Alkaline protease from Aspergillus oryzae: Esterase activity

Abstract

Kinetic data are presented for the hydrolysis of N-substituted amino acid esters by aspergillopeptidase B, an alkaline protease from Aspergillus oryzae. The rate of hydrolysis was followed with a pH-stat at pH 8.5, 25°C and ionic strength 0.1. The overall susceptibility of N-acetyl amino acid esters to hydrolysis decreases in the order of Phe, Tyr > Trp > Met > Leu > Lys > His ⪢ Val, Gly. The N-benzoyl derivatives of His, Lys and Arg methyl esters are approximately thirty times more reactive than the corresponding acetyl derivates. Amino acid esters with free α-amino groups are unable to serve as substrates. These data clearly suggest that before an amino acid ester can serve as a substrate for the alkaline protease of A. oryzae (a) the α-amino group must be blocked and (b) an aromatic group is required either in the amino acid side chain or as the N-terminal blocking group. It further suggests that the binding of substrate to the enzyme active site probably takes place through hydrophobic binding. The hydrolysis of N- benzoyl- l-arginine ethyl ester by aspergillopeptidase B showed a sigmoid pH-velocity profile which could be fitted with a calculated titration curve for a single group with a p K′ of 6.42 at 20°C and 6.02 at 35°C, giving a ΔH i of 9.1 kcal per mole at 0°C. These data suggest that the enzyme may require a non-protonated imidazole group for activity.