PARTIAL-PURIFICATION OF AN ENDOGLUCANASE FROM BIOMPHALARIA-GLABRATA

Previous studies have shown that Biomphalaria glabrata contains a comp lete cellulo-lytic system which includes an endoglucanase, an exogluca nase and a beta-glucosidase, Tn the present report, a scheme for the p urification of the endoglucanase from this invertebrate is proposed. T wo major problems were encountered during the study: 1) the presence o f a green-brownish pigment, which could not be eliminated by thermal s hock or ammonium sulfate precipitation and 2) relative instability of enzymatic activity. Various alternatives were tested and the best sequ ence of steps was: 1) a sample of the crude extract, obtained by homog enization of the digestive glands in 50 mM Tris-HCl buffer, pH 8.4, an d ultracentrifugation, was applied to a Q-Sepharose FPLC column (50 mM Tris-HCl buffer, pH 8.4; 10 mm x 22.2 cm column; now rate 1.5 ml/min; 0.1 to 0.5 M NaCl gradient); 2) the eluate peak containing activity w as dialyzed, lyophilized and eluted from a Superdex-75 gel filtration FPLC column (50 mM ammonium acetate buffer, pH 4.8; 16 mm x 60 cm colu mn; flow rate 1.0 ml/min). A low degree of purification (about 36-fold ) and recovery (about 12%) were observed, probably due to enzyme insta bility. SDS-electrophoresis of the active fraction showed a major peak of 30 kDa. In order to improve the purification scheme, further studi es are required to stabilize this enzyme during purification and stora ge.