Cell permeabilization of Kluyveromyces and Saccharomyces species to obtain potential biocatalysts for lactose hydrolysis

Yeast’s beta-galactosidase is an intracellular enzyme, through which it is possible to determine in vivo its activity as a biocatalyst in the lactose hydrolysis. Permeabilization process was used for transforming the microorganisms cells into biocatalysts with an enhanced enzyme activity. The potential application of this enzyme technology in industrial process depends mainly on the enzyme activity. Beta-galactosidase enzyme that hydrolyzes lactose, for instance, is largely dependent on the reaction time and its stability under different physical conditions, such as pH, temperature and enzyme concentration. The objective of this study was to optimize the cellular permeabilization process of Kluyveromyces marxianus CCT 3172 and Saccharomyces fragilis CCT 7586 cultured in cheese whey for lactose hydrolysis. Box-Behnken design was carried out for cell permeabilization with three independent variables, ethanol concentration, permeabilization time and temperature. The best permeability conditions for K. marxianus CCT 3172 were 27% (v v-1) ethanol, 3 min at 20ºC, with specific enzymatic activity of 0.98 U mg-1. For S. fragilis CCT 7586, a specific enzymatic activity of 1.31 U mg-1 was achieved using 45% (v v-1) of ethanol, 17 min. of reaction under 17ºC. Thus, it was concluded that cellular permeabilization with ethanol is an efficient process to determine beta-galactosidase activity.