No abstract

The gene encoding homodimeric β-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-β-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.

No abstract

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.