Characterization of a GH family 8 β-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4.

A β-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; its [corrected] molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-β-D-glucan > chitosan > carboxymethyl-cellulose > and lichenan. BGlc8H had a lower ratio of lichenase/barley-β-D-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-β-D-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3-G6) to G3 and G2 but not to G1. Ca(2+) increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.