Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease

Abstract

The invention discloses amethod for constructing a cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease, and belongs to the field of enzyme engineering. According to the method, coding genes provided with doubleactive protein of celluloseexonucleaseand endonucleaseare integrated to a position of an aspergillusniger heat shock protein gene with adoption of homologous recombination and genetic engineering technologies. Specifically, a homologous recombinant fragment whose nucleotide sequence is represented by SEQ ID NO.1 is synthesized, is digested by Sal I and connected with pWM1 plasmid,DH 5 alpha is transformed, and homologous recombinant plasmid is obtained; the homologous recombinant plasmid is transformed to agrobacterium tumefaciens, and a cellulase high-yielding aspergillusniger strain is obtained through agrobacterium tumefaciens mediated transformation. Expression of interest protein is increased remarkably by increasing the temperature through heat shockexpression, so that the activities of the celluloseexonucleaseand endonuclease are improved remarkably, and the enzyme activity of celluloseis improved.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a method for expressing cellulase exo- and endo-active proteins to construct cellulase-producing Aspergillus niger strains. Background technique [0002] Cellulose is the most abundant renewable resource on the earth. The cellulose produced by photosynthesis on the earth reaches about 10 billion tons every year. Using cellulose to produce bioenergy is the key to alleviating the energy crisis and realizing the sustainable development of human beings. [0003] The key to cellulose utilization is to degrade cellulose into fermentable sugar-glucose. The degradation of cellulose requires the joint action of exonuclease, endonuclease and glucosidase. Among them, exolytic enzymes degrade the crystal structure of cellulose, which is the rate-limiting step for cellulase to degrade cellulose, while endolytic enzymes degrade long fragments of cellulose into dimers, which is a ...

AI Technical Summary

Problems solved by technology

Mixing with high exonuclease activity cellulase itself increases the process of mixing and increases the cost of degradation

And the cost of cellulase with high exonuclease activity is also high

Mixed fermentation due to toxins produced by Trichoderma reesei greatly limits the scope of use of cellulase

[0006] The reported method of constructing a high-yield strain of Aspergillus niger cellulase is to express the introduced gene together with the existing cellulase gene of Aspergillus niger, but the expressed enzyme only has one activity, and cannot increase endoenzyme and exoenzyme at the same time Activity

At the same time, the existing genetically engineered bacteria cannot control the expression of a certain enzyme in a single way through the control of conditions (temperature, acidity, etc.), and have limited changes in the composition of the cellulase enzyme system.