Produção de proteases de Monacrosporium thaumasium (NF34a) e sua utilização no controle de larvas de Angiostrongylus vasorum

• Autor(a) principal: Soares, Filippe Elias de Freitas
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2451
• topic: Proteases; Monacrosporium thaumasium; Angiostrongylus vasorum; Protease; Monacrosporium thaumasium; Angiostrongylus vasorum; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::METABOLISMO E BIOENERGETICA

Angiostrongylus vasorum is a nematode that parasitizes domestic dogs and wild canids. The treatment of the definitive host (dogs) is accomplished with the use of anthelmintics, however, due to its importance in humans and animals, the study of alternative measures that may help in controlling this nematode is justified. In this context, the application of biological control performed with nematophagous fungi is an alternative that has great potential. Nematophagous fungi produce extracellular proteases which may be directly involved in various stages of infection of eggs and larvae. Thus, this study aimed to optimize production, purify, characterize and evaluate the application of protease of the nematophagous fungus Monacrosporium thaumasium (NF34a) in the control of first-stage-larvae of A. vasorum. Fungal mycelia were obtained by transferring culture disks of the isolated kept in 2% corn-meal-agar (2% CMA) and transferred to flasks containing 50 ml of liquid medium which had the composition optimized for the production of protease. Then, the protease was purified and characterized with respect to its pH, temperature, thermostability and influence of salts in the reaction medium. Finally, was evaluated the larvicidal activity of the crude extract and of the purified protease from M. thaumasium (NF34) on first-stage-larvae of A. vasorum. Yeast extract at the levels evaluated, showed a significant effect (p <0.05) on the production of protease. It was also observed that the variable pH was significant (p <0.1) on production of protease. A protease (Mt1) produced by NF34a was purified in only one step, using an ion exchange chromatography at pH 8.0. The molecular mass of purified enzyme (Mt1) was approximately 40 kDa. The maximum activity of protease was obtained at the pH range between 7.0 and 8.0 and 60 ° C. The ions Mg+2 and Zn+2 partially inhibited the activity of Mt1, whereas PMSF inhibited it completely. In contrast, Ca2+ caused a slight increase of protease activity. Regarding the enzymatic activity on A. vasorum L1 in the interval of 24 hours, the crude extract produced by NF34a, reduced in 77.4% the number of larvae (p <0.05) while Mt1 reduced in 23.9% the number of A. vasorum L1 (p <0.05), compared to control. Both the crude extract as Mt1, kept their biological activity intact after incubation at 28° C for 7 days. It was observed the production of Mt1 when M. thaumasium (NF34a) grew using A. vasorum L1 as the only source of carbon and nitrogen. These results demonstrate that the enzyme may have an important role in the infection of larvae. Thus, further studies about the molecular mechanism of the interaction of the fungus M. thaumasium with potential zoonotic nematodes are necessary.