Purification and properties of alpha-galactosidase from white-rot fungus Pleurotus florida.
alpha-Galactosidase was strongly induced in the white-rot fungus Pleurotus florida by arabinose than its natural substrates and was purified to homogeneity by acetone precipitation, ultrafiltration and DEAE-Sepharose chromatography. The enzyme was a monomeric protein with a molecular mass of approximately equal to 99 kDa, as revealed by native-PAGE and SDS-PAGE. alpha-Galactosidase was optimally active at 55 degrees C for the hydrolysis of p-nitrophenyl-alpha-galactopyranoside (PNPalphaG) and lost its 20% and 50% of original activity in 30 min at 60 degres C and 70 degrees C, respectively. The pH optimum of the enzyme was between 4.6 and 5.0. It was stable in a wide pH range (pH 4.0 to 9.0) at 55 degrees C for 2 h. The Ag+ and Hg2+ strongly inhibited the enzyme activity. Galactose, glucose, maltose and lactose also inhibited the enzyme activity, whereas N-bromosuccinimide treatment resulted in near total loss of acitivity. The Km and Vmax values of the enzyme for PNPalphaG were found to be 1.1 mM, and 77 micromol min(-1) mg(-1), respectively. alpha-Galactosidase immobilized in agar was more effective for the degradation of raffinose than in the sodium alginate. TLC results indicated its potential for the removal of raffinose and stachyose in soymilk.
Ramalingam
Department of Biotechnology, Kumaraguru College of Technology, Coimbatore 641 006, Tamil Nadu, India. gobiram@rediffmail.com
Saraswathy
N
N
Sadasivam
S
S
Subha
K
K
Poorani
N
N
D004790:Enzyme Induction; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D009598:Nitrophenylgalactosides / Q000737:chemistry; D020076:Pleurotus / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000302:isolation & purification
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011134:Polysaccharides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D000519:alpha-Galactosidase