Optimized nucleotide sequence of alkaline pectinase pell68s and high-level expression method thereof

Abstract

The invention provides and discloses an optimized nucleotide sequence of alkaline pectinase pel168s and a high-level expression method thereof. According to the method, a pel168 gene sequence (wherein the gene is Bacillus subtilis 168 the accession number of which in a GenBank is AL009126) is optimized by DNA works software, restriction enzyme cutting sites SalI and PmeI are shielded, a restriction enzyme cutting site EcoRI is added at the 3' end of a primer, and a restriction enzyme cutting site NotI is introduced at the 5' end of the primer. After the procedures of PCR (Polymerase Chain Reaction) amplification, connection transformation and sequencing verification, the optimized gene sequence of the alkaline pectinase pel168s is obtained. Recombinant plasmid pel168s-9k is constructed according to the sequence, and then is transformed into pichia yeast GS115, thereby obtaining a positive recombinant strain GS115 / pel168s-9k. According to the invention, when alkaline pectinase is produced by adopting the optimized nucleotide sequence of the alkaline pectinase pel168s and utilizing the pichia yeast, the target protein expression index is high, the purge process is simple, the production cost of the alkaline pectinase is reduced greatly, and the utilization rate of an enterprise on the alkaline pectinase is enhanced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an alkaline pectinase pel168s nucleotide optimized sequence and a high-efficiency expression method thereof. Background technique [0002] Alkaline high-temperature pectinase has an incomparable role in biological degumming. With the development of society, the country has higher and higher requirements for enterprises, and energy conservation and emission reduction has become a hot topic for enterprises. Biological degumming of ramie and bioscouring of fabrics is a green and environmentally friendly degumming method. Compared with chemical degumming, biological degumming has the advantages of improving the quality of dry hemp, no damage to fibers, and no pollution. Biological degumming technology has become a research hotspot for ramie researchers at home and abroad, and it is also the main development direction of ramie degumming in the future. [0003] However, the high-temperat...

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Problems solved by technology

[0003] However, the high-temperature alkaline pectinase required in the ramie degumming process is generally derived from Bacillus and Escherichia coli, and they can be expressed in large quantities in prokaryotic cells. If one wants to obtain a large amount of purer pectinase, one must carry out A series of complex purification processes

In order to facilitate purification, people began to use Pichia pastoris as an expression strain. Pichia pastoris only secretes very little self-protein, and there is only a small amount of protein in the minimal growth medium of Pichia pastoris, which means that the secreted foreign protein is The main component of protein in the medium, however, due to the problem of the source gene codon preference, the yield of alkaline pectinase is low

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