Cloning of a serine protease gene from the nematophagous fungus Esteya vermicola and expressed activity of the recombinant enzyme against Bursaphelenchus xylophilus

A serine protease gene Evsp was cloned from the nematophagous fungus Esteya vermicola with strong virulence against Bursaphelenchus xylophilus . The full-length cDNA of Evsp contains 2280 nucleotides with a 1656 bp ORF encoding a protein with 551 amino acids. The genomic Evsp includes two exons (396 bp and 1260 bp) separated by an intron (207 bp). There is only one copy of Evsp gene in the fungal genome. The deduced amino acids sequences of Evsp showed highly homology with the catalytic domains in subtilisin serine proteases. Phylogenetic analyses based on the protein sequences revealed that E. vermicola is separated from nematode-trapping fungi but close to other nematophagous and entomopathogenic fungi. The recombinant serine protease rEvsp was induced in Escherichia coli with expression vector pET28a(+). The tests of protease and nematicidal activities for the purified and refolded rEvsp indicated it is possibly involved in the fungal infection process against B. xylophilus .