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A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第1期11卷 41-51页

作者： Qin GUOt Wei ZHANG Liu-liu MA Qi-he CHEN Ji-cheng CHEN Hong-bo ZHANG Hui RUAN Guo-qing HE Department of Food Science and Nutrition Zhejiang University Hangzhou 310029 China Wenzhou Medical College Wenzhou 325035 China

The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

关键词： α-agglutinin Food-grade selection marker β-1 3-1 4-glucanase α-galactosidase Thermostability

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