Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium.
An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and 56 degrees (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (Km) and Vmax for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were 1.67x10-4 M and 0.6071 OD410 per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might play a role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mc1 showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mc1 possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.
Yang
Jin-Kui
JK
Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, China.
Ye
Feng-Ping
FP
Mi
Qi-Li
QL
Tang
Song-Qing
SQ
Li
Juan
J
Zhang
Ke-Qin
KQ
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009348:Nematoda / Q000382:microbiology*; D010802:Phylogeny; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D012697:Serine Endopeptidases