Handbook Part 2 - International Mycological Association

PS4-395-0086
Purification and characterization of an extracellular serine protease serving as the potential pathogenic
factor in Clonostachys rosea
Xiaowei Huang, Jun Li, Keqin Zhang
Laboratory for conservation and utilization of Bio-resources, Yunnan University, Kunming, Yunnan Province, China
The fungus Clonostachys rosea (syn. Gliocladium roseum) is a common saprophyte in the soil and it has been reported
to be toxic to nematodes such as Bursaphelenchus xylophilus. Since the nematode cuticle is a flexible exoskeleton
composed primarily of proteins, the involvement of protease in the infection should be reasonable. In our study, an
extracellular protease (PrC) was purified to homogeneity from an isolate of C. rosea by the methods of precipitation
with ammonium sulfate, hydrophobic interaction chromatography and anion-exchange chromatography
successively. The nematicidal activity of PrC was confirmed the fact that 80±5% of nematodes could be immobilized
and degraded after treating with PrC for 48h. The purified protease had a molecular mass of 33kDa and displayed
optimal activity at 60 ?, pH 9-10. Furthermore, the protease PrC hydrolyzed a broad range of substrates including
casein, gelatin and the purified nematode cuticle. The sequencing of N-terminal amino acid residues revealed the
protease of PrC should belong to serine protease family, which was consistent with the analysis of the protease
inhibitors. The multiple sequences alignments demonstrated that the purified PrC shared 32-80% homology with the
known cuticle-degrading protease from nematophagous or entomopathogenic fungi Vercillium lecanii, Trichoderma
harizianum, Metarhizium anisopliae, Athobotrys. oligospora, Pochonia chamydosporia, Lecanicillium psalliotae,
Paecilomyces lilacinus, implying PrC play a role in the infection against nematodes. Compared with the other serine
proteases, it was worth noticing that either biochemical characterizations or amino acid sequences of PrC showed
high similarity to VCP1, P32 pSP3, and Ver112, which were isolated from egg-parasitic or endoparasitic fungi P.
chlamydosporia, P. suchlasporia, P. lilacinus and L. psalliotae, respectively. However, PII and Aoz1, another tow serine
proteases isolated from nematode-trapping fungus A. oligospora, had the lower pI and higher molecular mass in
biochemical properties. The homologeous analysis based on amino acid sequences suggested that all of the serine
proteases mentioned above should be divided by two main clusters; but PII and Aoz1, by contrast with those from
egg-parasitic or endoparasitic fungi including PrC, were placed in the different subfamily.
PS4-396-0087
Water availability and metabolomic profiles of Epicoccum nigrum and Sarophorum palmicola grown in
solid substrate fermentation systems
N Magan, D Aldred, J Penn
Cranfield University, Bedford, United Kingdom
There has been interest in using environmental screening procedures for enhancing the metabolomic production
profiles by fungi which are of pharmaceutical interest. Surprisingly few attempts have been made to examine the
ecological context of isolated species to improve the potential for maximising metabolomic profiles and specific
metabolites based on a more effective eco-environmental approach.
This study examined two ecologically distinct species, Epicoccum nigrum and Sarophorum palmicola, in solid
substrate fermentation systems over a range of water availability conditions (water activity, aw). Total secondary
metabolite profiles were obtained by HPLC + diode array detection after 14 days incubation on cereal-based
substrates in relation to four aw treatments.
Temporal studies (24 d, E. nigrum and 18 d, S. palmicola) showed that metabolite production profiles varied markedly
between the two fungi. For E. nigrum, metabolite production generally increased with reduction in aw, and was
optimal in the range 0.99 – 0.97. In contrast to this, for S. palmicola, metabolite production was restricted to the highest
aw level used, 0.998, and declined to zero at 0.99 aw. Statistical analysis revealed that time, aw and their interactions
were significant in all cases (p