Rizzo, Andrea; (2025) Discovery and characterisation of C-C bond forming enzymes from genomic and metagenomic sources for applications in industrial biocatalysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bio-processes based on enzymatic catalysis will play a major role in the development of green, sustainable processes in the future. A key aspect to this approach is the discovery of new enzymes, which can be used to improve or replace traditional chemical processes and even discover new methods and techniques. Moreover, it is important to implement screening methods suitable for testing a large number of enzymes at the same time. A common way to achieve this, is to create large libraries of different enzymes belonging to the same class, which can be screened together in the pursuit of hits. To the best of our knowledge, this type of library is not available for a group of proteins that in the last decades has gathered a lot of interest: carbon-carbon bond forming enzymes. In addition, this broad group has not been fully explored yet and the discovery of novel enzymes is an important aspect to collect more data about them. In this work, we created two libraries, one containing both TDP-dependent enzymes and aldolases, two of the most common carbon-carbon bond forming enzymes, and one containing only 2-deoxy-D-ribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) and fructose-6-phosphate aldolases (FSAs, EC 4.1.2.13), two sub-classes of aldolases with broad substrate range. 48 of the enzymes added to this second library were novel sequences retrieved from metagenomic libraries, using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions using non-native substrates. The product of these reactions, aldols, can find applications as building blocks in the synthesis of many biologically active compounds. One enzyme, DERA-76, catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity and for this reason it was deeply characterized. In terms of activity, the reaction conditions and the substrate concentrations were optimized, and the reaction was scaled-up to 100 ml. Moreover, the catalytic kinetics towards furfural and the stereoselectivity of the reaction were addressed. The melting temperature of DERA-76 was measured to address its stability, and its structure was predicted and used as a starting point for the identification of the active site and the interaction with its substrates by molecular docking. The results suggest that this enzyme belongs to a cluster of DERAs that has not been fully explored yet, but that has interesting characteristics such as an uncommon stereoselectivity, a high activity towards non-native substrates and a good stability in the presence of organic solvents. A phylogenetic analysis showed how DERA-76 and similar proteins all shared part of these characteristics and they may pave the way towards the use of these enzymes in large-scale processes and cascades in the future.

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