Enzymatic characterization of six fungal GH5_5 endo-β1,4-glucanases and their performance on corn stover deconstruction

Sequence analysis indicates that fungal endo-β1,4-glucanases from subfamily 5 of family 5 glycohydrolases (GH5_5) further diverge into two clades, GH5_5_1 and GH5_5_2. A Trichoderma reesei endoglucanase (TrCel5A) from clade 1 is a major component in commercial enzyme mixtures for cellulosic biomass deconstruction. Previous research demonstrated that a Sporotrichum thermophile endoglucanase (StCel5A) from clade 2 gave higher yields of Glc and Xyl from pretreated corn stover compared to TrCel5A when incorporated into an otherwise identical 8-component synthetic enzyme cocktail. Three enzymes from each clade, including StCel5A and TrCel5A, were expressed and characterized. All six enzymes have N-terminal CBM modules and are glycosylated. Clade 2 endoglucanases have higher pH optima using carboxymethyl cellulose (CMC) as substrate, and mannanase activity. Only one of the six enzymes showed more than 30% inhibition by 1% lignin. Of the three clade 2 enzymes, only StCel5 showed enhanced Glc and Xyl yields from corn stover, and therefore superiority is not a general property of clade 2. Furthermore, relative activities on pure substrates such as CMC, oat β-glucan and filter paper did not predict activities on pretreated corn stover when tested in 8-component mixtures.