Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Oxidation-resistant amylase mutant and 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 efficient degradation , Strong oxidation resistance

Active Publication Date: 2012-07-04
JIANGNAN UNIV
View PDF1 Cites 4 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Oxidation-resistant amylase mutant and preparation method and application thereof
  • Oxidation-resistant amylase mutant and preparation method and application thereof
  • Oxidation-resistant amylase mutant and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Site-directed mutation analysis and method for oxidation resistance of amylase

[0022] Through the 3D spatial structure of amylase ( figure 2 ) Was analyzed to determine the Met residues (M135, M204, M219, M237) that were not resistant to oxidation in the catalytic region. At the same time, analysis of other Met residues on the surface of the enzyme structure molecule around the active site revealed that M307 is on the surface of the amylase enzyme molecule and is easily oxidized by oxidants, resulting in reduced or inactivated amylase activity.

[0023] According to the amylase sequence of Bacillus alcalophilus, it was completely synthesized by chemical synthesis and cloned into plasmid pET-22b(+) to construct recombinant plasmid pAAQ( figure 1 ).

[0024] For the site-directed mutagenesis of different Met sites, the corresponding site-directed mutagenesis primers were designed (Table 1). Using site-directed mutagenesis primers, amylase performs site-directed mu...

Embodiment 2

[0027] Example 2: Amylase at different concentrations of H 2 O 2 Determination of residual enzyme activity after treatment under conditions

[0028] DNS method for measuring alkaline amylase activity

[0029] 1) DNS reagent configuration: Weigh 2.5g 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, add 0.5g phenol, then dissolve 0.075g sodium sulfite, 2.5g sodium hydroxide, 50g potassium sodium tartrate, Transfer to a 500mL volumetric flask, shake up to a constant volume, store in a brown bottle and place in a refrigerator at 4°C for later use.

[0030] 2) Preparation of maltose standard curve: prepare maltose solutions with different concentrations of 0.2g / L-1.0g / L. Take 1 mL of maltose of different concentrations and mix with the same volume of DNS solution, and put it in a boiling water bath for 10 minutes. Cool with cold water, dilute to 10mL, A 540 Determine the absorbance value. Take the concentration of maltose as the abscissa and the absorbance as the o...

Embodiment 3

[0035] Example 3: Amylase in 500mM H 2 O 2 Determination of residual enzyme activity under different conditions

[0036] The recombinant amylase after oxidation-resistant site-directed mutagenesis in 500mM H 2 O 2 Under the conditions of 40 ℃ treatment for different time. Use catalase (1200U / mL) to degrade residual H 2 O 2 . Using pH 10.0 glycine-sodium hydroxide buffer, mix the buffer with soluble starch, and use the method 3) to determine the residual enzyme activity after amylase treatment (see Figure 4 ). After M135, M204, M219, M237, M307 are mutated into Ser, 500mM concentration H 2 O 2 Treated at 40°C for 30 min. After 5 hours of treatment, the residual enzyme activity was 63%, 70%, 54%, 46%, and 56% of that before treatment. The oxidation resistance of the amylase is greatly improved, and starch can be efficiently degraded in a strong oxidizing environment.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an oxidation-resistant amylase mutant and a preparation method thereof, and belongs to the field of genetic engineering. The preparation method of the invention adopts Bacillus alcalophilus JN21 (CCTCC NO: M 2011231) amylase as a female parent, and performs site-directed mutagenesis of the Bacillus alcalophilus amylase sequence by molecular biology technology. Under the reconstruction conditions, the Bacillus alcalophilus amylase is treated at 40 DEG C in 500 mM H2O2 environment for 30 min, and the enzymatic activity residue is increased from 18% of a control (before mutation) case to 92%; with the strategy, the oxidation resistance of the amylase is greatly increased, which lays a foundation for the application of the amylase in industrial production fields of weaving desizing, washing agent additives, and the like. The strategy has important guidance meaning for the reconstruction of oxidation resistance of amylase and other enzymes.

Description

Technical field [0001] The invention relates to a mutant resistant to an oxidation resistant amylase and a preparation method thereof, in particular to an oxidation resistant amylase mutant and a preparation method thereof. Background technique [0002] Alpha-amylase (EC 3.2.1.1) is an important industrial enzyme that degrades starch, mainly used in food, textile, medicine, detergent and other fields. Oxidation-resistant amylase can be used for textile desizing, detergent additives and viscosity modifiers using starch as a binder. Horikoshi first reported the alkaline amylase produced by the alkaliphilic bacteria A-40-2 in 1971. In 2007, Saxena et al. screened a strain that can produce alkaline amylase. 2010 Pancha et al. (Pancha I, Jain D, ShrivastavA, Mishra S, Shethia B, Mishra S, VP M, Jha BA thermoactive[alpha]-amylase from a Bacillus sp.isolated from CSMCRI saltfarm. International Journal of Biological Macromolecules, 2010 , 47(2): 288-291.) A strain producing temperatur...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/63C12N5/10C12N1/21C12N15/56C12N15/10C12N15/70D06M16/00C12S11/00C12R1/07
Inventor 陈坚堵国成刘龙李江华杨海泉
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com