Hydrolysis Mechanism of Multimodular Endoglucanases with Distinctive Domain Composition in the Saccharification of Cellulosic Substrates.

Two multimodular endoglucanases in glycoside hydrolase family 5, Re Cel5 and El Cel5, share 73% identity and exhibit similar modular structures: family 1 carbohydrate-binding module (CBM1); catalytic domain; CBMX2; module of unknown function. However, they differed in their biochemical properties and catalytic performance. Re Cel5 showed optimal activity at pH 4.0 and 70 °C, maintaining stability at 70 °C (>80% activity). Conversely, El Cel5 is optimal at pH 3.0 and 50 °C (>50% activity at 50 °C). El Cel5 excels in degrading CMC-Na (256 U/mg vs 53 U/mg of Re Cel5). Five domain-truncated (TM1-TM5) and four domain-replaced (RM1-RM4) mutants of Re Cel5 with the counterparts of El Cel5 were constructed, and their enzymatic properties were compared with those of the wild type. Only RM1, with El Cel5-CBM1, displayed enhanced thermostability and activity. The hydrolysis of pretreated corn stover was reduced in most TM and RM mutants. Molecular dynamics simulations revealed interdomain interactions within the multimodular endoglucanase, potentially affecting its structural stability and complex biological catalytic processes.