Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, k cat/K m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside