Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2 |
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Authors: | Huabiao Miao Yu Ma Yuanyuan Zhe Xianghua Tang Qian Wu Zunxi Huang Nanyu Han |
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Affiliation: | 1.Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Kunming 650500, China; (H.M.); (X.T.); (Q.W.);2.School of Life Science, Yunnan Normal University, Kunming 650500, China; (Y.M.); (Y.Z.) |
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Abstract: | Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KP691331","term_id":"856571581"}}KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications. |
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Keywords: | GH11 xylanase CBM9_1-2 fusion enzyme thermostability |
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