title: Characterization of two cold-sensitive mutants of the beta-galactosidase from Lactobacillus delbruckii subsp. bulgaricus.
abstract: Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.
authors.affiliation: Genencor Int., Inc., South San Francisco, California 94080.
authors.forename: R M
authors.initials: RM
authors.lastname: Adams
authors.forename: S
authors.initials: S
authors.lastname: Yoast
authors.forename: S E
authors.initials: SE
authors.lastname: Mainzer
authors.forename: K
authors.initials: K
authors.lastname: Moon
authors.forename: A L
authors.initials: AL
authors.lastname: Palombella
authors.forename: D A
authors.initials: DA
authors.lastname: Estell
authors.forename: S D
authors.initials: SD
authors.lastname: Power
authors.forename: B F
authors.initials: BF
authors.lastname: Schmidt
mesh_terms: D003080:Cold Temperature; D006868:Hydrolysis; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D007785:Lactose / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase; D008274:Magnesium; D007785:Lactose