|551-1||Optimization of the extracellular chitinase production from Metharizium anisopliae strains IBCB 167 and IBCB 425 under SSF|
|Autores:||Cynthia Barbosa Rustiguel (FFCLRP-USP - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) ; Joao Atilio Jorge Atilio Jorge (FFCLRP-USP - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) ; Luis Henrique Souza Guimaraes (FFCLRP-USP - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) |
Nowadays, it is estimated that around of 10% of all existing insects can be pests which should be controled. Entomopathogenic fungi, such as M. anisopliae, are able to control some insect pests. For an efficient control it is necessary to select the correct microorganism and to evaluate different biological and biochemical parameters, such as the enzyme production. The production of chitinases, among others, is related to the pathogenicity and virulence. According to this the aim of this study was to analyze the production of chitinases by the strains IBCB 167 and IBCB 425 of M. anisopliae. The cultures under SSF were obtained using 4 g of crushed dry silkworm chrysalis as substrate, moistened with different solutions (tap water, yeast extract solution, SR salt solution or Khanna salt solution) (3:1 m/v). The production was optimized by an experimental design 22 using the CCRD to obtain surface response graphs considering the cultivation time and humidity as independent variables. After incubation the cultures were added with 50 mL of distilled water, maintained under agitation for 30 min. and harvested using a vacuum pump. The free cell filtrate (extracellular crude extract) was dyalized overnight against distilled water at 4 °C and used for chitinase assay. The enzymatic activity was determined using 1 mM 4-nitrophenyl N-acetyl-&beta-D-glucosaminide as substrate in 100 mM sodium acetate buffer, pH 5.0. The optimized conditions found for chitinase production was of 9-12 days and 48% to 72% of moisture for both strains. The optimum of pH and temperature for chitinase activity for strain IBCB 167 was 5.5 and 50 °C, and for strain IBCB 425 was 5.0 and 60 °C, respectively. The IBCB 167 chitinase was stable at pH 5.5 and showed t50 of 8 min. at 50 °C, while the IBCB 425 chitinase was stable in a wide pH range, and at 40-60 °C. The electrophoretic profile (10% SDS–PAGE) for extracellular proteins of the strains IBCB 167 and IBCB 425 are similars. In conclusion the strains showed differences in the chitinase production, where the strain IBCB 425 produced 1.14 times more extracellular chitinase than the strain IBCB 167, reinforcing the data on the virulence potential, as already shown for Diatrea saccharalis.
Palavras-chave: Metarhizium anisopliae, chitinase, CCDR, enzymes