Purification and cloning of a novel serine protease from the nematode-trapping fungus Dactylellina varietas and its potential roles in infection against nematodes.
From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5 degrees C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method.
Yang
Jinkui
J
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, 650091, People's Republic of China.
Liang
Lianming
L
Zhang
Ying
Y
Li
Juan
J
Zhang
Lin
L
Ye
Fengping
F
Gan
Zhongwei
Z
Zhang
Ke-Qin
KQ
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009348:Nematoda / Q000187:drug effects*; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D005656:Fungal Proteins; D011480:Protease Inhibitors; D012697:Serine Endopeptidases