The purification and characterization of an exo-beta (1 going to 3)-glucanohydrolase from sea urchin eggs

Abstract

An exo-beta (1 going to 3)-glucanohydrolase (EC 3.2.1.6) of sea urchin eggs (Strongylocentrotus purpuratus) was purified to homogeneity by DEAE-cellulose chromatography followed by affinity chromatography on gluconolactone-Sepharose. The increase in specific activity is 800- to 1500-fold. The native enzyme exists in aggregated forms on nondenaturiung gels, with a subunit molecular weight on sodium dodecyl sulfate gels of 68,000. The enzyme is stabilized by polyethylene glycol and retains activity in 0.2% sodium dodecyl sulfate. With soluble laminarin as substrate (0.67 mg/ml), the temperature optimum is 60 degrees C (pH 6.0) and the pH optimum is 5.4 (40 degrees C). The egg glucanase does not exhibit Michaelis-Menten kinetics, exhibiting instead sigmoidal kinetics with a Hill coefficient of 1.6. The maximum velocity is 80 mumol of glucose released from soluble laminarin min-1 mg-1 protein. The half-maximum velocity (at pH 6.0, 40 degrees C) occurs at about 58 micrograms of laminarin ml-1. The glucanase is inhibited by D-1,5-gluconolactone, D-erythritol, and DL-threitol. This affinity method of glucanase purification may be important in the study of glucanases from other sources.