A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral with a molecular mass of 45.6 kDa. The optimum activity was at 45 degrees C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn(3), Leu(6), His(10)-Leu(11), Ala(14), Glu(21), after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P-1 position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K-m values of 0.858 and 2.363 mM for N-(3[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-G1y-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasem, azocoll, keratin azure and wool. (c) 2007 Elsevier Inc. All rights reserved.