Prospects and problems in enzyme immobilization methodology: comprehensive review.

The applications of immobilized enzymes in industry are progressively increasing. Methodological aspects of engineering enzymology are largely determined by immobilization approaches. Using enzymes as biocatalysts provides advantages such as mild reaction conditions, environmental biodegradability, and high catalytic efficiency. Nevertheless, the extreme conditions often found in industrial settings can destabilize enzymes, reducing their operational durability in manufacturing applications. Immobilization of enzymes is a common practice, mainly in order to minimize enzyme costs on the process economics by making it possible to reuse the enzyme many times and also minimize the operation cost as the immobilization technique may be modify the enzyme behavior, thus reducing the enzyme and product costs significantly. Many techniques have been used previously for enzyme immobilization, such as adsorption, entrapment, encapsulation, covalent binding, and cross-linking. This review provides a comparative analysis of covalent and non-covalent methods of enzyme immobilization, and briefly outlines the main issues and prospects of their practical use in both analytical practice and industrial biotechnology and medicine. These techniques alter enzyme characteristics by modifying the surrounding microenvironment and adjusting the extent of multipoint binding. As a result, analyzing the enzyme's structural changes after attachment to the support surface is essential. Advanced nanoscale characterization tools play a vital role in examining surface-immobilized enzymes, providing key qualitative and quantitative insights, such as morphological visualization, into their behavior. Such analytical techniques are critical for evaluating the success of immobilization methods and guiding the design of future enzyme stabilization strategies. Special attention is paid to computer methods of analysis of immobilized enzymes. Importantly, while simulations have been primarily performed to rationalize the molecular aspects of the immobilization event, their use to predict adequate protocols that can control its impact on the enzyme properties is, up to date, mostly missing. Artificial intelligence and machine learning have been also successfully applied for optimization of the immobilization process and predictive modeling of unmeasured parameters. Using advanced analytics, they optimize biocatalytic processes by analyzing experimental data, forecasting immobilization parameters, and developing high-performance enzyme designs. Thus, this review summarizes various new immobilization techniques, limitations, prospects and applications of immobilized enzymes.