Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

• Advanced characterization of immobilized enzymes is important for heterogeneous biocatalyst development.
• Structural and in-operando studies contribute to identification of factors governing the activity of immobilized enzymes.
• Protein visualization in solid supports is used to facilitate enzyme loading in high quantity and quality.
• Elucidation of structural features of immobilized enzymes on solid support remains challenging.
• In-operando opto-chemical internal sensing is prominently used to characterize the biocatalyst's internal environment.

Like in chemical catalysis, there is a clear trend in biocatalysis to carry out synthetic transformations at the manufacturing scale heterogeneously catalyzed. Recycling of insoluble catalysts is simplified, and continuous reactor development thus promoted. Heterogeneous biocatalysis usually involves enzymes immobilized on mesoporous solid supports that offer a large internal surface area. Unraveling enzyme behavior under the confinement of a solid surface and its effect on the catalytic reaction in heterogeneous environment present longstanding core problems of biocatalysis with immobilized enzymes. Progress in deepening the mechanistic understanding of heterogeneous biocatalytic conversions is often restrained by severe limitations in methodology applicable to a direct characterization of solid-supported enzymes. Here we highlight recent evidence from the analysis of protein distribution on porous solid support using microscopic imaging methods with spatiotemporal resolution capability. We also show advance in the use of spectroscopic methods for the analysis of protein conformation on solid support. Methods of direct characterization of activity and stability of immobilized enzymes as heterogeneous biocatalysts are described and their important roles in promoting rational biocatalyst design as well as optimization and control of heterogeneously catalyzed processes are emphasized.

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