Journal
JOURNAL OF ZHEJIANG UNIVERSITY-SCIENCE B
Volume 11, Issue 1, Pages 41-51Publisher
ZHEJIANG UNIV
DOI: 10.1631/jzus.B0900185
Keywords
alpha-agglutinin; Food-grade selection marker; beta-1,3-1,4-glucanase; alpha-galactosidase; Thermostability
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Funding
- Hi-Tech Research and Development Program (863) of China [2006AA10Z316]
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The aim of this work was to construct a novel food-grade industrial arming yeast displaying beta-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 (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The beta-1,3-1,4-glucanase gene from Bacillus subtilis was fused to alpha-agglutinin and expressed under the control of the GAL1 promoter. alpha-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the alpha-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 beta-1,3-1,4-glucanase. The analysis of beta-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that beta-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed beta-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 beta-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.
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