Mejora y evaluación de lipasas microbianas para la síntesis de biodiésel = Improvement and evaluation of microbial lipases for biodiesel synthesis

Abstract

La presente tesis se ha centrado en la mejora de lipasas de Pseudomonas sp. y en la evaluación de las lipasas microbianas para su aplicación en la síntesis de biodiésel. Diferentes aspectos de este proceso han sido investigados para promover la introducción de la biocatálisis enzimática en la nueva era de la industria, mediante el desarrollo de procesos rentables y sostenibles. Se aplicaron nuevas técnicas de mutagénesis basadas en el diseño racional para mejorar la termoestabilidad de una lipasa psicrófila de Pseudomonas sp. (LipC) con el fin de poderla aplicar en procesos con temperaturas más altas. La variante obtenida (LipCmut) y la proteína salvaje, además de otras lipasas de Pseudomonas sp., fueron eficientemente inmovilizadas en soportes hidrofóbicos. Se puso a punto un protocolo de inmovilización, basado en la adsorción de la enzima a soportes porosos, rápido, de fácil reproducción y de bajo coste, y, por lo tanto, adecuado para un eventual escalado del proceso. Las preparaciones enzimáticas fueron ensayadas en la transesterificación de trioleína como modelo a pequeña escala de síntesis de biodiésel. Cambiando el vector de expresión, mejorando el método de obtención de las lipasas y optimizando las condiciones de transesterificación, se incrementaron las bajas producciones obtenidas inicialmente. Se evaluó el contendido de agua como parámetro fundamental en la síntesis de biodiésel y se introdujo, con éxito, la novedosa alternativa de las lipasas solubles aplicadas en este proceso. Una nueva lipasa soluble desarrollada por Novozymes, Callera Trans L, se ensayó en la transesterificación de aceites crudos, como ejemplo de materia prima más barata. Finalmente, se desarrolló un sistema combinado donde los fosfolípidos (o gomas), presentes en los aceites crudos, son hidrolizados por unas fosfolipasas y los ácidos grasos liberados se esterifican con el metanol para la síntesis final de biodiésel conducida por una lipasa soluble. Esta combinación de desgomado y transesterificación lleva a un proceso de síntesis de biodiésel más barato, más respetuoso con el medio ambiente y, por lo tanto, más sostenible.

This thesis is focused on the improvement and evaluation of microbial lipases for biodiesel synthesis. First, the thermal stability of LipC, lipase from Pseudomonas sp. (P. aeruginosa ) was improved. LipC is an enzyme with an interesting psychrophilic behavior and its biochemical characteristics make it a very promising enzyme for biotechnological applications, nevertheless it shows a low thermal stability. Based on the relation structure-function of the protein, a mutagenesis by rational design and saturation of different amino acid positions was performed. A mutant, showing a 7-fold increase in thermal stability at 60°C respect to the wild-type LipC, was obtained. Moreover, this variant kept its cold-adapted properties presenting an optimal activity between 4 and 20°C. Further, immobilization by adsorption of these lipases on different porous supports was carried out. The immobilization is a technique for enzyme stabilization when they have to be applied in industrial biocatalytic processes, such as synthesis of biodiesel. For the immobilization by adsorption, a very fast, efficient, low cost technique, therefore suitable for an eventual process scale, two polypropylene matrices and a silica were tested. Immobilization was performed from the previously obtained mutant (LipCmut) the wild-type (LipC ), its mesophilic homologous (LipA) and another lipase (LipI.3) from Pseudomonas CR611 (P. fluorescens). All lipases were successfully immobilized on the supports tested defining polypropylene Accurel MP1000 as the best carrier. Subsequently immobilized preparations were tested in transformation of triolein into FAMEs (Fatty Acid Metyl Esters) as a small scale model of biodiesel synthesis. LipA, LipCmut and LipC resulted positive for this reaction, but with moderate efficiency. LipCmut and LipC FAMEs production was improved changing their expression vector, increasing the level of protein production and recovery and optimizing the transesterification conditions, in terms of water and methanol content. in this manner, an enormous increase in available lipolytic activity was achieved, corresponding to a higher activity of the immobilized preparations, which were successfully applied for transesterification of soybean oil. The water content in the transesterification reactions was studied in collaboration with Novozymes (Denmark ), where was evaluated a new soluble lipase, Callera Trans L, in the production of biodiesel. Water was found to be an essential factor in the transesterification reaction and a particular reaction mechanism for this lipase, depending on the water and on the free fatty acids, was described. The possibility to apply soluble lipases in a process, so far been carried out only with immobilized enzymes, was an innovation tested also for LipC and LipCmut. The growth culture supernatant, where these extracellular lipases are secreted, was prepared and characterized for the main properties necessary for a transesterification reaction (temperature and methanol resistance). LipC and LipCmut in soluble form resulted to be able to form of FAMEs starting from soybean oil. Moreover, soluble Trans Callera L was tested in the transesterification of crude soybean oil, a substrate difficult for this reaction due to its high content of impurities (free fatty acids and phospholipids). Callera Trans L, showed to be extremely efficient also with this raw material. This result opened the way for the next research which aimed to combine the transesterification with an enzymatic degumming process to remove phospholipids from crude soybean oil. The degumming was carried out with phospholipases that hydrolize phospholipids present in the raw material and prepare it for an efficient transesterification carried out by the lipase. The two processes were performed in the same step, which means an increased sustainability and a substantial reduction in the costs of biodiesel production process.