工业酿酒酵母重组β-1,3-1,4-葡聚糖酶部分酶学性质的研究

用刚果红法测定β-1,3-1,4-葡聚糖酶的酶活力，研究重组酿酒酵母(S.cerevisiae)菌株SC-βG分泌表达的重组β-1,3-1,4-葡聚糖酶的部分酶学性质，并与出发菌株枯草芽孢杆菌(B.subtilis)表达的原始酶的性质进行比较。结果表明，重组酶保持了与原始酶相同的底物专一性。 重组酶的最适反应温度为35℃，而原始酶为55℃。重组酶的热稳定性也发生了改变，40℃热处理20min只保留63.4%的最初酶活力，但温度再升高时对热处理敏感度降低，70℃的热处理20min仍保留45.9%的最初酶活力；而原始酶50℃时稳定，60℃以上的热处理酶活力损失很大。与原始酶相比，重组酶的最适pH值下降为pH5.0，而原始酶为pH6.5；相比原始酶在pH7.0有最大稳定性，重组酶在pH5.5时有最大稳定性。重组β-1,3-1,4-葡聚糖酶的最适反应条件与原始酶相比更接近啤酒的实际生产条件。

Enzymatic Properties ofβ-1,3-1,4-Glucanase from Recombinant Industrial S. cerevisiae

Enzymatic properties of β-1,3-1,4-glucanase secreted by recombinant S. cerevisiae strain SC-βG was examined using Congo-Red method and compared with the native β-1,3-1,4-glucanase from original strain B. subtilis. Results showed that obvious differences in enzymatic properties of β-1,3-1,4-glucanase from S. cerevisiae strain SC-βG and original strain B. subtilis except substrate specificity were observed. The optimal reaction temperature was 35 ℃ for recombinantβ-1,3-1,4-glucanase and 55 ℃ for native β-1,3-1,4-glucanase. As for thermal stability, recombinantβ-1,3-1,4-glucanase remained 63.4% activity after 40 ℃ heat treatment for 20 min and 45.9% activity after 70 ℃heat treatment; in contrast, the nativeβ-1,3-1,4-glucanase was stable below 50 ℃ and its activity exhibited a significant loss after heat treatment at 60 ℃. Moreover, the optimal reaction pH for both enzymes also exhibited an obvious difference, such as pH 5.0 for recombinant β-1,3-1,4-glucanase and pH 6.5 for native β-1,3-1,4-glucanase. Similarly, the optimal pH for stability of both enzymes was 5.5 for recombinant β-1,3-1,4-glucanase and 7.0 for native β-1,3-1,4-glucanase. Therefore, the optimal reaction conditions of recombinant β-1,3-1,4-glucanase are closer to beer brewing conditions so that this enzyme should reveal better effect on beer brewing.