PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR SERINE-PROTEASEFROM THE NEMATODE-TRAPPING FUNGUS ARTHROBOTRYS OLIGOSPORA

When grown in liquid cultures allowing the formation of nematode traps , the fungus Arthrobotrys oligospora produced two extracellular protea ses hydrolysing the chromogenic substrate Azocoll. The protease activi ty was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized t he free-living nematode Panagrellus redivivus indicating that the enzy me(s) might be involved in the infection of nematodes. A protease desi gnated PII was purified from FII to apparent homogeneity by hydrophobi c interaction and size-exclusion chromatography, resulting in an appro ximately 15-fold increase in specific activity. The purified enzyme wa s glycosylated, had a molecular mass of approximately 35 kDa (gel filt ration) and an isoelectric point of ph 4.6. PII immobilized P. rediviv us in bioassays and hydrolysed proteins of the purified cuticle. The e nzyme hydrolysed several protein substrates including casein, bovine s erum albumin and gelatin, but not native collagen. Examination of subs trate specificity with synthetic peptides showed that PII readily hydr olysed tripeptides with aromatic or basic amino acids including oyl-L- phenylalanyl-L-valyl-L-argine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and c inyl-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 autodegr adation. PII was inhibited by several serine protease inhibitors inclu ding phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. T he protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active si te histidine residue of the subtilisin family of serine proteases.