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 geneEvspwas cloned from the nematophagous fungusEsteya vermicolawith strong virulence againstBursaphelenchus xylophilus. The full-length cDNA ofEvspcontains 2280 nucleotides with a 1656 bp ORF encoding a protein with 551 amino acids. The genomicEvspincludes two exons (396 bp and 1260 bp) separated by an intron (207 bp). There is only one copy ofEvspgene in the fungal genome. The deduced amino acids sequences ofEvspshowed highly homology with the catalytic domains in subtilisin serine proteases. Phylogenetic analyses based on the protein sequences revealed thatE. vermicolais separated from nematode-trapping fungi but close to other nematophagous and entomopathogenic fungi. The recombinant serine protease rEvsp was induced inEscherichia coliwith 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 againstB. xylophilus.