Studying the effect of industrial operating conditions on enzyme kinetics and stability

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Using protein engineering to accelerate implementation of continuous biocatalysis for API synthesis

Biocatalysis is an invaluable tool for the synthesis of many active pharmaceutical ingredients1,2. Enzymatic reactions are frequently performed in batch, but continuous biocatalysis is gaining interest in industry3, not least because it would allow better integration of chemical and enzymatic reaction steps4. However, selecting a suitable reactor configuration for continuous biocatalysis is often challenging due to the common limitations of enzymes, such as poor kinetic constants towards industrially relevant substrates, substrate/product inhibition, the need for an aqueous reaction environment and/or pH control. For this reason, we have recently developed a tool5 that presents a variety of reactor configurations, combining continuously stirred tank reactors (CSTRs) and continuous plug-flow reactors (CPFRs), to overcome the aforementioned limitations and facilitate reactor selection. However, the continued development of protein engineering technologies6,7 has revealed one of the strongest advantages of biocatalysis over chemocatalysis, namely that the properties of the catalyst can be modified to remove limitations and better suit industrial production processes.8,9 In this presentation we highlight how protein engineering, guided by process-specific targets, can streamline process development. For example, reducing product inhibition by protein engineering could allow operation in a single CPFR where the possibility of complete conversion greatly simplifies downstream processing, improving the tolerance of enzymes towards organic solvents could allow water-free operation without mass transfer limitations between phases and improving pH tolerance could remove the need for acid/base addition to combat pH changes during operation. Such changes to the catalyst could greatly facilitate process development in an industry where reduction of time-to-market is often critical. (1) Pollard, D. J.; Woodley, J. M. Trends Biotechnol. 2007, 25, 66–73. (2) Devine, P. N.; Howard, R. M.; Kumar, R.; Thompson, M. P.; Truppo, M. D.; Turner, N. J. Nat. Rev. Chem. 2018, 2, 409–421. (3) Tamborini, L.; Fernandes, P.; Paradisi, F.; Molinari, F. Trends Biotechnol. 2018, 36, 73–88. (4) Rudroff, F.; Mihovilovic, M. D.; Gröger, H.; Snajdrova, R.; Iding, H.; Bornscheuer, U. T. Nat. Catal. 2018, 1, 12–22. (5) Lindeque, R. M.; Woodley, J. M. Catalysts 2019, 9, 262. (6) Arnold, F. H. Angew. Chem. Int. Ed. 2018, 57, 4143–4148. (7) Yang, K. K.; Wu, Z.; Arnold, F. H. arXiv:1811.10775 2018. (8) Woodley, J. M. Curr. Opin. Chem. Biol. 2013, 17, 310–316. (9) Woodley, J. M. Phil. Trans. R. Soc. A 2017, 376

Understanding the effect of Air-liquid interface on enzyme stability in the presence of hydrophobins

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The growing need to assess the kinetic stability of enzymes

1. Bommarius, A.S., Paye, M.F. (2013) Chem. Soc. Rev., 42, 6534. 2. Sheldon, R.A., Woodley, J.M. (2018) Chem. Rev., 118, 801. 3. Bhagia, S., Dhir, R., Kumar, R., Wyman, C.E. (2018) Sci. Rep., 8, 1350. 4. Thomas, C., Geer, D. (2011) Biotechnol. Lett., 33, 443. 5. Rehn, G., Toftgaard Pedersen, A., Woodley, J.M. (2016) J. Mol. Catal. B Enym., 134, 331

Improving KMO via enzyme engineering for industrally competitive oxidases

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The effect of ionic strength on the kinetic stability of NADH oxidase in a bubble column

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Reactor Selection for Effective Continuous Biocatalytic Production of Pharmaceuticals

Enzyme catalyzed reactions are rapidly becoming an invaluable tool for the synthesis of many active pharmaceutical ingredients. These reactions are commonly performed in batch, but continuous biocatalysis is gaining interest in industry because it would allow seamless integration of chemical and enzymatic reaction steps. However, because this is an emerging field, little attention has been paid towards the suitability of different reactor types for continuous biocatalytic reactions. Two types of continuous flow reactor are possible: continuous stirred tank and continuous plug-flow. These reactor types differ in a number of ways, but in this contribution, we focus on residence time distribution and how enzyme kinetics are affected by the unique mass balance of each reactor. For the first time, we present a tool to facilitate reactor selection for continuous biocatalytic production of pharmaceuticals. From this analysis, it was found that plug-flow reactors should generally be the system of choice. However, there are particular cases where they may need to be coupled with a continuous stirred tank reactor or replaced entirely by a series of continuous stirred tank reactors, which can approximate plug-flow behavior. This systematic approach should accelerate the implementation of biocatalysis for continuous pharmaceutical production