Purification and characterization of an extracellular acid proteinase from the ectomycorrhizal fungus Hebeloma crustuliniforme

Abstract

Hebeloma crustuliniforme produced an extracellular acid proteinase in a liquid medium containing bovine serum albumin as the sole nitrogen source. The proteinase was purified 26-fold with 20% activity recovery and was shown to have a molecular weight of 37,800 (as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 4.8 ± 0.2. The enzyme was most active at 50°C and pH 2.5 against bovine serum albumin and was stable in the absence of substrates at temperatures up to 45°C and pHs between 2.0 and 5.0. Pepstatin A, diazoacetyl-DL-norleucine methylester, metallic ions Fe2+ and Fe3+, and phenolic acids severely inhibited the enzyme activity, while antipain, leupeptin, N-α-p-tosyl-L-lysine chloromethyl ketone, and trypsin inhibitor inhibited the activity moderately. The proteinase hydrolyzed bovine serum albumin and cytochrome c rapidly compared with casein and azocasein but failed to hydrolyze any of the low-molecular-weight peptide derivatives tested.