raffinose and 4-nitrophenylgalactoside

alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from watermelon was covalently immobilized on chitin. The immobilized alpha-galactosidase exhibited an activity of 0.61 U per g of carrier and an activity yield of 67%. The properties of free and immobilized alpha-galactosidase were also searched and compared. The results showed that, optimum conditions for activity were not affected by immobilization. The optimum pH and temperature for free and immobilized enzyme found as pH 6.0 and 65 degress C, respectively. Compared with the free enzyme, the temperature and pH stabilities of the immobilized enzyme were similar. Both the enzymes were stable between pH 2-10 and below 50 degrees C. The Km values for free and immobilized enzyme were determined using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) and raffinose as substrates. Operational stability of the immobilized enzyme was investigated by using both substrates. The operational half-life (t 1/2) was calculated as 34 h for PNPG and 28 h for raffinose. The immobilized alpha-galactosidase was also utilized in the hydrolysis of raffinose. The immobilization procedure on chitin was cheap and also easy to carry out, and the immobilized enzyme had good properties that the potential for practical application is considerable.

Topics: alpha-Galactosidase; Chitin; Citrullus; Enzymes, Immobilized; Hydrogen-Ion Concentration; Hydrolysis; Imidazoles; Nitrophenylgalactosides; Photoaffinity Labels; Raffinose; Sulfhydryl Reagents; Temperature

To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes. Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity. The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations. These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter. The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease.

Topics: Amino Acid Substitution; Binding Sites; Biological Transport; Carbohydrate Metabolism; Carbohydrates; Cations, Monovalent; Escherichia coli; Kinetics; Melibiose; Membrane Transport Proteins; Methylgalactosides; Models, Molecular; Mutation; Nitrophenylgalactosides; Osmolar Concentration; Protein Structure, Secondary; Protein Structure, Tertiary; Proteolipids; Raffinose; Sodium; Spectrometry, Fluorescence; Substrate Specificity; Symporters; Thiogalactosides; Tryptophan; Tyrosine