When grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular
proteases hydrolysing the
chromogenic substrate Azocoll. The
protease activity was separated into two fractions (FI and FII) using
anion-exchange chromatography. In bioassays,
protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the
enzyme(s) might be involved in the
infection of nematodes. A
protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified
enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PII immobilized P. redivivus in bioassays and hydrolysed
proteins of the purified cuticle. The
enzyme hydrolysed several
protein substrates including
casein,
bovine serum albumin and
gelatin, but not native
collagen. Examination of substrate specificity with synthetic
peptides showed that PII readily hydrolysed tripeptides with aromatic or
basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-
Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-
Gly-Phe-NA). Mono-
peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several
serine protease inhibitors including
phenylmethylsulfonyl fluoride (PMSF),
chymostatin and
antipain. The
protease was N-terminally blocked, but the sequence of one internal
peptide showed a high homology with a region containing the active site
histidine residue of the
subtilisin family of
serine proteases.