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Soluble and immobilized beta galactosidase ec 3.2.1.23 different kinetics with p nitrophenyl beta d galactoside



Soluble and immobilized beta galactosidase ec 3.2.1.23 different kinetics with p nitrophenyl beta d galactoside



Biochimie 59(3): 247-256



A .beta.-galactosidase was purified approximately 1000-fold from the seeds of Saracen corn by successive chromatographic steps on Sephadex G75, DEAE-Sephadex A50 and hydroxyapatite. The enzyme preparation appeared homogenous with respect to the .beta.-galactosidase activity on the basis of 2 different criteria: MW and isoelectric pH. The enzyme was coupled to Sepharose 4B activated by cyanogen bromide and catalyzed the hydrolysis of several galactose containing polysaccharides; it could be a promising tool for structural studies. Some properties of the .beta.-galactosidase in solution in comparison with those of immobilized forms of the enzyme, using p-nitrophenyl-.beta.-D-galactoside as substrate are described. This substrate exibited a marked inhibitory effect towards soluble .beta.-galactosidase; this phenomenon was less marked with the insoluble forms of the enzyme when the coupling involved some primary amino-groups of the protein (.beta.-galactosidase-CNBr-Sepharose and .beta.-galactosidase-CH-Sepharose). The insertion of a spacer arm between the enzyme and the matrix was also important. When covalent attachment was accomplished through carboxyl groups (.beta.-galactosidase-AH-Sepharose) no differences were noted in the kinetics with p-nitrophenyl-.beta.-D-galactoside. Apparent Km values could be calculated from the slope of the linear part of the curve (Lineweaver plot), which corresponded to the low concentration regions. The Km values of the bound enzymes are slightly higher (2.4-5mM) than that of the free enzyme (1.8 mM), presumably as a result of diffusional limitations. The optimum pH of each form was approximately the same (pH 3.2-3.4). .beta.-galactosidase was bound to concanavaline A-Sepharose via the carbohydrate moiety and formed an enzymically active complex. The Lineweaver plot showed a change in the slope, with a downward inflexion at high substrate concentrations; the Km measured considerably higher (12.5 mM) than the value of soluble enzyme. As a tentative interpretation, the assumption of the existence of 2 kinds of binding sites in the .beta.-galactosidase molecule, with different affinities for p-nitrophenyl-.beta.-D-galactoside was retained. At low substrate concentrations the substrate molecules preferentially bind to the catalytic site. At higher substrate concentrations a 2nd substrate molecule binds to the Michaelis-Menten complex, on a 2nd (and inhibitory) site. It may be postulated that binding of the .beta.-galactosidase molecules via the amino groups, tended to hinder the access to the 2nd (and inhibitory) site for p-nitrophenyl-.beta.-D-galactoside.

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