CHARACTERIZATION OF 2 COLD-SENSITIVE MUTANTS OF THE BETA-GALACTOSIDASE FROM LACTOBACILLUS-DELBRUCKII SUBSP BULGARICUS

Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobac illus delbruckii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetical ly in order to determine the enzymatic consequences of these mutations . The kinetic parameters K-m and V-max on the synthetic substrate o-ni trophenyl-beta-D-galactopyranoside have been determined over a tempera ture range of 11-45 degrees C. Only the V-max of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The V-max of the L317F variant is reduced proportionat ely at all temperatures compared to the wild-type enzyme while the val ue of V,, for the P429S mutant deviates from wild-type only at lower t emperatures (in 2 mM Mg2+). This temperature-dependent effect on the V -max of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is al tered in the P429S variant such that its dissociation is increased by lowering the temperature.