Purification and Cloning of an Extracellular Serine Protease from the Nematode-Trapping Fungus Monacrosporium cystosporium

  • Yang, Jin-Kui (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University) ;
  • Ye, Feng-Ping (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University) ;
  • Mi, Qi-Li (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University) ;
  • Tang, Song-Qing (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University) ;
  • Li, Juan (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University) ;
  • Zhang, Ke-Qin (Laboratory for Conservation and Utilization of Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University)
  • 발행 : 2008.05.31

초록

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 $56^{\circ}C$ (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 ($K_m$) and $V_max$ for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were $1.67{\times}10^{-4}\;M$ and 0.6071 $OD_{410}$ 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 playa 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 Mcl showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mcl possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.

키워드

참고문헌

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