The major problem in pest control is increasing insect resistance to traditional pesticides. Naturally occurring entomopathogens are important regulatory factors of insect populations, therefore several species of entomopathogenic fungi are employed as biological control agents. Entomopathogenic soil fungus Conidiobolus coronatus (Entomophtorales) also demonstrates high efficiency in insect paralysis.

Penetration of insect cuticle by fungal pathogens is achieved through a combination of enzymatic and mechanical mechanisms. Entomopathogenic fungi produce a range of cuticle degrading enzymes including proteases, chitinases (exo- and endochitinases) as well as lipases that degrade major components of the cuticle and provide nutrients for the fungus.

N-acetylglucosaminidase (NAGase) belongs to exochitinases and cleaves olygomers of insect chitin to N-acetylglucosamine monomers. Assuming that chitinases play essential role in fungal virulence we decided to optimize reaction conditions for NAGase in order to determine the role of this enzyme in C. coronatus virulence.

NAGase activity in mycelial homogenate of C. coronatus was assayed spectrophotometrically at 405 nm with 4-Nitrophenyl N-acetyl-β-D-glucosaminide (pNAG, Sigma) as a substrate. Effect of pH on enzyme activity and stability was measured under 10mM Tris-HCl buffer at pH range 4-8. The stability of the enzyme at various pH levels was examined after pre-incubation for 30 minutes in a solution of tested pH at 20^oC (temperature optimal for growing C. coronatus) and 30^oC (temperature optimal for insect host, Galleria mellonella). Effect of temperature was assayed at 20-80^oC range. The thermal stability of the NAGase was tested after 30 minutes incubation at the temperature ranging from 20 to 60^oC. Effect of substrate concentration was examined with pNAG at various concentrations (6x10^-1 – 0.375x10^-1 mM).

The optimal conditions for assaying the C. coronatus NAGase are as follows: amount of fungal homogenat 4.25x10^-2 mg of fungal protein/ml, substrate concentration 3x10^-1 mM, reaction time 15 minutes. The pH optimal for NAGase activity was 7.0. Enzyme was more stable at pH 7.0 in the temperature optimal to cultivate the insect host G. mellonella (30^oC, 2.9x10^-2 dA/min) than in the temperature optimal to propagate C. coronatus (20^oC, 2.6x10^-2 dA/min). The optimal temperature for enzymatic activity was 60^oC. Activity of enzyme incubated for 30 minutes at 60^oC remained stable (100% activity), while incubation at 30 and 20^oC resulted in the 11 and 29% loss of NAGase activity, respectively.

This work was supported by the Ministry of Science and Higher Education, grant No. N303 027 31/0837

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1. Proteo-, chitino- and lipolytic enzymes production by entomopathogenic fungus Conidiobolus coronatus