osw2对酿酒酵母孢子固定化酶影响的分析

Analysis of osw2 Effect on Saccharomyces cerevisiae Spore Immobilized Enzyme

摘要:

构建了α-半乳糖苷酶（Mel1p）的多拷贝质粒pRS426-TEFpr-MEL1，转入酿酒酵母野生型AN120及osw2Δ突变株并进行固定化酶活性测定。实验分别选取蜜二糖、棉子糖（三糖）与水苏糖（四糖）3种低聚寡糖作为Mel1p的底物，检测两种菌株孢子固定化酶对这3种底物的催化活性，结果表明野生型AN120酵母孢子壁的孔径最大只允许介于三糖和四糖之间的底物通过，四糖不能通过；而osw2?驻突变株酵母孢子壁的孔径可以允许四糖通过。通过孢子固定化酶保护性实验，发现孢子固定化酶可以完全抵抗2% SDS的处理，也可部分抵抗10% SDS的处理。通过8 mol/L尿素处理后，酶活检测发现野生型AN120酵母孢子可阻挡尿素对孢子固定化酶的影响，而osw2Δ孢子不能；蛋白免疫印迹分析进一步表明，尿素处理后野生型AN120酵母孢子固定化酶的含量几乎不变，而osw2Δ突变株孢子固定化酶的含量明显降低，说明尿素可进入osw2Δ突变株孢子内部溶解并降低固定化酶的含量，但对AN120酵母孢子无影响。本研究也在多种突变株孢子中表达Mel1p，并进行活性比较，发现osw2，osw2Δydl186wΔ及osw2Δydr326cΔ突变株固定化酶均有很高的催化活性。

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.