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Oxidation resistive amylase mutant as well as preparation method and application thereof

A technology of oxidation resistance and amylase, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve the problems of difficult to obtain target strains, blindness, etc., and achieve shortened transformation time and high-efficiency degradation , Strong oxidation resistance

Inactive Publication Date: 2015-01-07
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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  • Oxidation resistive amylase mutant as well as preparation method and application thereof
  • Oxidation resistive amylase mutant as well as preparation method and application thereof
  • Oxidation resistive amylase mutant as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0019] By analyzing the 3D spatial structure of amylase, the Met residues (M135, M204, M219, M237) that are not resistant to oxidation in the catalytic region were determined. At the same time, the analysis of other Met residues on the surface of the enzyme structure molecule around the active site found that M307 is on the surface of the amylase molecule and is easily oxidized by oxidants, resulting in reduced or inactivated amylase activity.

[0020] According to the amylase sequence (SEQ ID NO.1) of Bacillus alcalophilus, the amylase sequence (SEQ ID NO.1) was fully synthesized by chemical synthesis and then cloned into the plasmid pET-22b(+) to construct the recombinant plasmid pAAQ ( figure 1 ).

[0021] For site-directed mutagenesis of different Met sites, corresponding site-directed mutagenesis primers were designed (Table 1). Using site-directed mutagenesis primers, amyl...

Embodiment 2

[0024] Embodiment 2: the mensuration of amylase enzyme activity

[0025] Determination of Alkaline Amylase Activity by DNS Method

[0026] 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.

[0027] 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 absorbance as the ordinate to make...

Embodiment 3

[0030] Embodiment 3: amylase is in 500mM H 2 o 2 Determination of residual enzyme activity under different conditions

[0031] In 500mM H 2 o 2 Treated at 40°C for different periods of time. Use catalase (1200U / mL) to degrade residual H 2 o 2 . Use pH10.0 glycine-sodium hydroxide buffer solution, mix the buffer solution with soluble starch, and use the method 2) to measure the remaining enzyme activity after amylase treatment (see figure 2 ). After M135, M204, M219, M237, M307 are mutated into Leu, the concentration of 500mM H 2 o 2 Treat at 40°C for 5h. After 5 hours of treatment, the remaining enzyme activity was 28%, 46%, 28%, 72%, and 43% of that before treatment. The oxidation resistance of the amylase is greatly improved, and the amylase can efficiently degrade starch in a strong oxidizing environment.

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Abstract

The invention discloses an oxidation resistive amylase mutant as well as a preparation method and application thereof, and belongs to the field of genetic engineering. According to the invention, the bacillus alcalophilus amylase sequence is mutated in directed site by using molecular biological technique based on bacillus alcalophilus JN21 (CCTCC NO:M2011231) amylase as a female parent. In the modifying condition, the bacillus alcalophilus amylas is treated for 5 hours at 40 DEG C in 500mM H2O2, and enzymatic activity residue is changed from 10% (before mutation) to 72%. The oxidation resistance of amylase can be greatly improved through the policy so as to lay a foundation in the industrial production fields such as spinning desizing and detergent and additive. The policy has an import guiding meaning for modifying the oxidation resistance of amylase and other enzymes.

Description

technical field [0001] The invention relates to an amylase mutant and a preparation method thereof, in particular to an oxidation-resistant amylase mutant and a preparation method thereof. Background technique [0002] α-amylase (EC3.2.1.1) is an important industrial enzyme for degrading starch, 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, Shrivastav A, Mishra S, Shethia B, Mishra S, VP M, Jha B.A thermoactive[alpha]-amylase from a Bacillus sp.isolated from CSMCRI salt farm.International Joumrnl of Biological Macromolecules , 2010, 47(2):288-291.) Screened out a thermostable amylase-producing strain. At present, the pret...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/26C12N15/63C12N15/56C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 陈坚堵国成刘龙李江华杨海泉
Owner JIANGNAN UNIV
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