Otimização das condições de cultivo e caracterização parcial da lipase produzida por Yarrowia divulgata

Abstract

Lipases are classified as hydrolases and have the function of catalyzing reactions of total or partial hydrolysis of triglycerones in diacylglycerols, monoglycerols and liberating free fatty acids and glycerol in an oil-water interface. These enzymes are obtained through solid culture or in submerged culture. The submerged fermentation is used in the production of enzymes due to the advantages of more efficient operation in relation to pH, temperature, oxygen ratio, carbon /nitrogen ratio. The objective of this work was to study the best conditions of submerged culture for the production of lipase at 30 °C and 150 rpm for 48 h of culture by yeast Yarrowia divulgata, using factorial experiments (DCCR) to evaluate the effect of the variables, oil concentration and pH, using olive oil and residual soybean oil as carbon sources. Lipase production was compared in the 2 oils and the best condition found was used for evaluation of lipase production in bench bioreactor at 28 °C for 72 h of culture with stirring variation between 200 and 400 rpm. The influence of the addition of surfactants and emulsifiers (Tween 20, soy lecithin and xanthan gum) on lipase production was also evaluated. The lipase produced was characterized as the activity and stability at different temperatures, pH, metal ions and the effect of solvents and surfactant on the enzymatic activity. The results obtained in the DCCR (22) indicate that, among the carbon sources tested, olive oil provided higher production, with activity of 67.0 U.mL-1 at pH5.3 and 9.97 g.L-1 of oil. Using residual soybean oil the lipase production was 36.0 U.mL-1 at pH 5.0 and 16.22 g.L-1 oil. In the comparison between the two oils, the activity was higher in olive oil (57.0 U.mL-1) compared to residual soybean oil (30.8 U.mL-1). The assays in bench-top bioreactor using olive oil as carbon source showed more expressive results of 300 rpm (2.939 U.mL-1) agitated lipolytic activity and the highest cell count (2.56x109 cells.mL-1) was obtained at 400 rpm, after 48 h of culture. The assays at 200 rpm showed less activity and less accumulation of cells during 72h of culture. EEB showed stability in the range of pH 3.0 to 8.0, with lipolytic activity above 50% and pH 3.0 to 6.0 with residual activity close to 100%. The Mn2+ ion was the only one responsible for providing EEB activation at concentrations of 5mM and 10mM, and the Mg2+, Zn2+ and Ca2+ ions presented in EEB activation effect only at 5mM concentration. Surfactants negatively influenced lipolytic activity, with the exception of Triton X-100 (1%). The organic solvents presented activation of the lipase only in 1% and DMSO had a positive influence in 1% and 10%.