Enzymatic Processing in Microfluidic Reactors

Abstract

Microreaction technology is an interdisciplinary area of science and engineering. It has attracted the attention of researchers from different fields in the past few years and consequently, several microreactors have been developed. Enzymes are organic catalysts used for the production useful substances in an environmentally friendly way, and have high potential for analytical applications. However, relatively few enzymatic processes have been commercialized because of problems in the stability of enzyme molecule, and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies and focus our discussion on enzyme microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared with the conventional reactors. Fundamental techniques for enzyme microreactors and important applications of this multidisciplinary technology in chemical processing are also included in our topics.     

Microfluidic combinatorial chemistry

Microreactors are finding increasing application in the field of combinatorial chemistry. In the past few years, microreactor chemistry has shown great promise as a novel method on which to build new chemical technology and processes. It has been conclusively demonstrated that reactions performed within microreactors invariably generate relatively pure products in high yield. One of the immediate and obvious applications is therefore in combinatorial chemistry and drug discovery.     

Microreactors for chemical synthesis.

The advances of the past few years in microreactors have demonstrated that the miniaturization of chemistry has significant advantages with respect to cost, safety, throughput, kinetics and scale-up. The use of chemical microreactors for catalytic oxidations, heterocyclic syntheses and photochemical reactions has illustrated the utility and benefits for both chemical discovery and chemical development applications.     

Expanding the boundary of biocatalysis: design and optimization of in vitro tandem catalytic reactions for biochemical production

Biocatalysts have been increasingly used in the synthesis of fine chemicals and medicinal compounds due to significant advances in enzyme discovery and engineering. To mimic the synergistic effects of cascade reactions catalyzed by multiple enzymes in nature, researchers have been developing artificial tandem enzymatic reactions in vivo by harnessing synthetic biology and metabolic engineering tools. There is also growing interest in the development of one-pot tandem enzymatic or chemo-enzymatic processes in vitro due to their neat and concise catalytic systems and product purification procedures. In this review, we will briefly summarize the strategies of designing and optimizing in vitro tandem catalytic reactions, highlight a few representative examples, and discuss the future trend in this field.     

Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

Multifunctional cargo systems for biotechnology

One of the challenges in the field of bio-nanotechnology is the development of nano-sized delivery systems comprising different functionalities. These systems should carry bioactive substances to predefined site and unload them in controlled manner. Capsules assembled layer-by-layer have been intensively studied in the past few years owing to their ability to be modified, their capacity to encapsulate a wide range of chemicals, their responsiveness to different factors, and the variety of functionalities with which they can be enhanced. Current research focuses on the development of carriers with remote guiding and activation (optical, magnetic or ultrasound), thereby addressing unique in vivo applications with multifunctional biomaterials. Submicron-sized capsules are good models to mimic biochemical processes in a confined geometry that imitates cell organelles. Moreover, the cellular and tissue-targeted delivery of the capsules might serve as an intracellular reporter or enzymatic reactor. However, several obstacles still have to be overcome before capsule technology can be implemented. This article discusses possible solutions as well as promising applications.     

Flow-through polymerase chain reactions in chip thermocyclers

The miniaturization of analytical devices by micromachining technology is destined to have a major impact on medical and bioanalytical fields. To meet the current demands for rapid DNA amplification, various instruments and innovative technologies have been introduced by several groups in recent years. The development of the devices was extended in different directions and adapted to corresponding applications. In this review the development of a variety of devices and components for performing DNA amplification as well as the comparison of batch-process thermocyclers with reaction chambers and flow-through devices for different purposes are discussed. The main attention is turned to a flow device concept for thermocycling using microfabricated elements for local heat flow management, for which simulations and considerations for further improvement regarding design, material choice and applied technology were performed. The present review article mainly discusses and compares thermocycling devices for rapid thermocycling made of silicon or of silicon and glass with a short excursion to the possibility of plastic chip devices. In order to perform polymerase chain reactions (PCRs) in the microreactors, special attention must be paid to the conditions of the internal surfaces. For microchips, surface effects are generally pronounced because the surface to volume ratio increases upon miniaturization. Solutions for solving this problem are presented. We propose an overview of layouts for batch-process thermocyclers with different parallelization of reaction chambers and also of different designs of continuous flow thermocycling chips, paying particular attention to the parameters which influence the efficiency of such chip devices. Finally we point out some recent issues for applications in the field of clinical diagnostics.     

Enzymatic microreactors in chemical analysis and kinetic studies

The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Patenting trends in enzyme related microfluidic applications

The miniaturization of continuous processes has been of interest in the academia and industry which is reflected by the increase in scientific publications and patent disclosures in the last decade. The aim of this study was to evaluate the patenting trends regarding enzyme related microfluidic applications in order to observe the progress of science and technology. The mapped patents have been classified as “immobilization method”, “biomolecule screening systems”, “integrated process development” and “microreactor design”. Half of the patent disclosures were filed by academia, whereas the other half was from industrial research which complies with the shift in microfluidics from academic and industrial research to commercial applications. Immobilization procedures carried out at room temperatures such as formulation of silica matrices using sol–gel technique, incorporation of novel hybrid materials, the integration of supercritical fluids and microfluidics, employing ionic liquids as wall-less microreactors, designing low cost, high performance microfluidic devices were the highlights which can pose challenges in various life science applications. The increasing trend is expected to continue and the presented state-of-the-art in enzyme related microfluidic applications have the potential to enhance industry's capabilities for designing innovative systems which would demonstrate significant economic, societal and environmental benefits.     

