Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora

• Published in: Microbiology (Society for General Microbiology) Vol. 140; no. 7; pp. 1687 - 1695
• Main Authors: TUNLID, ANDERS, ROSEN, STEFAN, EK, BO, RASK, LARS
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.07.1994; Society for General Microbiology
• Subjects: Amino Acid Sequence; Animals; Arthrobotrys oligospora; Biologi; Biological and medical sciences; Biological Sciences; Cuticle-degrading enzyme; Extracellular serine protease; Fundamental and applied biological sciences. Psychology; Glycosylation; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Helminth Proteins - metabolism; Isoelectric Point; Microbiology; Mikrobiologi; Mitosporic Fungi - enzymology; Molecular Sequence Data; Molecular Weight; Mycology; Natural Sciences; Naturvetenskap; Nematophagous fungus; Rhabditida - drug effects; Sequence Homology, Amino Acid; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Serine Endopeptidases - secretion; Substrate Specificity; Subtilisin; Purification; Serine endopeptidases; Enzyme; Biological control; Enzyme inhibitor; Extracellular; Predatory fungi; Fungi; Enzymatic activity; Substrate specificity; Hydrolases; Fungi Imperfecti; Proteinases; Nematophagous; Physicochemical properties; Thallophyta
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-140-7-1687

1 Department of Microbial Ecology, Lund University, Ecology Building, Helgonavägen 5, S-22362 Lund, Sweden 2 Department of Cell Research, Swedish University of Agricultural Science, Box 7055, 5-75007 Uppsala, Sweden Author for correspondence: Anders Tunlid. Tel: + 46 46109614. Fax: +46 46104158. e-mail: Anders.Tunlid{at}mbiockol.lu.sc . ABSTRACT 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. Keywords: Arthrobotrys oligospora. extracellular serine protease, nematophagous fungus, subtilisin, cuticle-degrading enzyme