Abdul Halim, AB; (2014) In vitro metabolic pathway construction in an immobilised enzyme microreactor (IEMR). Doctoral thesis (PhD), UCL (University College London).

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Abstract

The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilised enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesising a chiral molecule. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesised from simple achiral substrates using two enzymes, transketolase (TK) and ω-transaminase (TAm). This project involves the design and the development of an IEMR using His6-tagged TK and TAm immobilised onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The IEMR was first characterised based on the operational and storage stability. Furthermore, kinetic parameters of both enzymes were determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. For the multi-step enzyme reaction, two model systems were investigated. The first model investigated was the dual TK (pQR 791)-TAm (pQR 801) reaction. With initial 60 mM (HPA and GA each) and 6 mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 minutes at the lowest flow rate. On the other hand, the second model reaction comprises of three sequential enzyme reaction, TAm (pQR 1021)-TK (pQR 791)-TAm (pQR 1021). A 6% yield of ABT was produced from initial substrate mixture of 100 mM serine and 40 mM GA at flow rate of 0.5 μL/min. Further considerations to improve the system would allow for better yield of the target product and potentially make this IEMR system a powerful tool for construction and evaluation of de novo pathways as well as for rapid determination of various enzymes kinetics.

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