Studies on the inactivation of Lactobacillus bulgaricus beta-galactosidase by lactate

Batch cultures of Lactobacillus bulgaricus B031 displayed a transient pattern of {dollar}beta{dollar}-galactosidase activity with time. The activity increased steadily from 5 to 44 units/mg protein, peaking at the midlogarithmic phase of growth and then declined to 8 units/mg protein as the culture entered the stationary phase (A{dollar}sb{lcub}600{rcub}{dollar} of 2.0-2.5). Batch cultures grown with the pH maintained at 6.2 did not exhibit a large decline in {dollar}beta{dollar}-galactosidase activity as compared to cultures grown without pH control.; The intracellular lactate concentration during logarithmic growth increased from 0.6 M to 2.2 M, and by early stationary phase declined to 1.2 M. The intracellular pH ranged from 6.2 at the level of highest {dollar}beta{dollar}-galactosidase activity to 5.5 at a level of reduced activity. The intracellular protonated lactate concentration calculated from the intracellular lactate concentration and pH measurements was 9.7 mM at the point of highest activity, and 41 mM when {dollar}beta{dollar}-galactosidase activity was 31% of the maximum. {dollar}beta{dollar}-galactosidase activity was completely inactivated in permeabilized cells by the addition of 100 mM lactate at pH 4.0.; L. bulgaricus {dollar}beta{dollar}-galactosidase was purified and used in kinetic studies. The K{dollar}sb{lcub}rm m{rcub}{dollar} for {dollar}o{dollar}-nitrophenyl-{dollar}beta{dollar}-D-galactopyranoside was 1.33 mM, and K{dollar}sb{lcub}rm i{rcub}{dollar} values for D ({dollar}-{dollar}) lactate were K{dollar}sb{lcub}rm i{rcub}{dollar} (intercept), 3.85 mM; K{dollar}sb{lcub}rm i{rcub}{dollar} (slope), 4.81 mM. Lactate-inactivation studies using purified enzyme compared favorably with results involving permeabilized cells in that 100 mM lactate inactivated {dollar}beta{dollar}-galactosidase at pH 4.0. The degree of inactivation increased with an increase in incubation time and lactate concentration, and could not be reversed by dialysis.; {dollar}beta{dollar}-galactosidase inactivated by 100 mM lactate at pH 4.0 was quantitatively converted to a form which migrated faster on a nondenaturing polyacrylamide gel as compared to the active enzyme. Fluorescence spectroscopy analysis of purified {dollar}beta{dollar}-galactosidase revealed a change in the {dollar}lambdasb{lcub}rm max{rcub}{dollar} from 334 nm to 343 nm after lactate-inactivation, suggesting a conformational change in the enzyme. Lactate analogs such as pyruvate, acetate, 3-phosphoglycerate, and phenyllactate inactivated {dollar}beta{dollar}-galactosidase while glycerol and acetaldehyde did not.; As the {dollar}beta{dollar}-galactosidase activity declined in growing cells, the relative concentration of inactive forms increased, possibly the result of accumulation of intracellular lactate and a decline in the internal pH.