Purification, Characterization And Gene Cloning Of A Serine Protease From Hirsutella Rhossiliensis OWVT-1

Hisutella rhossiliensis is a nematophagous fungus producing adhesive sporesthat adhere to passing verminfOrm nematodes and subsequently infect and kill the host.lt is probably an obligate parasite in natUre and can parasitize various nematodes witha high percentage in some 1ocalities. Strain OWVTl, isolated frOm a soybean cystnematode supperssive soil in Minnesota, U.S.A., has showed high potential inbiocontrol of nematode.Based on the extensive studies of subtilisin-like protease (Prl) of MetarhiziumanisoPliae, extracellullar serine protease is suggested to be a key enZyme involved inthe fimgal penetration to invertebrates. The investigation of serine protease in thenematode infected by OWVTl may help to understand the mechanism ofnematophagous fimgi as biological control agents.A 3l kDa serine protease was isolated and purified from the liquid culture of Hrhossiliensis OWVTl challenged with nematode Panagrellus redivivus. Bioassayshowed that the second-stage juvenile (J2) could be killed by the raw extract of serineprotease. Serine protease was characterized after purification with ion exchangechromatograPhy and gel filtration chromatograPhy. The T profile, T stability profile,pH profile, substrate specificity and inhibitor were tested. The optimum T of theenzyme activity is 75C. The enZyme activity can be inhibited by enzyme inhibitorSSI and CI-2. h showed that the purified enzyme degraded AAPEII(Suc-A1a-Ala-Pro-Glu-PNA ). Primers were designed by the N-arnino acid sequenceAVIDTGVEASHPEF. The gene encoding the mature peptide was cloned from thetotal RNA of H rhossiliensis OWVT1 by RTPCR. Sequence analysis of the genewas described in this paPeL The amino acid sequence, as derived from the nucleotidesequence of a cDNA clone, had high homology with other subtilisin-like serineprotease of nematogenous fimgi.