Purification and properties of an α-d-galactoside galactohydrolase from the seeds of Trifolium repens (white clover)

Abstract

Five .alpha.-D-galactosidases (.alpha.-D-galactoside galactohydrolase; EC 3.2.1.22) were identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of T. repens (white clover). .alpha.-Galactosidase I was purified to homogeneity with an approximate 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH 3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose [epichlorohydrintriethanolamine-alkali treated cellulose] column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the .alpha.-D-galactosidic activity. The pH optimum was between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, and it was found that .alpha.-galactosidase I exhibits 2 enzymic activities, namely .alpha.-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick and Smith and by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis the MW was estimated to be 43,000 and 41,000, respectively. Apparently, .alpha.-galactosidase I is a monomeric protein and both enzymic activities associated with the enzyme reside on the same polypeptide chain.