Activity by DNS Method

[0021] 1) DNS reagent configuration: Weigh 2.5g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, add 0.5g of phenol, then dissolve 0.075g of sodium sulfite, 2.5g of sodium hydroxide, and 50g of potassium sodium tartrate, and transfer it to Put it into a 500mL volumetric flask, shake it up to volume, store it in a brown bottle and place it in a refrigerator at 4°C until use.

[0022] 2) Preparation of maltose standard curve: Prepare maltose solutions with different concentrations from 0.2g / L to 1.0g / L. Take 1mL of different concentrations of maltose 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 maltose as the abscissa and the abs...

Embodiment 3

[0027] Embodiment 3: Determination of catalytic efficiency of amylase before and after fusion

[0028] After the recombinant amylase fused with the short peptide is purified, use pH 10.0 glycine-sodium hydroxide buffer, mix the buffer with soluble starch, and use the method 3) to measure the amylase enzyme activity. After the alkaline amylase was purified, the protein concentration of the purified amylase before and after fusion was measured by Bradford method. Using the Eadie-hofstee curve method, determine the maximum reaction rate V of alkaline amylase max . Compared with the protein concentration corresponding to the enzyme, the catalytic efficiency constant (k cat ). After the fusion of short peptides, the catalytic efficiency is higher than that of the control (before mutation) (k cat ) by a factor of 3.5. The catalytic efficiency of the amylase is greatly improved, and the starch can be efficiently degraded.

[0029]

[0030]

[0031]

[0032]

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Abstract

The invention discloses an alkaline amylase mutant with a high catalytic efficiency as well as a preparation method and an application thereof, belonging to the field of genetic engineering. According to the invention, Bacillus alcalophilus JN21 (CCTCC NO: M2011231) amylase is taken as a female parent, and the sequence of the Bacillus alcalophilus amylase is subjected to oligopeptide fusion expression by using a molecular biological technology. Under such modification condition, the Bacillus alcalophilus amylase is improved by 4.1 times compared with a control case (before mutation) in terms of specific enzyme activity, and is improved by 3.5 times compared with the control case (before mutation) in terms of catalytic efficiency. With the adoption of the strategy, the catalytic efficiency of the amylase can be improved greatly, and a foundation is laid for the application of the amylase in industrial production fields such as textile desizing, detergents and additives. The strategy has an important guiding significance in improving the catalytic efficiency of the amylase and other enzymes.

Description

technical field [0001] The invention relates to an amylase mutant with high catalytic efficiency and a preparation method thereof. Background technique [0002] α-amylase (EC3.2.1.1) is an important industrial enzyme for degrading starch, which is mainly used in food, textile, medicine, detergent and other fields. Oxidation-resistant amylase can be used in textile desizing, detergent additives, and viscosity regulators using starch as a binder. Horikoshi first reported alkaline amylase produced by alkaliphile A-40-2 in 1971. In 2007, Saxena et al screened a strain that could produce alkaline amylase. In 2010, Pancha et al. (Pancha I, Jain D, ShrivastavA, Mishra S, Shethia B, Mishra S, VP M, Jha B.A thermoactive[alpha]-amylase from a Bacillus sp.isolated from CSMCRI salt farm.International Journal of Biological Macromolecules,2010 , 47(2):288-291.) A thermostable amylase-producing strain was screened out. At present, the domestic pretreatment enzyme preparation market is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28C12N15/70C12R1/07
Inventor 陈坚堵国成刘龙李江华杨海泉
Owner JIANGNAN UNIV