Isolement, purification et quelques caractéristiques physicochimiques de deux β‐hexosidases du suc digestif d' Helix pomatia

Two enzymes, G1 et G2, that hydrolyse o ‐nitrophenyl‐β‐ d ‐galactopyranoside (ONPG), were isolated from the digestive juice of the snail Helix pomatia . Preparation involved precipitation with 30 vol.‐% acetone at pH 5.4 and −12°, filtration on Sephadex G‐200, precipitation with 2.8 M ammonium sulfate, chromatography on CM‐cellulose for G1 and on DEAE‐sephadex for G2; yield is approximately 7% for G1 and 25% for G2 from total activity. Pure enzymes were free from β‐glucuronidase and N ‐acetyl‐glucosaminidase activities. Their homogeneity was established by criteria such as ultracentrifugation, immunoelectrophoresis and electrophoresis on cellulose acetate and in polyacrylamide or starch gel. Enzymes had a carbohydrate content of about 15‐16%, with galactose, mannose, glucose, fucose and glucosamine, but without sialic acid. Isoelectric points and electrophoretic mobilities of the two enzymes were also similar, but migration on starch gel and sedimentation coefficients (respectively 6.4 S and 10.4 S) were different. They had no immunological identity. The pH optima, in constant ionic strength 0.1 M sodium phosphate‐citric acid buffers, for hydrolysis of ONPG and lactose were respectively 5.4 for G1 and 5.2 for G2. Both enzymes attacked cellobiose, p ‐nitrophenyl‐β‐ d ‐glucopyranoside and p ‐nitrophenyl‐β‐ d ‐fucoside at the same pH as lactose or ONPG. Michaelis constants were determined by the method of Eadie. Values were lower for G2 than for G1. Evidences were produced that β‐galactosidase and β‐glucosidase activities reside in a single protein.