[Characterization of proteo-, chitino- and lipolytic enzymes of parasitic fungus Conidiobolus coronatus].

Abstract

The largest problem in limitation of insect pest population is increasing resistance of them to chemical pesticides. Alternative are entomopathogens, which regulate frequency of insect pests. Among them decisive role play entomopathogenic fungi, which possess the ability to active penetration through cuticle by mechanical pressure of invasive hypha and production of proteo-, chitino- (egzo- and endochitinases) as well as lipolytic enzymes, which provide nutrients for subsequent development of fungus. Entomopathogenic soil fungus Conidiobolus coronatus (Entomophtorales) is saprophyte fungus, which demonstrates a high efficiency in the paralysis of varied insects. Although leading investigations over mechanism of insect paralysis, we still do not know, what role fungal enzymes play in insect cuticle penetration. The main aim of research was establishment of optimal conditions for elastase, N-acetylglucosaminidase (NAGase), chitobiosidase as well as lipase. Optimal reaction parameters were determined: volume of reaction mixture, volume of homogenate, working pH and the substrate concentration. Having on aim a possible use of C. coronatus in pest control, two ranges of temperatures were chosen: 20 degrees C--optimal temperature for the fungus growing and 30 degrees C--optimal temperature for the cultivation of the great wax moth larvae, Galleria mellonella, on which examinations were performed. Also kinetic constants Km and Vmax were determined. Activity of elastase and N-acetylglucosaminidase of C. coronatus was measured spectrophotometrically at 410 nm (towards N-Succinyl-Ala-Ala-Pro-Leu-p-Nitroanilide) and 405 nm (towards 4-Nitrophenyl-N-acetyl-beta-D-glucosaminide), respectively. The following optimal conditions of elastase activity were established: the volume of reaction mixture 0.5 ml, volume of homogenate 1 microl, temperature 30 degrees C, pH 8, substrate concentration 40 mM. Optimal conditions of NAGase assay: the volume of reaction mixture 0.5 ml, dose of homogenate 12.5 microl, temperature 30 degrees C, pH neutral and 6 mM substrate concentration. The activities of chitobiosidase and lipase were measured spectrofluorometrically (Ex=360 nm, Em=450 nm) towards 4-Methylumbelliferyl beta-D-N-N'-diacetylchitobioside and 4-Methylumbelliferyl oleate, respectively. Chitobiosidase showed the highest activity in dose of 30 microl in 1 ml volume of reaction mixture, at the temperature of 30 degrees C, pH 7 and substrate concentration equal to 2 mM. Lipase showed the highest catalytic activity in 1 ml volume of reaction mixture, in 30 degrees C but 50 microl of homogenate, pH 10 and 10 mM substrate concentration were needed. Higher activity investigated enzymes in 30 degrees C than 20 degrees C indicated that they can take part in pathogenesis. It was suggested that as first in perforation of coats of insects body elastase and lipase take part. Indicated of it, large thermoresistance of both enzymes (only 10.5% decrease of elastase activity at 20 degrees C and 9.4% decrease of lipase activity in comparison with maximal activity at 30 degrees C), alkalophilicity of both proteins (elastase shows the alkaline optimal pH equal to 8 at pH 9 preserves 97% activity, and at pH 10 94% activity, respectively while lipase prefers the pH 10 and at pH 8 and pH 9 enzyme keeps 57 and 60% activity, respectively) as well as lack of repression by suitable substrates. Sigmoid character of curve concerning pH influence on the activity of both enzymes, also indicates similarity between elastase and lipase. On minor part of NAGase and chitobiosidase of fungus C. coronatus in perforation of coats of host body showed high sensibility of both enzymes on hydrogen ions concentration: both enzymes prefer neutral pH, in pH 6 and 8 lose over 35% activity but subjection to substrate repression and 3-4-fold growth of activity followed only in 30 degrees C. In the course of work it was found, that rich medium (LB) stimulates growth of mycelium and production of fungal lipases. So far nobody managed to isolate chitinolytic or lipolytic enzymes from C. coronatus homogenate. The majority of fungal enzymes were isolated from post incubation filtrates. In the literature of the subject lack of data about C. coronatus NAGase, therefore in examinations also the trial of isolation NAGase from C. coronatus homogenate was undertaken. Activity of NAGase showed only first fraction, which did not separate with none of used columns. Disappointing results of purification on cation exchanger CM, weak anion exchanger DEAE, and strong anion exchanger Q were obtained as well as after fractionation tests with the use of Microcon microcolumns. In aim of NAGase molecular mass estimation, two zymograms were made with Triton X-100 and casein and with the use of fluorescent substrate 4-Methylumbelliferyl N-acetyl-beta-D-glucosaminide. Molecular mass of NAGase from C. coronatus was established on ca. 60 kDa. This is the first report describing molecular weight of NAGase from C. coronatus. Examined NAGase has different properties than known NAGases from other entomopathogenic fungi. Although its molecular weight is equal to the Metarhizium anisopliae NAGase, optimal pH for both NAGases are different: neutral in the case of C. coronatus NAGase versus acidic in the case of M. anisopliae NAGase. Knowledge of molecular mass of the C. coronatus NAGase should allow to find a new method of this enzyme isolation from C. coronatus homogenate. Thanks to developed methods of assaying activities of elastase, NAGase, chitobiosidase and lipase, real becomes the understanding of mechanism of insects paralysis through C. coronatus fungus.