疏绵状嗜热丝孢菌外切β-葡聚糖酶的基因克隆与酶学特征

Gene cloning and biochemical properties of Thermomyces lanuginosus exo-β-glucanase

摘要:

【目的】疏绵状嗜热丝孢菌是一种嗜热丝状真菌，具有合成与分泌多种耐热酶的能力，从中找寻酶活高、耐热性能优良的β-葡聚糖酶。【方法】对疏绵状嗜热丝孢菌其中一个外切β-葡聚糖酶的编码基因glnB进行克隆，并在毕赤酵母GS115中表达。【结果】在摇瓶水平上重组菌的产酶活为11.5 U/mL，重组酶在SDS-PAGE中的大小约为48 kD。重组GlnB最适作用温度为65 °C，最适作用pH为5.0；在低于50 °C或pH 3.0?10.0之间具有良好稳定性。重组酶水解海带三糖，首先生成单糖和二糖，延长酶作用时间，可以进一步将其中的二糖部分水解为单糖。【结论】疏绵状嗜热丝孢菌来源的重组酶GlnB为外切β-葡聚糖酶，具有较好的耐热性能和pH稳定性。

Abstract:

[Objective] Thermophilic filamentous fungus Thermomyces lanuginosus is capable of secreting a host of thermostable enzymes. We are committed to find a high activity and thermostable β-glucanase gene. [Mehtods] a novel exo-β-glucanase gene (glnB) was cloned from T. lanuginosus and successfully expressed in Pichia pastoris GS115. [Results] SDS-PAGE shows that GlnB has a molecular weight of about 48 kD. Levels of recombinant GlnB reached 11.5 U/mL in the flask fermentation broth of recombinant Pichia cells. GlnB exhibited optimum temperature and pH at 65 °C and 5.0, respectively, and was stable below 50 °C and over the pH range 3.0?10.0. Hydrolysis of laminarin by the recombinant GlnB produced monosaccharide and disaccharide, and extended incubation resulted in partial hydrolysis of disaccharide. [Conclusion] The recombinant enzyme GlnB from T. lanuginosus is an exo-β-glucanase and has good command of thermo stability and pH stability.