(101c) Immobilisation of Thermophilic Enzymes within Miniaturised Flow Reactors for Biotransformations

Biocatalysis provides an appealing alternative for the synthesis of important pharmaceuticals due to the advantages of selectivity and stereospecificity offered by enzymatic reactions. Using micro reactors, optimisation of the biocatalytic reaction as well as screening an enzyme for potentially useful transformations can be achieved with less cost, and be more sustainable. With very small reaction volumes, the continuous flow system in miniaturized flow reactors allows the spatial and temporal control of chemical reactions, and facilitates high surface interactions which generate high product yields in smaller times compared to the conventional batch systems. This micro reaction system permits the use of a small amount of enzyme to be immobilised within the microfluidic devices, which can be reused, thereby reducing costs.

Miniaturised flow reactors containing L-aminoacylase (E.C. 3.5.1.14) from the thermophilic archaeon, Thermococcus litoralis were investigated for their potential utilisation for industrial biotransformation reactions. The enzyme was immobilised on polyglycidylmethacrylate-co-ethylenedimethacrylate monoliths formed within the micro reactors by the reaction between amino groups of the enzyme and epoxy terminal on the surface of the monolithic microchannels. The immobilised enzymes were assayed with the preferred substrate, N-benzoyl-L-phenylalanine. The enzymes remained stereospecific after being immobilised onto monolithic microchannels. Using the continuous flow micro reactor system, 100% substrate conversion was obtained at room temperature which is a significantly lower temperature than the optimal condition reported for the conventional batch system (85 C, Toogood et al., 2002). Highest enzyme activity at room temperature was observed at flow rates of 1-4 microlitre/minute and the operating window could be expanded by performing the reaction at higher temperature (50 C, flow rate up to 10 microlitre/minute). Stability tests showed that the enzyme retained optimal activity after being exploited for 120 hours. This demonstrates that the continuous-flow micro reaction system with this enzyme is a suitable tool for further development.

Substrate specificity of the immobilised enzymes was assayed using 10 mM substrates in 100 mM Tris-HCl, pH 8.0 inside the micro reactor using a flow rate of 1 microlitre/minute. The enzyme appeared to be very specific for N-benzoyl-L-phenylalanine, N-acetyl-L-phenylalanine and N-chloroacetyl-L-phenylalanine, which all showed 100% substrate conversion. The order of preferred activity decreased from N-benzoyl-L-threonine (68.3%) > N-benzoyl-L-leucine (52.2%) > N-acetyl-L-tyrosine (33.3%) > N-acetyl-L-tryptophan (7%).

Many biotransformation reactions need to be carried out in organic solvents due to the insolubility of the substrates to be used. Enzyme immobilisation onto solid supports is an approach to stabilise enzymes for use under these conditions. The activity of the immobilised enzyme within the monolithic microchannels in the presence of organic solvents (5-50%) was studied using dimethylformamide, dimethylsulfoxide, ethanol, methanol and acetone. The presence of solvents reduced the activity of the enzyme from 20-80% depending on type and amount of solvent used.

For future work, immobilised amidase within the monolithic microchannels will be investigated in order to achieve high throughput biocatalytic synthesis and screening in micro reactors. Additionally, the use of redox enzymes requiring co-factor regeneration for the stereoselective synthesis of high value compounds will be studied. The incorporation of enzymatic synthesis with in situ co-factor regeneration within a microenvironment will be carried out.

Keywords: enzyme immobilisation, L-aminoacylase, monolithic micro reactors, substrate screening, high-throughput

Reference

H. S. Toogood, E. J. Hollingsworth, R. C. Brown, I. N. Taylor, S. J.C. Taylor,

R. McCague, J. A. Littlechild, A thermostable L-aminoacylase from Thermococcus litoralis: Cloning, overexpression, characterisation, and applications in biotransformations. Extremophiles (2002) 6: 111-122.