ABSTRACT
The gene encoding homodimeric ß-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-ß-D: -galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4-42°C) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg²(+), Mn²(+), and Ca²(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.
Subject(s)
Bacillus/enzymology , Galactosidases/metabolism , Lactose/metabolism , Bacillus/genetics , Cloning, Molecular , Enzyme Activators/metabolism , Enzyme Inhibitors/metabolism , Enzyme Stability , Escherichia coli/genetics , Galactose/metabolism , Galactosidases/chemistry , Galactosidases/genetics , Galactosidases/isolation & purification , Gene Expression , Glucose/metabolism , Hydrogen-Ion Concentration , Kinetics , Metals/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , TemperatureABSTRACT
A novel heterodimeric beta-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U(oNPG)/mg protein. The K(m), k(cat) and k(cat)/K(m) values for lactose and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 38 mM, 20 s(-1), 530 M(-1).s(-1) and 1.67 mM, 540 s(-1), 325 000 M(-1).s(-1), respectively. The temperature optimum of beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of beta-(1-->3) and beta-(1-->6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Gal, beta-D-Galp-(1-->3)-D-Gal, and the trisaccharides beta-D-Galp-(1-->3)-D-Lac, beta-D-Galp-(1-->6)-D-Lac.
