Coconut α- d-galactosidase isoenzymes: isolation, purification and characterization

Abstract

α- d-Galactosidases (α- d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α- d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α- d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K m and V max of α- d-galactosidase A were obtained at 3.46 × 10 −4M and 1.38 × 10 −3 M p-nitrophenyl α-< d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α- d-galactosidase was localized in the 40–70 % (NH 4) 2SO 4 cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K m was obtained at 6.75 × 10 −4 M p-nitrophenyl α- d-galactoside while the V max was noted at 5.28 × 10 −3 M p-nitrophenyl α- d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α- d-galactosidase activity in makapuno is discussed.