Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2 |
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Authors: | He Jun Yu Bing Zhang Keying Ding Xuemei Chen Daiwen |
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Affiliation: | 1. College of Life Science, China JiLiang University, Hangzhou 310018, China;2. College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;3. College of Life Science, Zhejiang University, Hangzhou 310058, China |
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Abstract: | To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (α-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 °C that was 10 °C higher than the native Xyn2. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA. |
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