Abstract

Molecular characterisation of a lytic thermoactive ?-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 ± 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65°C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% ?-helix, 7% random coil, and 68% ?-sheet and ?-turn structure. Nuclear magnetic resonance spectra confirm the high content of ?-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively ?-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.