MOLECULAR CHARACTERIZATION OF A THERMOACTIVE BETA-1,3-GLUCANASE FROM OERSKOVIA-XANTHINEOLYTICA

Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27195.6+/-1.3 Da and an isoelectric point of 4.85 were determ ined by electrospray mass spectrometry and from its titration curve, r espectively. Its thermoactivity profile shows it to be a heat-stable e nzyme with a temperature optimum of 65 degrees C. The secondary struct ure content of the protein was estimated by circular dichroism to be a pprox. 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-tu rn structure. Nuclear magnetic resonance spectra confirm the high cont ent of beta-structure. Furthermore, the presence of a compact hydropho bic core is indicated by the presence of slowly exchanging amide hydro ens and the enzyme's relatively high resistance to proteolysis. The N -terminal sequences of the intact protein and of a tryptic peptide eac h exhibit significant similarity to family 16 of glycosyl hydrolases w hose overall fold is known to contain almost exclusively beta-sheets a nd surface loops. Moreover, the sequenced tryptic peptide appears to e ncompass residues of the Oerskovia xanthineolytica, glucanase active s ite, since it contains a portion of the family 16 active-site motif E- [L/I/V]-D-[L/I/V]-E.