Resumen de Ús d'alcohol deshidrogenasa i alcohol oxidasa per la conversió d'alcohols en dos productes valuosos: clorolactona i vanil·lina
Enzymes have some catalytic advantages over chemical catalysts used in classical chemical synthesis: specificity, selectivity and the possibility to work under mild conditions of temperature and pressure. However, they also have some limitations such as low stability and low productivity. This work combines two techniques aiming to optimise the target reactions: immobilisation and reaction engineering.
The target reactions of this work are redox reactions focused on the biosynthesis of molecules, of medium-high value, of industrial interest. In the first part of the thesis, an alcohol dehydrogenase (ADH99) was used, with an NAD(P)H oxidase (NOX) as a cofactor regeneration system, to oxidise a chlorolactol to chlorolactone. Chlorolactone is a precursor for the synthesis of statins which are drugs used to lower LDL-cholesterol by inhibiting the enzyme responsible for its biosynthesis. Both enzymes were efficiently immobilised on different supports, selecting the three that showed the highest retained activity. The stability of the immobilised derivatives under reaction conditions was studied and the maximum enzyme load for each enzyme also was determined. The use of immobilised NOX was discarded because no stability improvements were achieved with any support. The reaction conditions were optimised by design of experiments (DoE), using soluble ADH99 added at maximum loading onto an epoxy-agarose support. Finally, the reusability of the immobilised enzyme was studied, where both the total product obtained and the biocatalyst yield could be improved 1.5-fold. However, the best configuration resulted from the use of the two enzymes in soluble form.
The second part of this thesis was focused on the oxidation reaction of vanillyl alcohol to vanillin catalysed by eugenol oxidase (EUGO). Vanillin is the molecule that gives vanilla its organoleptic properties. Vanillin biotechnological synthesis is of high interest industrially because it is the second most expensive flavouring in the world and the product can be labelled as natural. Similar to the previous section, EUGO was efficiently immobilised onto different supports, selecting the three that retained most activity. These supports were used to study the stability of the immobilised enzyme and the maximum EUGO load that can be immobilised. In this case, the three immobilised derivatives were used to perform the target reaction, in order to select the most stable operationally. All immobilised derivatives could be reused 5 times maintaining a high conversion in the last cycle. Epoxy-agarose-UAB M2 was the support that showed the best stability, improving the biocatalyst yield 3-fold.
The encouraging results obtained in the second section of this work allowed us to deepen the study of this reaction. Therefore, in the third section, an optimisation of the reaction conditions was carried out to improve the process metrics and also aiming to make the process more environmentally sustainable. The EUGO activity and its stability were taken into account to choose the reaction conditions. Both conditions, maximum activity and maximum stability, were tested in the target reaction with soluble and immobilised EUGO. Using the new conditions, it was possible to improve the volumetric productivity 5.7 and 6.6-fold respectively, compared to the previous conditions. Finally, the reusability of the immobilised EUGO allowed us to perform 5 reaction cycles and 18 reaction cycles, with unoptimised and optimised reaction conditions respectively. This resulted in an improvement of the biocatalyst yield of 3.9 and 12.4-fold, respectively, compared to reactions with soluble enzyme under the same conditions.
