High-yield and temperature-resistant beta-dextranase pichia pastoris and construction thereof

Abstract

The invention discloses a pichia pastoris recombinant strain and application thereof in the field of gene engineering. The research room performs directed evolution with improved thermal stability on bacillus amyloliquefaciens producing beta-1,3-1,4-glucanase gene by in-vitro molecular evolution technology to obtain a plurality of mutants with improved thermal stability. In the invention, a beta-1,3-1,4-glucanase gene bg1 with most obviously improved thermal stability is expressed in a pichia pastoris system for the first time. The gene is cloned to a pichia pastoris expression vector pPICZalphaA and constructed to AOX I methanol inducible promoter downstream to obtain recombinant plasmid pPICZalphaA-his6-bg1; the recombinant plasmid is subjected to Pme I linearization to transform pichia pastoris GS115; and the bg1 gene is integrated on the pichia pastoris chromosome through homologous recombination and positioned on the downstream of yeast alpha-factor to realize heterogenous secretion expression. By optimizing the culture condition of the recombinant strain, the optimal expression conditions of the beta-1,3-1,4-glucanase are that the pH is 7.0, OD600 is 2.5, the daily induced addition amount of methanol is 1 percent and the culture time of the strain after the methanol induction is 2.5 to 3 days. The protein expression level for secreting the beta-glucanase to a culture medium under the condition is 190mg/L, and the specific activity of one milligram of protein reaches 4,312U. SDS-PAGE result shows that the size of the expressed protein is about 27KDa and matches with the size of theoretical molecular weight.

Description

technical field [0001] field of genetic engineering. Background technique [0002] In the beer industry, it is common for breweries to use β-glucanase in the saccharification process. Many foreign companies such as NOVO, Novozymes, and DMS have already sold commercial enzyme preparations. During the saccharification process, although most of the β-glucan in the barley endosperm cell wall is degraded during the malting stage, due to the poor temperature tolerance of plant β-glucanase, its remaining activity will be completely destroyed during saccharification, leaving β-glucan may still lead to excessive viscosity of wort, resulting in difficulty in filtration, prolonging the mash filtration time and reducing the content of extract. During fermentation, excess β-glucan will cause early sedimentation of the yeast, reducing the degree of fermentation. In finished beer, β-glucan will combine with protein, causing haze and gel precipitation, reducing the abiotic stability of be...

AI Technical Summary

Problems solved by technology

In finished beer, β-glucan will bind to protein, causing haze

Images