[Saturation mutagenesis of three amino acid positions consisting of the active site of an endoglucanase from termite Coptotermes formosanus].

Functional improvement to one component of the cellulase, endo-beta-1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo-beta-1,4-glucanase (NtEG PDB id = 1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56IIe showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.