Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Microreaction technology, which is an interdisciplinary science and engineering area, has been the focus of different fields of research in the past few years. Several microreactors have been developed. Enzymes are a type of catalyst, which are useful in the production of substance in an environmentally friendly way, and they also have high potential for analytical applications. However, not many enzymatic processes have been commercialized, because of problems in stability of the enzymes, cost, and efficiency of the reactions. Thus, there have been demands for innovation in process engineering, particularly for enzymatic reactions, and microreaction devices represent important tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies especially for enzyme immobilized microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared to conventional reaction devices. Fundamental techniques for enzyme immobilized microreactors and important applications of this multidisciplinary technology are also included in our topics.

Enzymes are biocatalysts. They are useful in environmentally friendly production processes and have high potential for industrial applications. However, because of problems with operational stability, cost, and catalytic efficiency, many enzymatic processes have limited applications. The use of cross-linked enzyme aggregates (CLEAs) has been introduced as an effective carrier-free immobilization method. This immobilization method is attractive because it is simple and robust, and unpurified enzymes can be used. Coimmobilization of different enzymes can be achieved. CLEAs generally show high catalytic activities, good storage and operational stabilities, and good reusability. In this review, we summarize techniques for the preparation of CLEAs for use as biocatalysts. Some important applications of these techniques in chemical synthesis and environmental applications are also included. CLEAs provide feasible and efficient techniques for improving the properties of immobilized enzymes for use in industrial applications.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

Immobilized enzymes possess catalytic activity as well as substrate specificity. Even though enzyme immobilization techniques for industrial applications are well established, the operational stability of immobilized enzymes often needs to be improved. Laccases have been applied for the degradation of endocrine-disrupting chemicals (EDCs) or organic synthesis. In this study, using the technique of poly-lysine (poly-Lys)-supported cross-linking, laccase molecules were for the first time immobilized as enzyme aggregates onto PEGA resin (Lac–PEGA) and the prepared Lac–PEGA was evaluated in its operational stability and ability to degrade the EDC bisphenol A (BPA). Lac–PEGA was prepared through a simple and inexpensive immobilization technique without any costly equipment. Lac–PEGA showed similar thermal and pH stabilities to those of free laccase, suggesting that immobilized laccase maintains the conformation required for its catalytic activity. Compared with free laccase and Lac–PEGA(−), which was prepared without addition of poly-Lys, Lac–PEGA was more stable in organic solvents and could be reused for 50 cycles without appreciable loss of activity. Moreover, Lac–PEGA could effectively remove BPA and its derivatives in a batch reactor. In the batch reactor, a BPA removal efficiency of 144 μM h⁻¹ was obtained at 30 °C. Due to its good operational stability, Lac–PEGA was successfully applied for reactions with high removal efficiency in a flow reactor. In the flow reactor, a BPA removal efficiency of 2880 μM h⁻¹ was obtained at 50 °C, much superior to other reported reactors by the laccase immobilized with traditional carrier-bound immobilization methods. These results demonstrated that Lac–PEGA with specific enzymatic activity and high operational stability can be used as catalysts not only for the efficient removal of BPA from the environment but also for promising applications in the fields of chemistry and biotechnology.