Construction Of A New Brewing Yeast Engineering Strain With ?-glucanase Activity

During the beer brewing, ?-glucan could increase the viscosity of fermentation and bring the difficulty for filtration. In order to decrease the amount of ?-glucan, enzyme supplementation had been added in beer brewery therefore the production processes and costs would be increased. In this paper, with the method of molecular biology and gene engineering, a new Saccharomyces cerevisiae engineering strain which could produce?-glucanase and had no change in other fermentation performance was constructed. The main contents and results of the research were listed as follows.1 Screen of starting strain which could produce ?-glucanaseA strain with the activity ?-glucanase was screened after preliminary screening and secondary screening according to the performance of ?-glucan. The strain was identified as Bacillus subtilis and named as Bacillus subtilis strain SK-1.2 Cloning and expression of ?-glucanase gene in S. cerevisiaeThree sequences from S. cerevisiae genome, including PGK1, MPa2 and ADH4 were cloned and inserted into YEp352. Then the expression plasmid include BGL3 gene was constructed and transformed into S. cerevisiae. After screening, a recombinant S.cerevisiae yeast strain which could produce ?-glucanase was selected. The enzyme activity of ?-glucanase produced by this strain could reach the maximum when cultured for 60 h.3 Determination of enzyme activity of ?-glucanaseThe enzyme activity of ?-glucanase in Bacillus subtilis strain SK-1 and recombinant yeast engineering strain were determined and contrasted by DNS method. It showed that the optimum temperature of ?-glucanase in both strains were 50?. But the range of the higher activity in recombinant yeast was smaller. The thermal stability of the enzyme was also changed. Contrasted to the enzyme in the starting strain, the residual enzyme activity of ?-glucanase in recombinant yeast reduced quickly at 50?.In addition the optimum pH and the pH stability of the enzyme were changed with which in starting strain was pH 7.0and in recombinant yeast was pH5.0~6.0.4 Determination of fermentation properties of recombinant yeastThe fermentation properties of recombinant yeast were determined including of aggregation property, death temperature, appearance fermentation degree, real fermentation degree, fermentation rate, diacety production and growth curve. Results showed that the physiological characteristics of recombinant yeast engineering strainwere smillar to the starting strain.