Comparison of two models of surface display of xylose reductase in the Saccharomyces cerevisiae cell wall.
In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to β-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1, or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the Km values for NADPH of both immobilized enzymes were somewhat higher than the Km of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization.
['Hossain|Amir Sk|AS|', 'Teparić|Renata|R|', 'Mrša|Vladimir|V|']
D000135:Acid Phosphatase D002473:Cell Wall D050542:D-Xylulose Reductase D005686:Galactokinase D005936:Glucans D017261:Glycosylphosphatidylinositols D010957:Plasmids D011401:Promoter Regions, Genetic D021382:Protein Sorting Signals D012441:Saccharomyces cerevisiae D029701:Saccharomyces cerevisiae Proteins D014994:Xylose
Surface display Xylose reductase Cell wall protein Genetic immobilization Yeast cell wall