Citrate Inhibition of Aminopeptidase in Commercial Fungal Protease Preparations Used to Accelerate Cheese Ripening

Abstract The potential for inhibiting leucine-aminopeptidase activity by citrate was examined using commercial proteases (Rhozyme P-41, Corolase PN, Corolase PS) from Aspergillus oryzae . Leucine-aminopeptidase activities of the first two enzyme preparations were reduced by 20- to 25-fold in citrate-phosphate buffer, 2.2- to 2.4-fold in Tris-maleate buffer, and 10.4- to 10.6-fold in sodium acetate buffer. All buffers were .01 M and at pH 5.4 and 37°C except the acetate buffer. Corolase PS was more sensitive to buffer type and lost all activity in the citrate-phosphate buffer. Leucine-aminopeptidase activities were reduced by 96 to 98% in the presence of .01 M citrate in Tris-maleate buffer, which approximates the concentration in the moisture phase of cheese if none is metabolized by lactic bacteria. Activity was restored by adding Mg ++ , suggesting that chelation of essential divalent cations caused inactivation. However, only 6.8% of activity was restored by 5m M Mg ++ , equal to the total Mg concentration in milk. The data suggest that cheese ripening could be affected by citrate or other cation- chelating agents in cheese.