Substrate specificity of alkaline protease from alkaliphilic feather-degrading Nesterenkonia sp. AL20

The substrate specificity of an alkaline protease, produced by Nesterenkonia sp. AL20 grown on chicken feather as the nutrient source, was assessed using oxidized insulin B-chain and derivatized peptide substrates. The initial cleavage of the insulin chain was determined to be at Tyr16-Leu17 and Tyr26-Thr27, followed by Gln4-His5, Phe25-Tyr26 and Leu15-Tyr16 bonds. An additional cleavage site at Ser9-His10 was found during hydrolysis for a long time. Among the peptide substrates, the enzyme exhibited activity mainly with tetrapeptide substrates with hydrophobic residues located at P1 site in the order Tyr > Phe > Leu. Decreasing the size of the peptide resulted in a drastic reduction of activity, suggesting the enzyme to possess relatively narrow substrate specificity in comparison to several other serine proteases. AL20 protease showed good activity towards casein and hemoglobin as substrates, low activity with keratin azure, and poor activity with elastin–orcein.