Development of flow-based biocatalytic reactors

Enzymes are highly valued in biochemical processes for their ability to function efficiently under mild conditions while achieving rapid reaction rates. Their utilization facilitates a more environmentally sustainable production route compared to traditional chemical synthesis methods. However, their limited long-term stability and inefficient reusability hindered the widespread adoption of soluble enzymes. Immobilization of enzymes emerges has as a crucial strategy to overcome these challenges, offering enhanced stability and enabling efficient reuse, thus finding widespread application in industrial settings. Electrochemistry provides a controlled, facile, and rapid method of immobilizing enzymes on conductive supports. The properties of the enzyme-modified films can be controlled by altering the potential of the electrode and/or adjusting the electrodeposition conditions. Electrochemical methods of enzyme immobilization have primarily focussed on applications such as biosensors and biofuel cells. This approach can also be used to immobilize enzymes for applications in, e.g., biocatalysis, where the electrode is used as a support rather than an electrode perspective. The development of enzymatic cascades reflects the wide range of applications in synthetic chemistry, particularly in flow systems. Electrochemistry can be used for the immobilization of enzymes involved in enzymatic cascades in the flow system. For this, suitable reactors need to be developed to incorporate conductive surfaces inside. In light of these considerations, this project investigated electrochemistry for enzyme immobilization on the conductive surface, which was further used to design enzymatic cascades in the flow system, for which a suitable reactor was designed.

Peroxidases catalyze various synthetically valuable reactions, including halogenation, oxidation, and epoxidation, with high regioselectivity and enantioselectivity. However, the requirement for H2O2 as a co-substrate, particularly at high concentrations, can hamper the use of peroxidases. In general, this problem can be tackled by the in situ production of H2O2 using enzymatic production of H2O2 by glucose oxidase and removing the excess of H2O2 from the solution by catalase. For this purpose, initial efforts centred on the targeted electrochemical immobilization of glucose oxidase on the conductive surface for controlled H2O2 production, exploring various methods such as diazonium coupling, electrodeposition of silicate layers, and electropolymerization. A flow reactor incorporating modified graphite rods was designed and constructed to run H2O2 production in the flow system.

Next, the constructed flow reactor was characterized by computational fluid dynamics. And a three-enzyme cascade system comprising glucose oxidase (a H2O2 generator), chloroperoxidase/horseradish peroxidase (a H2O2 dependent enzyme), and catalase (a H2O2 scavenger) was developed in a reactor to maintain the activity of peroxidases in the flow system. Immobilization of the enzymes on a graphite rod was achieved through electrochemically driven physical adsorption followed by cross-linking with glutaraldehyde. A three-enzyme cascade was successfully used in halogenation and oxidation reactions.

Further studies explored electrochemical methods for lactate dehydrogenase (LDH) and horseradish peroxidase (HRP) immobilization. PEDOP electrodeposition successfully immobilized LDH for an NAD+regeneration system, achieving high conversion rates and operational stability. HRP immobilization using silica gel and chitosan electrodeposition revealed varying stability, with chitosan showing better operational stability.

In the field of biotechnology processes, the utilization of water-miscible organic solvents has emerged as a highly promising approach for conducting enzymatic reactions. Through the substitution of water in enzymatic reactions with an organic solvent, reactions can achieve enhanced efficiency in converting hydrophobic substrates. However, increasing the concentration of an organic solvent in the reaction medium may result in enzyme inactivation because of reversible alterations in the protein structure. Enzyme immobilization onto porous, insoluble supports can increase the stability of enzymes in organic solvents. In light of this, the project further focused on using enzymes in organic solvents. The study focused on designing insoluble supports in organic solvents for enzyme immobilization, with a particular emphasis on peroxidases. Different porous, insoluble supports were designed, and the best porous structure was found to be chitosan beads modified with phytic acid. This is the first study using the combination of chitosan and phytic acid for enzyme immobilization. Immobilized horseradish peroxidase in chitosan beads modified with phytic acid exhibited excellent stability and performance in 95% organic solvents during 2-methoxyphenol oxidation. This research contributes valuable insights into the design and application of biocatalytic flow reactors and the stability of immobilized enzymes in various environments, paving the way for innovative approaches in enzymatic reactions.