Anaerobic synthesis of extracellular proteases by the soil bacterium Bacillus sp. AM-23: Purification and characterization of the enzymes

Abstract

The anaerobic synthesis, purification and characterization of proteases from soil bacteria were investigated. We isolated a facultative anaerobe, Bacillus sp. AM-23, which synthesized extracellular proteases under anaerobic conditions. The bacterium generated two proteases (Protease I and II) on anaerobic culture, which were separated from each other by chromatography on DE-52 cellulose and purified to homogeneity by successive chromatographic techniques. The two proteases were similar to each other in molecular and catalytic properties. The molecular masses of Protease I and II were 20 kDa by gel filtration and 21 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two enzymes showed the maximal activity at pH 7.6 and were stable up to 55°C in the presence of Ca 2+. Protease I and II were stable in the range of pH 6.0–8.2 and 6.5–8.5, respectively. Because diisopropyl fluorophosphate strongly inhibited the two proteases, the enzymes were presumed to be serine proteases. The two enzymes showed high hydrolytic activities for peptides with hydrophobic amino acid residues. The results revealed that there were several differences in molecular mass and substrate specificity between these “anaerobic” proteases and the serine proteases from aerobic microorganisms and animals.