Seleção de lipase microbiana para aplicação em reações de hidrólise e de esterificação do óleo da amêndoa de inajá (Maximiliana maripa (Aubl.) Drude) visando produção de biodiesel

Abstract

The reactions of hydrolysis and esterification are of great importance in the have been used as steps in a new technology for the production of biodiesel esters. Biodiesel from oilseeds is an important strategy for the sustainable use of biomass, understood as main source of raw materials for generating renewable fuels. The state of Amazonas has several species of native oil palm still exploited, but with potential for agroenergy production. Among them, the palm of Inajá (Maximiliana maripa (Aubl.) Drude) has been reported as potential to form the basis of the production chain of this biofuel. Through some disadvantages of the chemical process, such as the difficulty of separating products and need to use high levels of energy in the reaction, in recent years significantly increased interest in biocatalysis using lipases. Against In addition, the objective of this study was to determine lipase production among 44 isolates to select the one with the best hydrolytic activity by means of submerged fermentation combining the use of almond oil from the inajá, refined and saturated as substrate. Initially, the material was obtained and its chemical and constitutive characterization in fatty acids. Posteriorly, tests were performed with the bacteria in solid culture medium with rhodamine B for the detection of lipolytic halos. The enzymatic activity was determined by the of hydrolysis of p-nitrophenyl laurate (p-NPL) and confirmed by the titration method using the oil of inajá. Among the bacteria tested, the producer of LIPB17 presented the best affinity for inajá oil and its crude preparation showed activity hydrolytic 349.8 U / mL and specific activity 8587.8 U / mg. This lipase is about 45kDa (SDS-PAGE) was selected for inhalation oil hydrolysis tests and esterification of lauric acid. Tests were carried out to study the effect of temperature, time, pH and percentage of enzyme, in the hydrolysis reactions of the oil of inajá, using a complete factorial design 24 with triplicate of the central point. The reaction reached a conversion of 57.49% at the temperature of 28 ° C, time of 48 hours, pH 6 and the enzyme maximum (7.5%), the temperature being the most significant variable (p <0.05). In the case of esterification, a central composite (DCCR) 23 for the analysis of the influence of temperature, enzyme (E%) and RM lauric acid: ethanol. The reaction reached conversion of 55.59% of lauric acid in ethyl esters 30 ° C, with 7.5% of lyophilized lipase and acid: ethanol (1: 4) molar ratio, once again the temperature and also the molar ratio were the most significant variables (p <0.05). LIPB17 was characterized as a Gram-negative bacillus and showed 99.5% identity with Pseudomonas fluorescens strain KC30 (KF733015). These results showed that the non-commercial lipase of P. fluorescens (LIPB17) has potential to be applied as biocatalyst in reactions of hydrolysis and synthesis of ethyl esters, which allows conclude that these enzymatic processes are effective means of obtaining monoalkyl esters (biodiesel).