Enzyme immobilization has been used an alternative approach to enzyme engineering to produce robust biocatalytic products, as well as to improve the enzyme longevity in industrial applications. Among the various immobilization methods, enzyme entrapment exhibits advantages in versatility and high enzyme loading. However, entrapped enzymes can experience limitations in mass transfer efficiency, especially when diffusion-limited enzymes are entrapped. In this study, a robust biocatalytic matrix with controlled liquid transport properties was fabricated to address this problem. Catalase, an enzyme that catalyzes peroxide decomposition (H2O2 H2O + O2) at room temperature with ultrafast kinetics (107 s-1 turnover rate), was entrapped in a thin chitosan layer at a mild condition and then applied to a cellulosic fibrous support to examine the impact of controlled liquid transport on catalytic efficiency. Mild immobilization conditions and good affinity between the two polysaccharides with opposite charges minimized enzyme inactivation during the preparation steps and prevented enzyme from leaching during peroxide decomposition testing and washing, providing a novel, durable and versatile enzyme immobilization strategy. The catalytic efficiency of entrapped diffusion-limited enzymes in a reaction containing solid, liquid and gas phases was facilitated when the fibrous matrix was used as a flow-through configuration, where dissolved enzyme substrate was transported by liquid flowing through the coated fibrous structure. The flow-through configuration decomposed at least two times more peroxide in a twenty-times smaller reaction zone volume compared to a stirred tank configuration, and the mechanism of the improved catalytic efficiency was revealed by a neutron imaging technique. This new class of sustainable and flexible biocatalytic matrices has beneficial multifunctional properties that are applicable for numerous small- and large-scale applications including controlled flow reactors and reactive filtration.

Biocatalytic reactor with improved catalytic efficiency and longevity for entrapped diffusion-limited enzyme

ACS Fall 2021

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