Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase

文献类型： 外文期刊

第一作者： Yang, Hong

作者： Yang, Hong;Zhang, Yueqi;Li, Xinxin;Bai, Yingguo;Xia, Wei;Ma, Rui;Luo, Huiying;Shi, Pengjun;Yao, Bin

作者机构：

关键词： Pichia pastoris; Cellulase; Disulfide bonds; Thermostable; Folding and refolding

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

ISSN： 0175-7598

年卷期： 2018 年 102 卷 21 期

页码：

收录情况： SCI

摘要： A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley -glucan were 769, 498, and 486U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0-6.0 and 60-65 degrees C with barley beta-glucan and CMC-Na as substrates and had a melting temperature (T-m) of 68.4 degrees C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 degrees C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and T-m values of 50 and 60.2 degrees C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

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