Method for heterologous expression and purification of trichoderma harzianum acidic protease P6281

Abstract

The invention discloses a method for heterologous expression and purification of trichoderma harzianum acidic protease P6281. Trichoderma harzianum RNA is extracted and is subjected to reverse transcription to obtain cDNA; an acidic protease gene is obtained by PCR and is inserted into an expression vector; a recombinant plasmid is electro-transformed and introduced into pichia pastoris GS115 and induced expression is performed; and then a relatively pure enzyme product is obtained by a nickel column affinity chromatography and molecular sieve chromatography. The trichoderma harzianum acidic protease P6281 is successfully expressed and purified for the first time; the method improves the yield of the P6281 protease; and the enzyme activity of the obtained acidic protease is much higher than that of conventional trichoderma acidic protease.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a heterologous expression and purification method of Trichoderma harzianum acid protease P6281. Background technique [0002] Trichoderma harzianum, a species of Trichoderma fungi present in almost all soils, readily colonize plant roots and some strains are rhizosphere active, ie able to grow on roots as they develop. At the same time, Trichoderma harzianum will also obtain nutrients by attacking and parasitizing other fungi. Over the years of evolution, they have developed many mechanisms for attack by other fungi and for enhancing plant and root growth. As an economical, effective, safe and sustainable fungal agent, Trichoderma harzianum is also used as a fungicide. It is currently the most important biocontrol fungus widely used in the biological control of plant fungal diseases. Its antibacterial mechanism and application research has received much attention. It is...

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Problems solved by technology

However, so far, no one has isolated and purified the P6281 protease from Trichoderma harzianum, and its properties have not been clearly studied

[0004] However, in the current reports, the activity of Trichoderma acid protease obtained by heterologous expression and purification is generally not high. For example, in 2007, the enzyme activity of Trichoderma harzianum SA76 acid protease fermented by Liu in Saccharomyces cerevisiae was 10.5 U / mL In 2010, the activity of N. crassa acid protease fermented by Guo in Saccharomyces cerevisiae was 6.8 U / mL; in 2013, the fermented activity of Trichoderma aculeatus acid protease expressed by Yang in Pichia pastoris was 18.5 U / mL. In 2014, the pure enzyme activity of Trichoderma aculeatus ASP55 acid protease expressed by Dou in Escherichia coli was 9.52U / mL

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