Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis.

Sporulating cells of Bacillus brevis ATCC 9999 produced a high level of an intracellular serine protease when grown in nutrient medium. The protease activity in the crude extracts of this strain appeared at hour 5 (t5) after the end of exponential growth and increased gradually during sporulation, reaching a maximum at t12 to t13. The enzyme isolated in a partially purified state showed a pH optimum between 7.3 and 9.0 and had an apparent molecular weight of about 60,000. The activity was completely inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, EDTA, and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The protease possessed a high activity for azocoll and low activities for azocasein and 14C-labeled hemoglobin. It cleaved the cyclic decapeptide gramicidin S specifically at the peptide linkage between valine and ornithine and hydrolyzed the oxidized insulin B-chain mainly at peptide bonds 4-5 (Glu-His), 6-7 (Leu-CysSO3H), and 15-16 (Leu-Tyr). No catalysis of bond cleavage by the enzyme on a variety of small peptides or esters was detected. Unlike other Bacillus species, B. brevis ATCC 9999 grown in nutrient medium excreted no extracellular proteases.