重组阿拉伯糖苷酶在毕赤酵母中的分泌表达及其活性分析

假密环菌是一种果树腐病菌，具有较强的分解代谢纤维素的能力。本文根据已克隆出的阿拉伯糖苷酶基因序列（Accession Number AJ620046）设计引物从假密环菌的mRNA中克隆出阿拉伯糖苷酶的ORF片段，构建重组质粒pPIC9-AF，与组氨酸标签融合，电转化毕赤酵母菌GS115并进行诱导表达。所得到的重组阿拉伯糖苷酶在30-35℃时有较高的酶活，最适反应活性pH值在6.0-8.0之间，而在pH4.0－8.0范围内酶活基本保持在80%以上，比大多已报道的阿拉伯糖苷酶最适pH范围宽。本研究工作对于高效表达重组阿拉伯糖苷酶以及对其进一步的酶学性质研究提供了一个良好的基础。

Cloning, Expression and property analysis of Arabinosidase in Pichia pastoris

SMART-RACE was performed after Isolating the total RNA of Armillariella tabescens to amplify the full-length cDNA of arabinosidase（GenBank Accession No. AJ620046）. Bioinformatics analysis was used to analyze the code frame of arabinosidase, to predict its structure and function. Recombinant plasmid pPIC9-AF was constructed and then electroporated into methylotrophic yeast Pichia pastoris GS115. The secreted 6×His fusion protein was purified to analyze its enzymology property. This arabinosidase had high activity at 30℃-35℃ under acid condition, and was stable within wide range of pH and temperature. It maintained about 80% activity at the range of pH4.0-8.0 and 20℃-40℃，which was wider than many other cloned arabinosidase. So it was worthy to go step further to study this enzyme. And recombinant expression provided a chance of highly expressing arabinosidase.