ScienceAsia 21 (1995): 183-196 |doi: 10.2306/scienceasia1513-1874.1995.21.183

PURIFICATION AND PROPERTIES OF -GALACTOSIDASE FROM HIBISCUS SABDARIFFA L. VAR. ALTISSIMA

WIPA SUGINTA^1,2 AND M.R. JISNUSON SVASTI^2,3

ABSTRACT: -Galactosidase was purified from the seeds of Thai jute (Hibiscus sabdariffa L. var. altissima) by ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-100 column chromatography, Lactosyl-Sepharose chromatography, and a second DEAE-cellulose chromatography step. The final product was purified 868-fold with a yield of 13%, and gave a single major band of molecular weight 66,000 on SDS-PAGE, similar to the native molecular weight of 55,000 determined by gel filtration. The K_m values for various substrates were as follows: 0.80 mM for p-NP--D-galactoside, 12.8 mM for o-NP-{3-D-galactoside and 84.7 mM for -lactose, and the relative Vmax values were 100% for p-NP-{3-D-galactoside, 26.7% for o-NP--D-galactoside and 9.4% for {3-lactose. Hydrolysis of p-NP-{3-D-galacroside was strongly inhibited by various substances including D-galactal, galactono-1,4-lactone, methyl--D-galactoside, methyl--D-galactoside, HgCI₂ and p-hydroxymercuribenzoate.

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1 School of Chemistry, Institute of Science, Suranaree University of Technology, Nakorn Ratchasima 30000, Thailand
2. Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
3 Laboratory of Biochemistry, Chulabhorn Research Institute, Vibhavadee Rangsit Rd., Bangkok 10210, Thailand.

Received April 11, 1995