Preparation and properties of β-galactosidase confined in cells of Kluyveromyces sp.

Abstract

Lactase (β- d-galactosidase EC 3.2.1.23) was immobilized in yeast cells of the genus Kluyveromyces. Comparison of various solvents or polycations led to the selection of a chloroform-ethanol mixture for cell permeabilization. The lactase was confined inside the ghosts, with a minimum deactivation, by treatment with 0.4% glutaraldehyde. The combined procedure, permeabilization by chloroform-ethanol and confinement by glutaraldehyde, was applied to strain, K. lactis CBS 683, yielding a biocatalyst with an activity of 3.8 units ONP per milligram dry weight as calculated from V max. The biocatalyst could be stored in suspension at 4°C for 14 months with 20% deactivation. The K m, 4.2 m m, was comparable to the values published for the free enzyme, and the biocatalyst was found free of diffusion limitation for the hydrolysis of ONPG in the range of concentration from 0.3 to 50 m m. The pH activity profile showed a sharp optimum around pH 6.5; the storage stability in buffer at 35°C was very good for pH values above 6.1. The biocatalyst was used and recovered by centrifugation seven times without loss of activity, for the hydrolysis of 5% lactose solution in buffer at 30°C, with a conversion of about 80%.