Protease Production During Growth And Autolysis Of Submerged Metarhizium Anisopliae Cultures

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.302107113Alfonso, C., Martinez, M.J., Reyes, F., Degradation of chitosan in the autolysis of mucorales (1991) Mycol. Res., 95, pp. 217-219Braga, G.U.L., Messias, C.L., Vencovsky, R., Estimates of genetic parameters related to protease production by Metarhizium anisopliae (1994) J. Invertebr. Pathol., 64, pp. 6-12Campbell, R.K., Perring, T.M., Barnes, G.L., Eikenbary, R.D., Gentry, C.R., Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae on media containing various amino acids (1978) J. Invertebr. Pathol., 31, pp. 289-295Goettel, M.S., St. Leger, R.J., Rizzo, N.W., Staples, R.C., Roberts, D.W., Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca sexta) cuticle (1989) J. Gen. Microbiol., 135, pp. 2233-2239Kalisz, H.M., Moore, D., Wood, D.A., Protein utilization by basidiomycete fungi (1986) Trans. Br. Mycol. Soc., 86, pp. 519-525Kucera, M., Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae (1980) J. Invertebr. Pathol., 35, pp. 304-310Kucera, M., The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture (1981) J. Invertebr. Pathol., 38, pp. 33-38Kucera, M., Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease (1984) J. Invertebr. Pathol., 43, pp. 190-196. , 1984Lahoz, R., Ibeas, J.G., The autolysis of Aspergillus flavus in an alkaline medium (1968) J. Gen. Microbiol., 53, pp. 101-108Lahoz, R., Miralles, M., Influence of the level of the carbon source on the autolysis of Aspergillus niger (1970) J. Gen. Microbiol., 60, pp. 271-276Lahoz, R., Reyes, F., Martinez, M.J., Jimeno, L., Effect of the pH on the degree of autolysis of Aspergillus niger (1979) Can. J. Bot., 57, pp. 1901-1903Nelson, N., A photometric adaptation of the Somogy method for the determination of glucose (1944) J. Biol. Chem., 153, pp. 375-380Oliveira, N.T., Messias, C.L., Variability of strains of Metarhizium anisopliae Var. anisopliae (Metsch.) Sorokin in terms of protein content and spore germination (1996) Rev. Microbiol., 27, pp. 268-272Pontecorvo, G., Roper, J.A., Hemmons, L.M., McDonald, K.F., Bufton, A.W.J., The genetics of Aspergillus nidulans (1953) Adv. Gen., 5, pp. 141-238Reyes, F., Calatayud, J., Martinez, M.J., Chitinolytic activity in the autolysis of Aspergilus nidulans (1988) FEMS Microbiol. Lett., 49, pp. 239-243Reyes, F., Lahoz, R., Variation in lysis of walls of Sclerotinia fructigena with age of culture (1977) J. Gen. Microbiol., 98, pp. 607-610Reyes, F., Lahoz, R., Cornago, P., Autolysis of Neurospora crassa in different culture conditions and release of ß-N-Acetyl-Glucosaminidase and chitinase (1977) Trans. Br. Mycol. Soc., 68, pp. 357-361Reyes, F., Lahoz, R., Moreno, A.V., Synthesis of 1,3-ß-glucanase and ß-N-acetylglucosaminidase during autolysis of Neurospora crassa (1981) J. Gen. Microbiol., 126, pp. 347-353Rosato, Y.B., Messias, C.L., Azevedo, J.L., Production of extracellular enzymes by isolates of Metarhizium anisopliae (1981) J. Invertebr. Pathol., pp. 1-3Santamaria, F., Reyes, F., Proteases produced during autolysis of filamentous fungi (1988) Trans. Br. Mycol. Soc., 91, pp. 217-220Söderhäll, K., Unestam, T., Properties of extracellular enzymes from Amphanomyces astaci and their relevance in the penetration process of crayfish cuticle (1975) Physiol. Plant., 35, pp. 140-146St. Leger, R.J., Charnley, A.K., Cooper, R.M., Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae (1987) Arch. Biochem. Biophys., 253, pp. 221-232St. Leger, R.J., Cooper, R.M., Charnley, A.K., Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Colliphora vomitoria and Manduca sexta (1987) J. Gen. Microbiol., 133, pp. 1371-138

Protease production during growth and autolysis of submerged Metarhizium anisopliae cultures Produção de protease durante o crescimento e análise de culturas submersas de Metarhizium anisopliae

The growth and autolysis of two strains of the entomopathogenic deuteromycete fungus Metarhizium anisopliae var. anisopliae were evaluated in medium containing casein or glucose as carbon source. Parameters such as economic coefficient and degree of autolysis were determined for each strain. Protease production was determined throughout the growth and autolysis phases of the cultures on medium under conditions of protease induction (in the presence of casein as sole source of carbon and nitrogen). The fungus was shown to utilize casein as a carbon/energy source in a more efficient manner than glucose. The autolysis shown by the strains was intense under both types of growth conditions, reaching up to 62.7% of the dry mass produced and started soon after the depletion of the exogenous carbon source. The relationship between the proteolytic activities of the two strains evaluated varied significantly (a maximum of 19.78 on the 5th day and a minimum of 2.03 on the 16th day of growth) during the various growth and autolysis phases, clearly showing that the difference between the growth curves and the difference in the kinetics of enzyme production may decisively affect the process of strain selection for protease production.<br>O crescimento e a autólise de duas linhagens do deuteromiceto entomopatogênico Metarhizium anisopliae var. anisopliae foram avaliados em meio contendo caseína ou glicose como fonte de carbono. Foram determinados parâmetros como o coeficiente econômico e o grau de autólise apresentado pelas linhagens. A produção de protease foi determinada durante todas as fases do crescimento e da autólise das culturas, em meio indutor da produção de proteases (meio contendo caseína como única fonte de carbono e de nitrogênio). Pôde-se verificar que o fungo foi capaz de utilizar a caseína como fonte de carbono/energia de maneira mais eficiente do que a glicose. A autólise apresentada pelas linhagens foi intensa em ambas as condições de crescimento, alcançando até 62,7% da massa seca produzida, e se iniciou logo após o esgotamento da fonte exógena de carbono. A relação entre as atividades proteolíticas apresentadas pelas duas linhagens avaliadas variou de maneira significativa (máxima de 19,78 no 5° dia e mínima de 1,55 no 16° dia de incubação) durante as diversas fases do crescimento e da autólise, deixando claro que as diferenças verificadas entre as curvas de crescimento e entre as cinéticas da produção das enzimas podem influenciar decisivamente no processo de seleção de linhagens para a produção de proteases