Entomopathogenic fungi posses the ability to invade insects actively, by penetration of their cuticle. Key-role in this process plays the production and secretion of cuticle degrading enzymes such as proteases, chitinases and lipases.

C. coronatus colony 59 (causing 100% mortality of Galleria mellonella host) was grown for 30 days in liquid medium Luria Broth (LB). After that time the medium was filtered through filter paper, and mycelium was collected and homogenized by sonication. The activities of elastase, chymotrypsin, N-acetyloglucosoaminidase (NAGase), chitinase and lipase were compared in fungal homogenate and in post incubation medium. The enzymatic activities were measured by spectrophotometry and fluorimetry with the use of suitable synthetic substrates.

In the homogenate the activities of three enzymes were detected: elastase, NAGase and lipase, whereas in the post incubation medium the activities of all five examined enzymes were present.

The activity of elastase was significantly higher in the post incubation medium (5.63 ΔA/min/mg) than in the homogenate (8.6x10^-2 ΔA/min/mg). The activity of chymotrypsin was minute and detected only in the medium (0.18x10^-2 ΔF/min/mg). The activity of NAGase was similar in both, the homogenate and the incubation medium (0.68 ΔA/min/mg and 0.52 ΔA/min/mg, respectively). On the contrary, the activity of chitinase was present only in the medium (1.14x10^-2 ΔF/min/mg). The lipolytic activity in the homogenate was ten times higher than in the medium (2.30x10^-2 ΔF/min/mg and 0.26x10^-2 ΔF/min/mg, respectively).

To sum up, the activities of proteolytic enzymes were higher in the post incubation medium than in the homogenate. Release of proteases to the environment reflects adaptation to effective insects’ cuticle digestion, an essential part of mechanism allowing fungal invasive hyphae to penetrate insect cuticle. Extracellular presence of NAGase and chitinase reflects this adaptation as well. The insects’ cuticle contains 70% of proteins, while chitin together with lipids and fatty acids composes app. 30% of it. The mode of action of lipases in infection process was not intensively studied yet. In this experiment lipase activity remained mainly in intracellular compartment.

This work was financed by The Ministry of Education and Science, grant No. 2P04C 002 28

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1. N-acetylglucosaminidase activity of entomopathogenic fungus Conidiobolus coronatus: optimization of an assay