Endo-beta-1,3-glucanase using affinity inhibition

Synthetic 2',3'-epoxypropyl-1-thio-beta-D-glucopyranoside selectively modifies a catalytically essential nucleophylic group in the active site of beta-1,3-glucanase LIV from the marine mollusc Spisula sachalinensis, the inactivation being as high as 95%. The properties of native and epoxypropylthioglucopyranoside-inhibited glucanase LIV were compared, using UV-spectroscopy, SDS polyacrylamide gel electrophoresis and isoelectrofocusing. It was found that the addition of laminarine and laminarioligosaccharides to a solution of the inhibited enzyme induces UV-differential spectra typical for the tryptophanyl residue involved in the formation of the enzyme-inhibitor-substrate complex. The glucone-1,5-lactone does not produce such spectrum. It was shown that epoxypropylthioglucopyranoside protects the accessible tryptophanyl residues in the enzyme active center against the oxidation by N-bromosuccinimide.