Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha(s1)-casein

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha(s1)-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P-1 and P'(1) positions but showed a preference for hydrophobic residues at the P-2, P-3, P-4, P'(2), P'(3), and P'(4) positions.