Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization |
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Authors: | Cesar V Mujert Dolores A Ramirez Evelyn Mae T Mendoza |
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Institution: | Biochemistry and Genetics Laboratories, Institute of Plant Breeding, University of the Philippines at Los Ban?os, College, Laguna 3720, Philippines |
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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 Km and Vmax 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 % (NH4)2SO4 cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its Km was obtained at 6.75 × 10?4 M p-nitrophenyl α-d-galactoside while the Vmax 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. |
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Keywords: | Palmae coconut enzyme purification regulation mutant endosperm |
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