Original Research Paper

Biotechnology Letters

, Volume 32, Issue 12, pp 1869-1875

First online:

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum

  • Zhuo-Lin YiAffiliated withChengdu Institute of Biology, Chinese Academy of SciencesGraduate University of the Chinese Academy of Sciences
  • , Zhong-Liu WuAffiliated withChengdu Institute of Biology, Chinese Academy of Sciences Email author 

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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.


CelA Clostridium thermocellum Endoglucanase Family shuffling Thermostability