Characterisation and kinetic modelling of a novel convergent enzymatic cascade for epsilon-caprolactone synthesis employing a double-smart cosubstrate with in situ polymerisation
Technical Chemistry
Final Report Abstract
Epsilon-caprolactone (ECL) is amongst others a precursor for the biodegradable plastic polycaprolactone (PCL), used in diverse application areas, one of which is biomedicine. PCL can be processed easily via 3D printing and is biodegradable, therefore if not needed anymore will rot without polluting the environment with plastic waste. On the way to producing PCL in an environmentally friendly way, that makes it safe for biomedical applications, the precursor should be produced without toxic or explosive compounds. This can be achieved by using enzymes, proteins that can perform chemical reactions under mild conditions (room temperature, in water, atmospheric pressure). Within this project the synthesis of ECL in an enzymatic cascade, involving two enzymes namely a Baeyer-Villiger monooxgenase (BVMO) and an alcohol dehydrogenase (ADH), with theoretically no waste generation was further developed. The convergent cascade utilizes only two different substrates and oxygen from air to produce ECL, while merely water is generated as waste product. The most labile component in the cascade is the BVMO cyclohexanone monooxygenase from Acinetobacter calcoaceticus (CHMOAcinet). Within the first part of the here presented study, stabilized variants of CHMOAcinet were screened and characterized to find the best candidate for the cascade. Afterwards, the convergent cascade was optimized via ‘Design of Experiments’, increasing the product yield from 6.8 % to 27 %. At the same time the cofactor amount was reduced 2-fold and the by-product formation decreased 52-fold (originated by ADH-catalyzed reduction of the main substrate cyclohexanone). Furthermore, it was discovered that the best variant of CHMOAcinet, CHMO M16 DS, had a 25-fold increased total-turnover number (TTN) compared to the wild-type enzyme, therefore being 25-fold more effective than the wild-type enzyme. Consequently, CHMO M16 DS was implemented in the convergent cascade together with the alcohol dehydrogenase TeSADH from Thermoanaerobacter ethanolicus. The cascade was characterized via progress curve analysis and a mathematical model was developed. Within the development process, it was discovered that a chemical side-reaction with the utilized Tris-buffer was existing. The product from the chemical reaction was then further converted by TeSADH to another by-product. The model was also describing the side reaction and could be applied to various BVMO concentrations. Hence, it could be used to some extent to further optimize the enzyme cascade without the need of further experiments. Nevertheless, it was not possible to describe the side reactions to an extent that a robust model could be created. Consequently, further optimization of the model is necessary in the future to create a robust model that can be used to predict the performance of the cascade under various conditions. The main achievements of this project were the finding and characterization of a stabilized CHMOAcinet variant and the modelling of the enzymatic cascade reaction for ECL synthesis. Although the latter still needs further improvement, knowledge about the cascade reaction was gained, which will be advantageous for the further research on this topic.
Publications
- Enzyme and Microbial Technology 2017, 106, 11-17
K. S. Mthethwa, K. Kassier, J. Engel, S. Kara, M. S. Smit, D. J. Opperman
(See online at https://doi.org/10.1016/j.enzmictec.2017.06.017) - ChemCatChem 2019
J. Engel, A. Cordellier, L. Huang, S. Kara
(See online at https://doi.org/10.1002/cctc.201900976) - Mol. Catal. 2019, 468, 44-51
J. Engel, K. S. Mthethwa, D. J. Opperman, S. Kara
(See online at https://doi.org/10.1016/j.mcat.2019.02.006)
