Endo‐β‐mannanases in the endosperm of germinated tomato seeds

Three forms of galactomannan‐hydrolyzing enzymes (dyed galactomannan as substrate) were partially purified from germinated tomato [Lycopersicon esculentum (L.) Mill.] seed. Two of the enzymes were of the same molecular mass, 38 kDa, as judged by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), but the points of elution from a CM‐Toyopearl column by a pH‐gradient were different between the two (pH 5.15 and 5.45. respectively). The molecular mass of the third form was slightly less (37.5 kDa) than that of the other two. These 3 enzymes showed no α‐galactosidase (EC 3.2.1.22) or β‐mannosidase (EC 3.2.1.25) activity. Thin‐layer chromatography (TLC) revealed that the products of the reaction were oligosaccharides and that free galactose and mannose were not released. These results indicate that the 3 galactomannan‐hydrolyzing enzymes are endo‐β‐mannanases (EC 3.2.1.78). Polyclonal antibodies raised against the 37.5‐kDa polypeptide cross‐reacted with the two 38‐kDa polypeptides, indicating that the 3 endo‐β‐mannanases are immunologically homologous. Activity staining and immunoblotting of native PAGE of endosperm extracts revealed that only two (38‐kDa. elution point pH 5.15 and 37.5‐kDa proteins) of the 3 forms were major endo‐β‐mannanases present in the endosperm of germinated tomato seeds.