PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR ASPARATE PROTEASE FROM PHYCOMYCES-BLAKESLEEANUS

De Vicente, J. I., De Arriaga, D., Del Valle, P., Soler, J., and Eslav a, A. P. 1996. Purification and Characterization of an Extracellular A spartate Protease from Phycomyces blakesleeanus. Fungal Genetics and B iology 20, 115-124. An acid protease has been found in the culture bro th of Phycomyces blakesleeanus growing under standard conditions, It h as been induced up to 70-fold with several complex growth media and th e enzyme has been purified to homogeneity and characterized, The molec ular mass of the native enzyme was estimated by gel filtration to be 4 0 kDa, The acid protease of Phycomyces migrated as a single band on so dium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa, The glycoprotein nature for the acid p rotease was deduced from its binding to a concanavalin A-Sepharose 4B column, The carbohydrate moiety is composed of mannose and rhamnose, I ts amino acid composition was determined, and its isoelectric point wa s estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum te mperature was 70 degrees C, using hemoglobin as a substrate. The enzym e showed thermal stability between 37 and 50 degrees C, The thermodyna mic parameters for hemoglobin hydrolysis and thermal inactivation were calculated, With Lys-Pro-Ile-Glu-Phe-Phe(4-NO2)-Arg-Leu as the substr ate, the K-m, k(cat), and V-max values were 8.78 mu M, 1.25 s(-1), and 2.12 mu mol min(-1) mg(-1), respectively. The protease was insensitiv e to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide , iodoacetamide, ethylenediaminetetraacetate, [ethylenebis(oxyethylene nitrilo)]tetraacetic acid, and trypsin inhibitor, However, pepstatin A established a strong competitive inhibition against it, with a K-i va lue of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase. (C) 1996 Academic Press, Inc.