Method for improving cellulase activity, cellulase mutant 5I77-M and application

A technology of 5I77-M and cellulase, applied in the field of agricultural biology, can solve problems such as limited research and difficulty in obtaining expected technical effects by mutation schemes

Active Publication Date: 2020-09-18
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the following enzyme mutation strategies have been disclosed, due to the limited research on the relationship between the amino acid sequence and function of the enzyme, the mutation scheme designed based on the enzyme mutation strategy is difficult to obtain the expected technical effect

Method used

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  • Method for improving cellulase activity, cellulase mutant 5I77-M and application
  • Method for improving cellulase activity, cellulase mutant 5I77-M and application
  • Method for improving cellulase activity, cellulase mutant 5I77-M and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 The cellulase mutant recombinant carrier that catalytic activity improves pPIC9r-5I77-M preparation of

[0033] The cellulase wild-type (before mutation) sequence fragment (remove the signal peptide) was cloned into the expression vector pPIC-9r, and the recombinant vector was named pPIC9r-5I77 . recombinant vector pPIC9r-5I77 As a template, it is amplified by primers carrying mutation sites to obtain recombinant vectors carrying mutant sequences, named as pPIC9r-5I77-M .

[0034] Table 1 Cellulase mutants with improved catalytic activity 5I77-M specific primer

[0035]

Embodiment 2

[0036] Example 2 Preparation of cellulase mutants with improved catalytic activity.

[0037] (1) Cellulase mutants 5I77-M Mass expression at the shake flask level in Pichia pastoris

[0038] The mutant gene will be obtained 5I77-M recombinant plasmid pPIC9r-5I77-M Transform Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / 5I77-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 4000 g for 5 min, discard the supernatant, and use 200 g for precipitation. mL of BMMY medium containing 0.5% methanol was resuspended, and placed 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.

[0039] (2) Purification of recombinant protease

[0040] The recombinant cellulase supernatant expressed in the shake flask...

Embodiment 3

[0041] Example 3 Activity Analysis of Cellulase Mutants and Wild Types with Improved Recombinant Catalytic Activity

[0042] The basic enzymatic properties of recombinant endocellulase were determined by dinitrosalicylic acid (DNS) method. The specific method is as follows: at pH 4.0, 75 °C, 1 mL of reaction system includes 100 µL of appropriate diluted enzyme solution, 900 µL of substrate, reacted for 10 min, added 1.5 mL of DNS to terminate the reaction; boiled in boiling water for 5 min and cooled to At room temperature, the OD value was measured at a wavelength of 540 nm. 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). All enzyme solutions used in the study of enzymatic properties must be of electrophoretic purity.

[0043] (1) Comparison of optimal pH analysis

[0044] The purified cellulase 5I77 expressed in Example 2 and the mut...

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Abstract

The invention relates to the technical field of agricultural biology, in particular to a method for improving the cellulase activity, a cellulase mutant 5I77-M and an application. A T300P / D307P mutantis obtained by carrying out site-specific mutagenesis on a T300 / D307 site of wild cellulase with an amino acid sequence as shown in SEQ ID NO:1. Results show that compared with the wild cellulase, the cellulase mutant has the advantages that the optimum pH value and the optimum temperature of the mutant do not change, and when sodium carboxymethylcellulose is used as a substrate, the specific activity of the mutant is improved by about 60% compared with that of the wild cellulase.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a method for improving cellulase activity, cellulase mutant 5I77-M and its application. Background technique [0002] Cellulose is the main component of plant cell walls, the most abundant organic matter on earth, and the largest renewable biomass resource today. In the existing cellulose conversion technology, cellulase is considered to be a key factor, and has gradually become one of the research hotspots. [0003] Cellulase is a class of hydrolytic enzymes that can degrade cellulose into cellooligosaccharides or glucose, and can hydrolyze β-1,4-glucosidic bonds, that is, chemical bonds connecting glucose units in cellulose molecules. According to existing research, most of the degradation and conversion of cellulose in nature is completed by fungi and bacteria, and these microorganisms have developed a variety of mechanisms for efficiently degrading natural crystalli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12P19/14C12P19/02C12R1/84
CPCC12N9/2437C12N15/815C12P19/02C12P19/14
Inventor 罗会颖郑洁姚斌王亚茹柏映国黄火清苏小运王苑涂涛张杰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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