产外切葡聚糖酶真菌的筛选鉴定及毕赤酵母中的表达

Screening and identification of exo-1,4-β-D-glucannase-producing fungi, and its expression in Pichia pastoris

摘要:

【目的】外切葡聚糖酶是纤维素酶组分中一类对结晶纤维素有降解作用的酶类，如何提高外切葡聚糖酶活力是研究的关键问题。【方法】从筛选鉴定得到的一株产外切葡聚糖酶酶活较高的黑曲霉Asp-524菌株出发，通过PCR技术克隆得到外切葡聚糖酶基因序列，生物学信息分析后，构建了毕赤酵母诱导型表达载体，实现了该基因在毕赤酵母中的成功表达。【结果】抗性筛选得到的阳性转化子，用终浓度为1%甲醇诱导5 d后，酶活达到4.74 U/mL。酶学性质分析显示重组外切葡聚糖酶最适pH为5.0，pH稳定性分析显示在pH为4.0?6.0范围内相对稳定，酶活能保持在最高酶活力的80%以上，最适反应温度为50 °C，经60 °C保温1 h后，酶活仍能保持80%以上。【结论】结果说明该外切葡聚糖酶具有较好的热稳定性和pH稳定性，这一研究为纤维素酶的实际应用奠定了一定基础。

Abstract:

[Objective] Exoglucanases is a class of cellulase which could degradation crystalline cellulose. Nowadays, how to improve the exoglucanase activity is of importance. [Methods] In this study, strain ASP-524, possessing higher level of cellobiohydrolase, was identified as Aspergillus niger. The cbhB gene was cloned from strain ASP-524, and expressed in Pichia pastoris GS115. [Results] After the induction of 1% methanol for 5 d, the expression strain reached a cellulase activity of 4.74 U/mL. The recombinant exoglucanase was a 57 kD protein with an optimum catalytic activity at pH 5.0 and 50 °C. [Conclusion] The enzyme was stable. The recombinant exoglucanase was relatively stable in a broad pH range, from 4.0 to 6.0, and retained 80% activity.