Biochemical and structural characterization of a β-1,3-1,4-glucanase from Bacillus subtilis 168

β-1,3-1,4-Glucanases (E.C. 3.2.1.73) hydrolyze linked β-d-glucans, such as lichenan and barley β-glucan. Recombinant β-1,3-1,4-glucanase from Bacillus subtilis expressed in Escherichia coli and purified by Ni-NTA chromatography exhibited optimum activity at 50 °C and pH 6.0. The catalytic half-life at 60 °C decreased from 90 to 5 min when the enzyme was incubated in the presence and absence of Ca2+ respectively. The kinetic parameters of lichenan hydrolysis were 2695, 3.1 and 1220 for Vmax (μmol/min/mg), Km (mg mL−1) and Kcat (s−1), respectively. Analysis by DLS, AUC and SAXS demonstrated the enzyme is monomeric in solution. Chemical denaturation monitored by ITFE and far-UV CD yielded ΔGH2O values of 9.6 and 9.1 kcal/mol, respectively, showing that the enzyme has intermediate stability when compared with other Bacillus β-1,3-1,4-glucanases. The crystal structure shows the anti-parallel jelly-roll β-sheet conserved in all GH16 β-1,3-1,4-glucanases, with the amino acid differences between Bacillus sp. enzymes that are likely determinants of stability being distributed throughout the protein.