Design and characterisation of a prototype immobilised enzyme microreactor for the quantification of multi-step enzyme kinetics

The large number of novel biocatalyst candidates available due to advances in protein engineering and evolution has driven research on automated microwell techniques for rapid catalyst evaluation and quantification of enzyme kinetics and stability. Interest in the further reduction in volume to the microfluidic scale has complemented these microwell approaches for the development of bioprocess operations due to their potential as inexpensive analytical tools with minute volumes and high throughput as well as for their potential for mass replication. This project involves the design and characterisation of a prototype immobilised enzyme microreactor (IEMR) on the inner surface of a 200 μm ID fused silica capillary. Immobilisation is achieved through affinity-based interaction between His6- tags engineered on the transketolase (TK) and transaminase (TAm) enzyme variants and Ni-NTA groups on the derivatised capillary surface. The microreactor concept was validated with two reactions, namely the transketolase-catalysed conversion of hydroxypyruvate (HPA) and glycolaldehyde (GA) to produce L-erythrulose followed by the conversion of erythrulose to 2−amino−1,3,4−butanetriol (erythrulose−aminotriol) in the presence of methybenzylamine (MBA) by CV (Chromobacterium violaceum)-derived ω- transaminase. These keto- and aminodiol synthons are synthetically very useful in the production of a range of compounds with pharmaceutical application. The principles of stop-flow (batch) kinetics were initially investigated with respect to the catalytic performance of both enzymes, where the reaction was shown to depend on substrate concentration and residence time. TK kinetic parameters, evaluated based on a Michaelis−Menten model, in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol.min-1, Km(app) = 26 ± 4 mM) were shown to be comparable to those measured in free solution. Furthermore, the kcat for the microreactor of 2.1 s−1 was similar to the value of 3.9 s−1 for the bioconversion in free solution. This was attributed to the controlled orientation and monolayer surface coverage of the His6−immobilised TK. Furthermore the quantitative elution of the immobilised TK and the regeneration and reuse of the derivatised capillary over 5 cycles were also demonstrated. Whilst slower than TK, the TAm reaction in the IEMR showed similar catalytic performance to a standard reaction in glass vials. Stopped−flow bioconversion results were complemented by continuous flow kinetics of the TK reaction with on-line UV detection (ActiPix, Paraytec), where the dependence of reaction kinetics on flow conditions was investigated. The Km(app), evaluated based on a continuous flow kinetic model, was shown to increase with flow rate, with the optimal being at the lowest flow rates used (0.2 μL.min-1). Furthermore, the value of Km(app) was shown to approach the value of the Michaelis constant of the free enzyme under zero flow (∼25 mM). The prototype microfluidic system was then implemented for the quantitative evaluation of multi-step TK-TAm bioconversion kinetics and the formation of chiral amino diol 2-amino-1,3,4- butanetriol (ABT) product from achiral substrates was demonstrated. The rate of accumulation of ABT (also referred to as EAT) by TAm was 0.02 mM.min-1. μgTAm -1, which was 4× slower than the rate of the TK−catalysed step. Demonstration of the synthesis of the product via the dual reaction and the monitoring of each component provided a full profile of the little known bioconversion and demonstrated the potential for creating novel multi−enzyme pathways in lab−on−a−chip systems, which further enables multi-substrate screening and screening of libraries of evolved enzymes of interest to be achieved rapidly and economically. This in vitro study of multi-step enzyme kinetics provides insight into the behaviour of these de novo engineered pathways to aid incorporation into suitable host cells.