Candida spp. redox machineries: An ample biocatalytic platform for practical applications and academic insights

The use of oxidoreductases as biocatalysts for the production of a wide number of chiral building blocks is presently a mature (bio-)technology. In this context some industrial applications are currently performed by means of those enzymatic approaches, and new examples are expected to be realized. Moreover, oxidoreductases provide an interesting academic platform to undertake fundamental research in enzymology, to acquire a better understanding on catalytic mechanisms, and to facilitate the development of new biocatalytic applications. Within this area, a wide number of oxidoreductases from genus Candida spp. have been characterized and used as biocatalysts. These enzymes are rather diverse, and are able to carry out many useful reactions, like highly (enantio)selective keto-reductions, (de)racemizations and stereoinversions, and promiscuous catalytic imine reductions. In addition, some Candida spp. dehydrogenases are very useful for regenerating the cofactors, with the aid of sacrificial substrates. Addressing those features, the present paper aims to give an overview of these enzymes, by focusing on practical applications that these biocatalysts can provide. Furthermore, when possible, academic insights on the enzymatic performances will be discussed as well.     

多孔纳米材料固定化酶研究进展

酶是一种天然生物催化剂,有催化效率高、底物选择性强和绿色环保等优点,但酶结构不稳定且重复利用率低,制约了其产业化应用。随着技术的发展,酶的固定化可以提高酶的活性和稳定性,为生物酶的工程化应用带来了新的机遇。多孔纳米材料具有比表面积大、孔隙率高、机械和化学性能稳定等特点和优异的成本效益,是理想的固定化酶载体。本文综述了近些年来金属有机框架、共价有机框架和多孔微球等纳米材料固定化酶的研究进展和应用,重点介绍了载体固定酶的方式,并总结了每种载体的特点,最后讨论了多孔纳米材料固定化酶面临的挑战和发展趋势。     

Bioorganic reactions in microemulsions: the case of lipases

Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. Important examples are the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. This review illustrates the biotechnological applications of microemulsions as media for bioorganic reactions. The principal focus is on lipase catalyzed processes.     

Cytochrome P450 monooxygenases: perspectives for synthetic application

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.     

Charge-transfer processes and redox reactions in planar lipid monolayers and bilayers

Supported bilayer lipid membranes (BLMs) and lipid monolayers have been known for quite sometime and are attracting sustained interest since they open new research vista and offer practical approaches in biosensor development and molecular device applications. Central to these areas of interest are electric processes and redox reactions where the movement of ions and electrons plays a pivotal role. In this paper an overview of the major findings in this field is presented. Further, we summarize the work on planar lipid bilayers and monolayers that have been done in the past few years in a number of laboratories. Supported planar BLMs and their closely related systems provide the foundation for a variety of lipid bilayer-based molecular sensors that are sensitive, versatile, as well as potentially inexpensive (i.e., disposable), and open to all sorts of experimentation.     

Impact of enzyme motion on activity

Experimental and theoretical data imply that enzyme motion plays an important role in enzymatic reactions. Enzyme motion can influence both the activation free energy barrier and the degree of barrier recrossing. A hybrid theoretical approach has been developed for the investigation of the relation between enzyme motion and activity. This approach includes both electronic and nuclear quantum effects. It distinguishes between thermally averaged promoting motions that influence the activation free energy barrier and dynamical motions that influence the barrier recrossings. Applications to hydride transfer in liver alcohol dehydrogenase and dihydrofolate reductase resulted in the identification and characterization of important enzyme motions. These applications have also led to the proposal of a network of coupled promoting motions in enzymatic reactions. These concepts have important implications for protein engineering and drug design.     

Responsive systems for cell sheet detachment

Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.     

Application of microorganisms towards synthesis of chiral terpenoid derivatives

Biotransformations are a standard tool of green chemistry and thus are following the rules of sustainable development. In this article, we describe the most common types of reactions conducted by microorganisms applied towards synthesis of chiral terpenoid derivatives. Potential applications of obtained products in various areas of industry and agriculture are shown. We also describe biological activity of presented compounds. Stereoselective hydroxylation, epoxidation, Baeyer-Villiger oxidation, stereo- and enantioselective reduction of ketones, and various kinetic resolutions carried out by bacteria and fungi have been reviewed. Mechanistic considerations regarding chemical and enzymatic reactions are presented. We also briefly describe modern approaches towards enhancing desired enzymatic activity in order to apply modified biocatalysts as an efficient tool and green alternative to chemical catalysts used in industry.     

Development of applications of industrial enzymes from Malaysian indigenous microbial sources

Malaysian enzyme industry is considered almost non-existence, although the import volume is large. Realizing the importance of enzymes, encompassing a wide range of applications in bioindustry, the development of home grown technologies for enzyme production and applications becomes one of the national priorities in industrial biotechnology. Enzyme production from indigenous microbial isolates was performed either by submerged or solid state fermentation processes. Based on its wide and unique spectrum of properties, enzymes have been developed for wide applications in various industrial processes. The development of the enzyme catalysed applications is based on the modification of the reaction systems to enhance their catalytic activities. Some of the applications of the industrial enzymes include the fine chemicals production, oleochemicals modification, detergent formulation, enzymatic drinking of waste papers, animal feed formulation and effluent treatment processes. Enzymes have also shown to be successfully used as analytical tool in the determination of compounds in body fluids. Although, most of these enzyme catalysed reactions were performed in aqueous phase, the use of enzymes in organic solvents was found to be significant for the production of new chemicals.