Abstract
Protease production by fourteen M. anisopliae isolates differing in geographical origin and host insect were investigated. Highest protease activity was observed during 4–8 days of culture incubation. Pr1 and Pr2 activity was investigated in various media containing different carbon and nitrogen source to evaluate the induction mechanism of these enzymes. Basal levels of Pr1 and Pr2 activity were observed in minimal medium suggesting constitutive production. Casein (1%) as an exogenous protein supplement was not able to induce significant release of Pr1 and Pr2 enzymes, whereas high levels of activity were observed in the medium containing colloidal chitin (2%) as sole carbon and nitrogen source. The pH, ammonia and oxalic acid production in in vitro conditions was also investigated and the alteration in pH for protease production was not significant in the different media used except for the medium containing casein (1%) as a supplement.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Whipps JM, Lumsden RD (2001) Commercial use of fungi as plant disease biological control agents: status and prospectus. In: Butt TM, Jackson CW, Magan N (eds) Fungi as biocontrol agents-progress problems and potential. CAB International, Wallingford, pp 23–69
Anderson SO (1979) Biochemistry of insect cuticle. Ann Rev Entomol 24:29–61
St. Leger RJ, Cooper RM, Charnley AK (1987) Production of cuticle-degrading enzymes by the Entomopathogen Metarhizium anisopliae during infection of cuticles from Colliphora vomitoria and Manduca sexta. J Gen Microbiol 133:1371–1382
St. Leger RJ, Joshi L, Bidochka MJ, Roberts DW (1995) Protein synthesis in Metarhizium anisopliae growing on host cuticle. Mycol Res 99:1034–1040
St. Leger RJ, Bidochka MJ, Roberts DW (1994) Isoforms of the cuticle degrading Pr1 protease and production of a metalloproteinase by Metarhizium anisopliae. Arch Biochem Biophys 313:1–7
St. Leger RJ, Durrands PK, Cooper RM, Charnley AK (1988) Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae. Arch Microbiol 150:413–416
St. Leger RJ, Nelson JO, Screen SE (1999) The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology 145:2691–2699
St. Leger RJ, Joshi L, Bidochka MJ, Rizzo NW (1996) Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl Environ Microbiol 62:1257–1264
Arst HN, Cove DJ (1969) Methyl ammonium resistance in Aspergillus nidulans. J Bacteriol 98:1284–1293
Cohen BL (1972) Ammonium repression of extracellular protease in Aspergillus nidulans. J Gen Microbiol 71:293–299
Kunitz M (1947) Crystalline soybean trypsin inhibitor. II. General properties. J Gen Physiol 30:291–310
St. Leger RJ, Charnley AK, Cooper RM (1987) Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Arch Biochem Biophys 253:221–232
Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Chaney AL, Marbach EP (1962) Modified reagents for the determination of urea and ammonia. Clin Chem 8:130–132
Yan ZY, Xing GM, Li ZX (2004) Quantitative determination of oxalic acid using victoria blue B based on a catalytic kinetic spectrophotometric method. Microchim Acta 144:199–205
Braga GUL, Destefano RHR, Messias CL (1999) Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures. Rev Microbiol 30:107–113
Kucera M (1971) Toxins of the entomophagous fungus Beauveria bassiana. II. Effect of nitrogen sources on formation of the toxic protease in submerged culture. J Invertebr Pathol 17:211–215
Cohen BL, Drucker M (1977) Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation. Arch Biochem Biophys 182:601–613
Bidochka MJ, Khachatourians GG (1988) Regulation of extracellular protease in the entomopathogenic fungus Beauveria bassiana. Exp Mycol 13:161–168
De Bernardis F, Muhlschlegel FA, Cassone A, Fonzi WA (1998) The pH of the host niche controls gene expression in and virulence of Candida albicans. Infect Immun 66:3317–3325
Drucker H (1972) Regulation of exocellular proteases in Neurospora crassa: induction and repression of enzyme synthesis. J Bacteriol 110:1041–1049
Dias BA, Neves PMOJ, Furlaneto-Maia L, Furlaneto MC (2008) Cuticle degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle. Braz J Microbiol 39:301–306
Acknowledgments
We acknowledge the financial support provided by Department of Science and Technology (Project No. SR/FT/L-144) and Ministry of Human Resource and Development (Project No. F. 26-11/2004 TS V). We also thank Dr. Richard A. Humber (USDA-ARS) for providing the fungal cultures.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Dhar, P., Kaur, G. Cuticle-Degrading Proteases Produced by Metarhizium anisopliae and Their Induction in Different Media. Indian J Microbiol 50, 449–455 (2010). https://doi.org/10.1007/s12088-011-0098-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12088-011-0098-1