Hydrolysis Mechanism\nof Multimodular Endoglucanases\nwith Distinctive Domain Composition in the Saccharification of Cellulosic\nSubstrates

Two multimodular endoglucanases in glycoside hydrolase\nfamily 5, <i>Re</i>Cel5 and <i>El</i>Cel5, share\n73% identity and\nexhibit similar modular structures: family 1 carbohydrate-binding\nmodule (CBM1); catalytic domain; CBMX2; module of unknown function.\nHowever, they differed in their biochemical properties and catalytic\nperformance. <i>Re</i>Cel5 showed optimal activity at pH\n4.0 and 70 °C, maintaining stability at 70 °C (>80% activity).\nConversely, <i>El</i>Cel5 is optimal at pH 3.0 and 50 °C\n(>50% activity at 50 °C). <i>El</i>Cel5 excels in\ndegrading\nCMC-Na (256 U/mg vs 53 U/mg of <i>Re</i>Cel5). Five domain-truncated\n(TM1–TM5) and four domain-replaced (RM1–RM4) mutants\nof <i>Re</i>Cel5 with the counterparts of <i>El</i>Cel5 were constructed, and their enzymatic properties were compared\nwith those of the wild type. Only RM1, with <i>El</i>Cel5-CBM1,\ndisplayed enhanced thermostability and activity. The hydrolysis of\npretreated corn stover was reduced in most TM and RM mutants. Molecular\ndynamics simulations revealed interdomain interactions within the\nmultimodular endoglucanase, potentially affecting its structural stability\nand complex biological catalytic processes.