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

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 freeliving nematode Panagrelhs redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PI1 was purified from FII to apparent homogeneity by hydrophobic interaction and sizeexclusion 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. PI1 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 PI1 readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-~-phenylalanyl-~-valyl-~-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succi n y I-g l ycy I-g l ycy l-~-phen y la lani ne-4-n itroani I ide (Suc-G I y-G I y-Phe-N A).Mono-peptides were hydrolysed a t considerably slower rates. PI1 had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PI1 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.

Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans , the causative agent of white-nose syndrome

“…Nematophagous fungi use a variety of methods to capture and kill nematodes, which are subsequently digested by the fungi (26). The subtilisin-type peptidases promote penetration and digestion of nematode cuticles, and are key enzymes in nematophagous species for killing of their prey (26,31,(40)(41)(42). A subtilisin-like serine peptidase was also recently identified in Batrachochytrium dendrobatidis, a chytrid fungus responsible for a global decline in amphibian species.…”

Characterization and Production of Thermostable and Acid-stable Extracellular Fibrinolytic Enzymes from Cordyceps militaris

“…Extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis was 31 kDa . The molecular weights were determined for the proteaes from A. oligospora (35.2 kDa) (Tunlid et al, 1994), A. frondosa (20 kDa), A. mellea (32 kDa), D. varietas (30 kDa) , Perenniporia fraxina mycelia (42 kDa) (Kim et al, 2008) and Fusarium sp CPCC480097 (28 kDa) (Wu et al, 2009). As a result, it is obvious that entomopathogenic fungi and nematophagous fungi produce various proteolytic enzymes to degrade insect cuticle.…”

Improving the Pathogenicity of a Nematode-Trapping Fungus by Genetic Engineering of a Subtilisin with Nematotoxic Activity

“…(ATCC 24927) was maintained on CMA7-containing cornmeal agar (Difco) supplemented with 2 g of KH 2 HPO 4 per liter. Trap-containing mycelium was grown in a liquid medium containing 0.01% soya peptone (neutralized; Oxoid) supplemented with 0.05 g of phenylalanine and 0.05 g of valine per liter (27). Two auxotrophic pyrG mutant strains of A. niger were used for heterologous expression of PII: AB1.13pcl3FOA17 (designated AB1.13pclA), which is deficient in both aspergillopepsin A and aspergillopepsin B (due to a regulatory mutation, prt-13) (14) and with the pclA gene deleted (processing enzyme) (P. J. Punt, unpublished data), and A. niger AB1.18, which is mutated in the pepA gene encoding aspergillopep-sin A (14).…”

“…We have identified two putative pathogenicity factors in the common nematophagous fungus Arthrobotrys oligospora, a carbohydrate binding protein (lectin) and an extracellular serine protease designated PII (1,20,26). PII belongs to the subtilisin family of serine proteases (27). Extracellular subtilisins have also been isolated from other species of nematophagous fungi (3,13,21).…”