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Bioreactor production, purification and characterization of microbial lipases


Lipases are enzymes belonging to the group of hydrolases and catalyze the conversion of triglycerides to free fatty acids and glycerol. However, lipases are distinguished by their ability to catalyze hydrolysis reactions not only but also synthesis in aqueous-limited media, a property that has expanded the potential industrial applications of these enzymes. The Laboratory of Biochemistry and Bioprocess (FCLA-UNESP, Assis) has been dedicated to select producers of lipases microorganisms. Among the microbial strains already studied three stood out either by economic characteristics related to culture conditions for enzyme production, either by high activity, either by the properties of the enzyme. Thus, they are promising strains fungi Fusarium sp. FCLA-MA-41 and Trichoderma harzianum LBBIO TH1 and the bacteria Burkholderia lata LBBIO BL2. The aim of this study is to determine the most important culture parameters for enzyme production in bioreactor by these strains, establish purification protocols and characterize kinetics and biochemically lipase each strain after purification. The microorganisms are grown in a bioreactor of 7.5 L in the culture medium whose formulation was determined in the previous step in shake flasks, being evaluated parameters such as agitation speed (100 to 1000 rpm), aeration rate (0.5 to 2.0 vvm) and pH of the medium (pH 5.0 to 8.0). For development of purification protocols will be used chromatographic methods with gel permeation columns and hydrophobic interaction. Kinetic characterization will be studied by measuring the activity and stability in organic solvents at different pH (2.3 to 10,0 t), temperature (20 to 70 ° C), protein concentration (1 to 40 mg / ml) substrate concentration (0.1 to 10 mmol / L) and the presence of metal ions, chelating agents or detergents. (AU)

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