High-expression cellulose endonuclease gene as well as recombinant vector and protein thereof

Abstract

The invention relates to a high-vitality cellulose endonuclease artificially synthetic gene shown in a nucleotide sequence in SEQ ID NO.1 in a sequence table or a sequence which has 90% homology or above and coding the same biological functional protein with the nucleotide sequence shown in the SEQ ID NO.1. The nucleotide sequence shown in the SEQ ID NO.1 in the sequence table can use pichia pastoris constitutive expression plasma pGAPZaA as a vector and realize high-level recombination expression of target protein in pichia pastoris host bacteria X33. Meanwhile, a screened high-level expression transformant can realize high-level secretion of recombinant protein by use of inorganic salt high-density fermentation, and by simple nickel affinity chromatography purification, a novel recombinant cellulose endonuclease protein pure product with high vitality can be obtained. The recombinant protein has very high capacity for hydrolyzing cellulose substances and producing glucose and has important application value in biological energy source, feed and food industries.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a novel high-expression cellulase gene derived from poria cocos and its recombinant carrier and protein. Background technique [0002] The annual output of crop straw alone in my country is as high as 6×10 8 ~7×10 8 tons, but these valuable cellulose resources have not been effectively utilized. Using cellulase to decompose cellulose is an efficient and environmentally friendly method. The use of cellulase to degrade cellulose and convert it into fuel, food and chemicals, such as sugar, ethanol, feed protein, etc., has great economic significance for alleviating the energy crisis and the shortage of food and feed resources. Cellulase will be used in food , Feed, environmental protection, energy and resource development and other fields play an important role. According to the structure of cellulase, cellulase can be divided into two categories: cellulase complex ...

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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