A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Vi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86 degrees C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85 degrees C. The results provide valuable thermostability-related structural information on this thermophilic enzyme. (C) 2010 Elsevier Ltd. All rights reserved.