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

Method for improving mannase activity of bifunctional cellulase, cellulase mutant RMX-M and application

A technology of mannanase and cellulase, applied in the field of agricultural biology, can solve the problems that the mutation scheme is difficult to obtain the expected technical effect, and the research between sequence and function is limited.

Active Publication Date: 2020-09-18
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The improvement of enzyme molecules by means of protein engineering is also a research hotspot in the field of enzyme engineering. However, due to the limited research on the relationship between the amino acid sequence and function of enzymes, it is difficult to obtain the expected technical effect by the mutation scheme designed based on the enzyme mutation strategy.

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
  • Method for improving mannase activity of bifunctional cellulase, cellulase mutant RMX-M and application
  • Method for improving mannase activity of bifunctional cellulase, cellulase mutant RMX-M and application
  • Method for improving mannase activity of bifunctional cellulase, cellulase mutant RMX-M and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cellulase mutant recombinant vector with altered substrate specificity pPIC9r-RMX-M preparation of

[0032] The wild-type cellulase coding gene before mutation (removing the signal peptide coding sequence) was cloned into the expression vector pPIC-9r, and the recombinant vector was named pPIC9r-RMX ; with recombinant vector pPIC9r-RMX As a template, it is amplified by primers carrying mutation sites to obtain recombinant vectors carrying mutant sequences, named as pPIC9r-RMX-M .

[0033] Table 1 Cellulase mutants with altered substrate specificity RMX-M specific primer

[0034] Primer sequence (5' — 3') a

Embodiment 2

[0035] Example 2 Preparation of cellulase mutants with altered substrate specificity.

[0036] (1) Cellulase mutants RMX-M Mass expression at the shake flask level in Pichia pastoris

[0037] Mutant genes containing altered substrate specificity will be obtained RMX-M recombinant plasmid pPIC9r-RMX-M Transform Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / RMX-M . Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1 L Erlenmeyer flask with 300 mL of BMGY medium, and culture it on a shaker at 220 rpm at 30°C for 48 h; centrifuge the culture solution at 4,000 g for 5 min, discard the supernatant, and use 200 mL for the precipitation. Resuspend in BMMY medium containing 0.5% methanol, and place again at 30°C, 220 rpm to induce culture. 1 mL of methanol was added every 12 h, and the supernatant was taken for enzyme activity detection.

[0038] (2) Purification of recombinant cellulase

[0039] The recombinant cellulase supernata...

Embodiment 3

[0040] Example 3 Activity Analysis of the Cellulase Mutant and Wild Type Improved by Recombinant Mannanase Activity

[0041] The basic enzymatic properties of recombinant cellulase RMX and mutant RMX-M were determined by dinitrosalicylic acid (DNS) method. The specific method is as follows: at pH 5.0, 75 °C, 1 mL of reaction system includes 100 µL of appropriate diluted enzyme solution, 900 µL of substrate, react for 10 min, add 1.5 mL of DNS to terminate the reaction; boil in boiling water for 5 min and then cool down To room temperature, measure the OD value at a wavelength of 540 nm. Mannanase assay method is the same as cellulase. Definition of endo-cellulase activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate to generate 1 μmoL glucose per minute is 1 activity unit (U). Definition of mannanase activity unit: Under certain conditions, the amount of enzyme required to decompose a substrate to generate 1 μmoL mannose per minute...

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 relates to the technical field of agricultural biology, in particular to a method for improving the mannase activity of bifunctional cellulase, a cellulase mutant RMX-M and an application. An E218H mutant is obtained by implementing site-specific mutagenesis on an E218 site of wild cellulase with an amino acid sequence as shown in SEQ ID NO:1. Results show that the optimum pH value of an enzymatic reaction is not changed and the optimum temperature is reduced by 5 DEG C when two substrates, namely sodium carboxymethylcellulose and carob bean gum, are used for determination; whenthe carob bean gum is used as the substrate, the mannan specific activity of the mutant is improved by about 80% compared with that of the wild cellulase; and when the sodium carboxymethylcellulose isused as the substrate, compared with the cellulose specific activity of wild RMX, the specific activity of the mutant RMX-M is slightly reduced, and the capability of degrading the hemicellulose substrate mannan is improved on the basis of relatively small cellulase activity loss.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a method for improving the mannanase activity of bifunctional cellulase, cellulase mutant RMX-M and its application. Background technique [0002] Non-starch polysaccharide (NSP) is a general term for carbohydrates in plants other than starch, including cellulose, hemicellulose and pectin. Non-starch polysaccharides are the main components of feed fibers. Because these fibers surround the nutrients in the feed inside the cells, they inhibit the degradation and absorption of nutrients by animals to a certain extent. Enzymes that can degrade non-starch polysaccharides widely exist in nature, including cellulase, mannanase, xylanase, glucanase, etc. These enzymes can effectively degrade NSP in feed, improve the nutritional value of feed and Improve animal growth performance. [0003] The degradation of non-starch polysaccharides in feed often requires the compound action ...

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
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2437C12N9/2494C12N15/815C12Y302/01078
Inventor 罗会颖郑洁姚斌黄火清王亚茹柏映国苏小运王苑涂涛张杰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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