A food-grade industrial arming yeast expressing beta-1,3-1,4-glucanase with enhanced thermal stability.
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.
Guo
Qin
Q
Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China.
Zhang
Wei
W
Ma
Liu-liu
LL
Chen
Qi-he
QH
Chen
Ji-cheng
JC
Zhang
Hong-bo
HB
Ruan
Hui
H
He
Guo-qing
GQ
D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D044644:Endo-1,3(4)-beta-Glucanase / Q000378:metabolism*; D005686:Galactokinase / Q000096:biosynthesis; D005822:Genetic Vectors; D007218:Industrial Microbiology / Q000379:methods; D008957:Models, Genetic; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013696:Temperature; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D004247:DNA; D005686:Galactokinase; D010735:Phosphoglycerate Kinase; D000519:alpha-Galactosidase; D044644:Endo-1,3(4)-beta-Glucanase