High level expression of extracellular secretion of a 尾-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris

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• 作者：Qiaojuan Yan^a ; ¹ ; Chengwei Hua^b ; ¹ ; Shaoqing Yang^b ; Yinan Li^b ; Zhengqiang Jiang^b ; ^{zhqjiang@cau.edu.cn}
• 关键词：Paecilomyces thermophila ; 尾-Glucosidase ; Cloning ; Expression ; Pichia pastoris
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：July, 2012
• 卷：84
• 期：1
• 页码：64-72
• 文件大小：1949 K

摘要

A novel 尾-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized 尾-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular 尾-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for 尾-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 掳C, and it was stable up to 60 掳C. The enzyme exhibited high specific activity toward pNP-尾-d-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminarin. The present results should contribute to improving industrial production of 尾-glucosidase.