Ingeniería de biocatalizadores inmovilizados de lipasas: captura de agregados bimoleculares por técnicas de inmovilización

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de Lectura: 10-06-2021El objetivo principal de esta Tesis Doctoral es estudiar la relación entre las diferentes estrategias de inmovilización de lipasas y las propiedades de los diferentes derivados obtenidos: actividad, estabilidad, selectividad. Inicialmente observamos que a concentraciones moderadamente altas de lipasas (p.e. 2 mg/mL) las enzimas solubles se encuentran mayoritariamente como agregados bimoleculares de dos estructuras abiertas de la enzima. A partir de este resultado desarrollamos estrategias para inmovilizar la forma abierta y la forma cerrada de la lipasa de Thermomyces lanuginosus (TLL). La forma abierta se inmovilizó por adsorción interfacial sobre soportes hidrofóbicos, por adsorción iónica del agregado bimolecular y por inmovilización covalente del agregado bimolecular. La forma cerrada se inmovilizó covalentemente en presencia del surfactante CTAB (bromuro de hexadeciltrimetilamonio). Todos los derivados de las formas abiertas eran bastante más estables que el derivado de la forma cerrada. El derivado más interesante (adsorción hidrofóbica más recubrimiento con polialilamina) era 10 veces más activo que la enzima soluble diluida y 1800 veces más estable en experimentos de inactivación térmica. Diseñamos una nueva estrategia para fijar e inmovilizar selectivamente los agregados bimoleculares de TLL y de otras lipasas (Cándida antárctica fracción B, Rhizomucor miehie, Lecitase). En todos los casos, los derivados de los agregados eran bastante más estables que el derivado de la forma cerrada. El derivado más estable era el de la Lecitase que 100 veces más estable que el derivado de la Lecitasa cerrada.The main objective of this Ph.D. Thesis is the study of the relationship between different strategies for immobilization of lipases and the properties of the different derivatives. Firstly, it was observed that, by using moderate or high concentrations of soluble lipases (e.g., 2 mg/mL), the soluble enzymes are mainly forming bimolecular enzyme aggregates of two open structure of the lipases. From these results, different strategies for the immobilization of the open and closed structure of Thermomyces lanuginosus lipase (TLL) were developed. The open structure was immobilized by adsorption of the isolated enzyme on hydrophobic supports, by ionic adsorption of the bimolecular aggregate on anionic exchangers and by covalent immobilization of bimolecular aggregates. The closed structure of TLL was immobilized by covalent immobilization of the isolated enzyme in the presence of CTAB (Cetyl trimethylammonium bromide). All immobilized derivatives of the open structure of TLL were much more stable than the derivative of the isolated closed enzyme. The most interesting derivative (hydrophobic adsorption plus coating with poly-allyl-amine) was 10 fold more active (intrinsic activity) tan the diluted soluble enzyme and 1800 fold more stable in experiments of thermal inactivation. Finally, a new strategy for selective fixation and immobilization of bimolecular lipase aggregates was developed. TLL and other lipases were studied (Candida antarctica fraction B, Rhizomucor miehei, Lecitase). In all cases, these derivatives were quite more stable than the derivative of isolated closed enzyme. Bimolecular Lecitase was 100 fold more stable than the isolated closed enzyme

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

[Background] Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.[Results] Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.[Conclusions] Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.This work was sponsored by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012–36861 and CTQ2015–70348). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19,260) funded by the Spanish Ministry of Economy, Industry and Competitiveness. We also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for granting the scholarship to Erick Abreu Silveira.Peer reviewe

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012–36861 and CTQ2015–70348)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2.605 JCR (2017) Q2, 62/160 Biotechnology and Applied MicrobiologyUE

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100–200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.Spanish Ministry of Economy, Industry and Competitiveness (BIO2012-36861; CTQ2015-70348; IJCI-2014-19260)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (2016/10636-8; 2015/10530-2; 2013/20826-0)3.503 JCR (2019) Q2, 47/156 Biotechnology & Aplied Microbiology0.992 SJR (2019) Q1, 60/359 BiotechnologyNo data IDR 2019UE