Determination of hydrolases secreted by filamentous fungus Lasiodiplodia theobromae

• Grant number: 16/05905-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2016
• End date: May 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Valéria Marta Gomes de Lima
• Grantee: Paula Zardetto Ciciliato
• Host Institution: Faculdade de Ciências e Letras (FCL-ASSIS). Universidade Estadual Paulista (UNESP). Campus de Assis. Assis , SP, Brazil

Abstract

Filamentous fungus Lasiodiplodia theobromae is a plant pathogen that has the ability to secrete exopolysaccharides (EPS) of ²-glucan type, with potential for industrial application. However, for this, this bioprocess needs to be economically viable, requiring appropriate studies, such as use of low cost components for composition of culture medium. Previous studies have shown that the residual biomass after EPS production by L. theobromae can be used as a supplement in subsequent crops for production of this polysaccharide due to its nutritional value. The interest of this study is to determine the hydrolytic potential of the fungus L. theobromae, targeting both understand and improve the degradation capacity of the biomass by the fungus as evaluate hydrolases secreted by the fungus which have industrial application. The microorganism, initially preserved at 4°C, will be inoculated into VGA medium, containing minimum Vogel salts (1956) (SMV), glucose (10g/L) and agar (15g/L), and maintained at 28°C for 5 to 7 days. The pre-inoculum will be composed of SMV and 0.5 g/L of glucose, with pH adjusted to 5.8 and will be kept at 28ºC under stirring of 180 rpm for 48 hours. The standard culture will consist of SMV and 50 g/L of sucrose, pH 5.8, at 28 ° C and will be kept under stirring of 180 rpm for 12, 24, 36 and 48 hours. Crops will also be carried out in a medium containing biomass (40 g/L, from standard culture, lyophilized) and SMV, without addition of sucrose, incubated and samples removed in the same standard culture conditions. After stopping the cultivation, the cells will be separated by centrifugation at 3000 g for 20 min and the supernatant will be assayed for enzymatic activity. It will be determined glucanases activity (²1-4;²1-3/²1-6;±1-4;±1-4/±1-6), glycosidases (± and ²), invertase, proteases and lipases.