Het gebruik van biokatalyse biedt een interessante mogelijkheid om de chemische industrie te verduurzamen. Tijdens dit project streefden Aminoverse B.V. en Zuyd Hogeschool ernaar om een nieuwe enzymfamilie te identificeren die veelbelovend zou kunnen zijn voor commerciële toepassingen in de biokatalyse. Specifiek werd er gefocussed op Fe(II)/α-ketoglutaraat-afhankelijke oxygenasen, oftewel KGO's, die theoretisch veel potentieel vertonen om als katalysator op te treden in één van een van de meer uitdagende reacties die toegepast worden in de organisch chemische industrie: nl. selectieve functionaliseringen van inerte C-H-bindingen.
Tijdens de gehele projectperiode zijn maar liefst 20 verschillende wild-type KGO's getest op 26 diverse substraten waaronder 20 aminozuren, 3 benzeenverbindingen en 3 terpenen. Alle combinaties werden gescreend op enzymatische activiteit met behulp van hogedruk vloeistofchromatografie (HPLC) en dunnelaagchromatografie (TLC). De resultaten gaven weer dat verscheidene KGO's reactiviteit vertoonden ten opzichte van verschillende substraten. Daaropvolgende structurele karakterisering van de gegenereerde eindproducten, met behulp van vloeistofchromatografie-massaspectrometrie (LC-MS/MS), onthulde diverse succesvolle hydroxylaties van specifieke substraten. Als gevolg hebben de projectresultaten een positieve bijdrage geleverd om het potentieel van KGO’s als biokatalysatoren voor de chemische industrie aan te tonen.
In the context of sustainability, the use of biocatalysis in organic synthesis is increasingly observed as an essential tool towards a modern and ‘green’ chemical industry. However, the lack of a diverse set of commercially available enzymes with a broad selectivity toward industrially-relevant substrates keeps hampering the widespread implementation of biocatalysis. Aminoverse B.V. aims to contribute to this challenge by developing enzymatic screening kits and identifying novel enzyme families with significant potential for biocatalysis.
One of the most important, yet notoriously challenging reaction in organic synthesis is site-selective functionalization (e.g. hydroxylation) of inert C-H bonds. Interestingly, Fe(II)/α-ketoglutarate-dependent oxygenases (KGOs) have been found to perform C-H hydroxylation, as well as other oxyfunctionalization, spontaneously in nature. However, as KGOs are not commercially available, or even extensively studied in this context, their potential is not readily accessible to the chemical industry.
This project aims to demonstrate the potential of KGOs in biocatalysis. In order to achieve this, the following challenges will be addressed: i) establishing an enzymatic screening methodology to study the activity and selectivity of recombinant KGOs towards industrially relevant substrates, ii) establishing analytical methods to characterize KGO-catalyzed substrate conversion and product formation. Eventually, the proof-of-principle demonstrated during this project will allow Aminoverse B.V. to develop a commercial biocatalysis kit comprised of KGO enzymes with a diverse activity profile, allowing their application in the sustainable production of either commodity, fine or speciality chemicals.
The project consortium is composed of: i) Aminoverse B.V, a start-up company dedicated to facilitate chemical partners towards implementing biocatalysis in their chemical processes, and ii) Zuyd University, which will link Aminoverse B.V. with students and (bio)chemical professionals in creating a novel collaboration which will not only stimulate the development of (bio)chemical students, but also the translation of academic knowledge on KGOs towards a feasible biocatalytic application.
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