Computational protocol: Homodimeric β-Galactosidasefrom Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillusplantarum and Biochemical Characterization

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Protocol publication

[…] Steady-state kinetic data for the substrates lactose or oNPG were obtained at 30 °C in 50 mM sodium phosphate buffer, pH 6.5, with concentrations ranging from 0 to 600 mM for lactose and from 0 to 25 mM for oNPG. Furthermore, the inhibition of the hydrolytic activity of LacZ by d-glucose as well as d-galactose was investigated by adding these sugars into the assay mixture in concentrations ranging from 10 to 300 mM, and the respective inhibition constants were determined. The kinetic parameters and the inhibition constants were calculated using nonlinear regression, fitting the observed data to the Henri–Michaelis–Menten equation using SigmaPlot (SPSS, Chicago, IL).The pH dependence of β-galactosidase activity was evaluated in the range of pH 3–10 using Britton–Robinson buffer (containing 20 mM each of phosphoric, acetic, and boric acid adjusted to the required pH with NaOH). The temperature dependence of β-galactosidase activity was assessed by measuring activity in the range of 20–90 °C for 10 min. The catalytic stability of β-galactosidase was determined by incubating the enzyme in 50 mM phosphate buffer (pH 6.5) at various temperatures and by subsequent measurements of the remaining enzyme activity (A) at various time points (t) using the standard oNPG assay. Residual activities (At/A0, where At is the activity measured at time t and A0 is the initial activity) were plotted versus the incubation time. The inactivation constants kin were obtained by linear regression of ln(activity) versus time. The half-life values of thermal inactivation τ1/2 were calculated using τ1/2 = ln 2/kin.To study the effect of various cations on β-galactosidase activity, the enzyme samples were assayed at 30 °C for 10 min with 22 mM oNPG (10 mM Bis-Tris, pH 6.5, or 50 mM sodium phosphate buffer, pH 6.5) as the substrate in the presence of various cations added in final concentrations of 1–50 mM. The measured activities were compared with the activity blank of the enzyme solution determined under identical conditions but without added cations using the standard oNPG assay. Unless otherwise stated, the nontagged enzyme LacZ was used for these characterization experiments. […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002, Lactobacillus plantarum WCFS1, Lactobacillus delbrueckii subsp. bulgaricus
Chemicals Glycosides, Lactose