Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase

Abstract

The effect of CO2" on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelting agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 ± 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium.

Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination

“…Stocks were maintained on defined minimal medium agar plates at 4°C (23). Inoculation and culture growth in a defined minimal salts (DMCo2") medium was described previously (23). In labeling experiments [355]methionine (10 ,uCi/ml) and cobalt (0.1 mM) were added when the alkaline phosphatase activity reached 0.2 U/ml.…”

“…The alkaline phosphatase (orthophosphoric monoester phosphohydrolase [alkaline optimum]) from Bacillus licheniformis MC14 exists as a membrane-associated enzyme (9) and as a soluble secreted enzyme (7,23). The membraneassociated form has been solubilized with 1 M Mg2+ and purified by two methods (9,20).…”

“…Glynn et al (7) showed that cationic detergents were as effective as Mg2e in solubilizing the membrane-associated enzyme and that the enzymatic activity was stable in concentrations as high as 1%. When cells are grown in a defined medium (23), they produce 35-fold more enzyme and the percent of membrane alkaline phosphatase extracted with 1 M Mg2+ varies from 60 to 80% depending on the membrane preparation. Tie extraction with cationic detergents is routinely above 90%o.…”

Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus

“…Two structural genes (phoA and phoB) have di¡erent developmental regulation and share over 60% identity at both the DNA and the deduced amino acid sequence levels [6]. B. subtilis APases, which were secreted e¤ciently into the culture medium, also differ from other APases in that they contain Co P and the apoenzyme is more e¡ectively reactivated with cobalt than with zinc [8]. All of these procaryotic APases had a molecular mass of 45 kDa.…”

Synergistic effect of cobalt on the induction by A‐factor of the formation of aerial mycelium and anthracyclines by a blocked mutant of Streptomyces griseus

“…Obviously, a proper supply of the growing inycelium with this trace metal during surface cultivation appears to be an important factor both with regard to the production of secondary metabolites and the activation of complex nutrients (WBIN-BERG 1978, SPENCER et al 1981.…”