Galactose and glucose inhibition kinetics of lactose hydrolysis by b-galactosidase in permeabilized cell mass of kluyveromyces lactis

Abstract

The effect of different galactose concentrations, glucose and of galactose mixture and glucose in the kinetics of lactose hydrolysis for bgalactosidase using permeabilized cell mass of Kluyveromyces lactis as biocatalisador was accomplished. The experiments were accomplished in room temperature (25ºC), using a suspension of 1mg/ml of permeabilized cell mass in phosphate buffer in the reaction mixture. For the galactose effect in the activity of the enzyme, they were different combined lactose concentrations (0, 1, 5, 10, 20, 30 and 40 mM) with different galactose concentrations (0, 1, 5, 10, 20, 30 and 40 mM).The reaction was accompanied in spectrophotometer DU in 510 nm for the measure of liberated glucose.The units of absorvance were transformed in glucose (mM), through a curve of appropriate calibration. During the studies of the galactose effect about the activity of the b-galactosidase, was used a statistical program of computer that aided in the calculation of the kinetic coefficients (V_máx, K_m and K_i) in a necessary and correct way and it also helped in the choice and definition of a model mechanistical mathematical applicable in the understanding of the process of lactose hydrolysis taking into consideration the inhibition form caused by the galactose presence.The inhibition competitive or no-competitive was defined by the smallest value presented by the residual sum of squares using the Marquardt iterative method of non-linear regression (NLIN) program of the statistical analysis system (SAS). The kinetic coefficients V_máx., K_m and K_i also dear for the same program, corresponded respectively to 0.291 + 0.01mM/min; 2.536 + 0.412 mM and 10.88 + 8.86 mM in the interval of trust of 95%. The inhibition type caused by the galactose presence was competitive for b-galactosidase of Kluyveromyces lactis.

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