Abstract
We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.
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Acknowledgments
This work was supported by the Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences, the Province Science Foundation of Sichuan, China (No. 08ZQ026-023 & 2010SZ0128) and the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX1-YW-11B2).
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Purpose of work CelA is one of the major endoglucanases in the cellulosome of thermophilic bacteria Clostridium thermocellum for cellulosic biomass degradation. The stabilizing effect of critical residues of CelA remain have now been studied.
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Yi, ZL., Wu, ZL. Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum . Biotechnol Lett 32, 1869–1875 (2010). https://doi.org/10.1007/s10529-010-0363-0
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DOI: https://doi.org/10.1007/s10529-010-0363-0