Oxidoreductive bioprocess intensification through reaction engineering and enzyme immobilization

• Autores: Jordi Solé Ferré
• Directores de la Tesis: Gloria Caminal Saperas (dir. tes.), Marina Guillén Montalbán (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2019
• Idioma: español
• Tribunal Calificador de la Tesis: Francisco Valero Barranco (presid.), Francisco José Plou Gasca (secret.), Martin Rebros (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
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• Resumen

  • The research performed and disclosed in this thesis deals with the reaction engineering and the enzyme immobilization principles as tools to improve biocatalyzed oxidoreductive reactions.

    The enzymes, considered “natural catalysts”, confer some advantages over tradition chemical routes: specificity for a single substrate, selectivity for one target reaction and the intrinsic property to perform in mild conditions in terms of temperature, pressure and/or harsh reagents. Once an enzyme is selected to catalyze a desired reaction, the conditions must be optimized in order to prove its potential profitability. Furthermore, enzyme immobilization can serve as a powerful tool to improve the biocatalyst capabilities.

    On a first stage, the co-immobilization of the P450 BM3 monooxygenase together with a NADPH cofactor regeneration enzyme, the glucose dehydrogenase (GDH-Tac), was studied. The best derivates were obtained when using two agarose supports, an epoxy functionalized (83% and 20% retained activity respectively) and an amino functionalized (28% and 25% retained activity respectively). Later on, the re-cycling of the immobilized enzymes was tested in reaction cycles using one of the natural substrates of the P450 BM3, the sodium laurate.

    Once it could be demonstrated that re-cycling of both P450 BM3 and GDH-Tac was possible, both enzymes were studied in two of the project’s target reactions, the hydroxylation of α-isophorone and the hydroxylation of diclofenac. In the first case, the optimization of the reaction conditions had to be performed prior to the reaction cycles. The reactor configuration, the oxygen income or the glucose concentration were adjusted. However, when the reaction was performed using the co-immobilized enzymes, the P450 BM3 was deactivated and it could not be re-used. The same happened with the hydroxylation of diclofenac. On the other hand, the reaction using soluble enzymes, resulted in 86.2% conversion for the α-isophorone (50 mM initial concentration) and 100% for the diclofenac (3.5 mM initial concentration).

    The product resulting from the hydroxylation of α-isophorone, the 4-hydroxy-isophorone, can be further oxidized to keto-isophorone, an intermediary for the synthesis of carotenoids and vitamin E. In order to enzymatically perform this step, an alcohol dehydrogenase and a NADPH oxidase, as a cofactor regenerator, were employed. When used in their soluble form, after 24 hours, 95.7% yield and a space time yield of 6.52 g L-1 day-1 were achieved. Moreover, the alcohol dehydrogenase was immobilized on epoxy-agarose and 58.2% retained activity was obtained. When re-used, the derivate could operate for 96h (4 cycles) improving the biocatalyst yield 2.5-fold compared with the reaction with soluble enzymes.

    The hydrogenation of α-isophorone results in 3,3,5-trimethylcyclohexanone, an industrial interesting substrate due to the polymers that can be obtained from its oxidized product, the trimethyl-ε-caprolactone. This compound is obtained by the Baeyer-Villiger insertion of an oxygen atom into the carbon ring. For this purpose, a cyclohexanone monooxygenase together with a commercial glucose dehydrogenase (GDH-01) were used. Different parameters of the reaction were optimized such as the biocatalyst formulation, the substrate addition rate or the biocatalyst loading. Afterwards, the reaction was scaled up to 1 liter first and then up to 100 liters. In this last pre-industrial reaction, 85% conversion, a space time yield of 2.7 g L-1 h-1 and a biocatalyst yield of 0.83 g g-1cww could be obtained.

    Finally, this same reaction was performed using both enzymes immobilized and re-cycling was intended. The cyclohexanone monooxygenase could be immobilized following a previously described method and 62.4% retained activity was achieved. In the GDH-01 case, different supports were screened albeit at the end, it was also the amino functionalized agarose that resulted successful. A retained activity of 62.6% was obtained. In the reaction cycles, the immobilized enzymes were used either separately or both together. In the best case scenario, after six cycles of reaction (132.5 mM initial substrate) 3.6-fold and 1.9-fold higher biocatalysts yields were obtained for the monooxygenase and the GDH-01, respectively.

    As a conclusion, this thesis has served to broaden the enzyme immobilization portfolio and it has conferred new optimized conditions to perform oxidoreductive reactions. In the case of the 3,3,5-trimethylcyclohexanone, the results reported have exceeded the target metrics set by the project partners. Its implementation at industrial scale is feasible.