High-activity poria cocos cellulose incision enzyme gene and protein and recombinant vector thereof

Abstract

The invention relates to a high-activity cellulose incision enzyme artificially synthesized gene. The gene is shown as a nucleotide sequence of SEQ ID NO.1 in a sequence table; or the gene is as shownin the sequence of a biological function protein with 90 or above homology with the nucleotide sequence and same coding. According to the high-activity cellulose incision enzyme artificially synthesized gene, such as the nucleotide sequence of SEQ ID NO.1 in the sequence table, a pichia pastoris constitutive expression plasmid pGAPZalpha can be used as a carrier, and high-level recombinant secretory expression of a target protein in pichia pastoris host bacteria X33 can be realized. Meanwhile, screened high-level expression converters can realize high-level serection of a recombinant proteinby using high-density fermentation of an inorganic salt, and novel high-activity recombinant cellulose incision enzyme protein sterling can be obtained through simple nickel affinity chromatography purification. The recombinant protein has the high ability of hydrolyzing cellulose substances and generating glucose, and has important application value in aspects such as biological energy sources, fodder and food industries.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a novel high-activity cellulose endonuclease gene and encoded protein derived from Poria cocos, and a recombinant vector constructed by the gene. Background technique [0002] Cellulase widely exists in natural organisms, such as bacteria, fungi, plants, animals and many other organisms can produce cellulase. These raw materials can be utilized by adding cellulase produced by living organisms to cellulose raw materials, so how to mass-produce cellulase has become the main bottleneck restricting the utilization of cellulose. With the development of biotechnology, the use of genetic engineering to develop cellulase resources with high activity and high yield has attracted more and more attention from scholars at home and abroad. At present, great progress has been made in the cloning and expression of cellulase genes, but there are few reports on the successfu...

AI Technical Summary

Problems solved by technology

In the application numbers 201811240264.9 and 201811281667.8, pET28 and pET32 were used as vectors to express and purify endocellulase in the Escherichia coli system to obtain a certain amount of recombinant protein with good activity, but the proteins expressed by the above two vectors were both Intracellular protein, the bacteria need to be broken first when purifying, the purification steps are cumbersome and the recovery rate is low

Moreover, expression in E. coli may cause toxicity due to the presence of LPS, so it is often necessary to analyze and determine the toxicity of the expressed purified product

Finally, obtaining high-purity proteins often requires multi-step purification operations. The more purification steps, the lower the protein yield and the more likely it will lead to the inactivation of the target product

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