Analysis of osw2 Effect on Saccharomyces cerevisiae Spore Immobilized Enzyme

Abstract:

In this study，the multicopy plasmid pRS426-TEFpr-MEL1 expressing α- galactosidase enzyme（Mel1p） was transformed into the Saccharomyces cerevisiae AN120 wild type and osw2Δ mutant strains. Three different oligosaccharides（melibiose，raffinose and stachyose） were used as substrates to detect the catalytic activity of immobilized enzyme on yeast spores. The results indicated that the pore size of AN120 wild-type spore wall was larger than trisaccharide but smaller than tetrasaccharide；however，the pore size of osw2Δ mutant yeast spore wall was larger than tetrasaccharide. The protective assay of immobilized enzyme suggested that enzyme immobilized on wild type and osw2?驻 mutant spores can completely resist 2% SDS treatment and partially resist 10% SDS treatment. Wild type spore can resist the treatment by 8 mol/L urea，whereas osw2Δ mutant spore was sensitive to urea.Furthermore，western blot analysis showed that the amounts of enzyme in the wild type spores were stable after urea treatment，but the amounts of enzyme onosw2Δ mutant spores decreased significantly. These results demonstrated that urea could enter into osw2Δ mutant spores and dissolve the enzyme，leading to the decrease of the amount of enzyme. Moreover，the immobilized enzymes on osw2Δ，osw2Δydl186Δand osw2Δydr326cΔspores all have high catalytic activity.