Enzyme Reactor Research Articles

Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production

Lipases catalyze a wide range of industrially important reactions, including the transesterification of triglycerides with alcohols for biodiesel production, and the stabilization of lipases are critical to achieve their recycled uses. Here, nanoscale enzyme reactor (NER) of lipase from Rhizopus oryzae (LP) was prepared via a simple two-step process, comprising of enzyme adsorption into magnetically-separable mesoporous silica and follow-up crosslinking of adsorbed enzymes. In aqueous phase, the specific hydrolysis activity of NER-LP was 4.7 times lower than that of free LP. On the other hand, however, the specific transesterification activity of NER-LP (130.4 μmol/min/mg LP) in organic phase for biodiesel production was 50 times higher than that of free LP (2.6 μmol/min/mg LP). These results reveal that the enzyme crosslinking for the preparation of NER does not interfere with the interfacial activation of LP molecules, opening the lid of LP active site under an optimal hydrophobic environment provided by the combination of organic solvent and mesoporous silica. Magnetic separation and optimized washing protocol facilitated the recycled uses of NER-LP. Highly stable and active NER-LP in magnetically-separable mesoporous silica has demonstrated its great potentials as an environmentally-friendly nanobiocatalyst for various lipase applications, including plasticizers, biosurfactants, functional fatty acids, as well as recyclable biodiesel production.

Laccase immobilization in polyelectrolyte multilayer membranes for 17α-ethynylestradiol removal: Biocatalytic approach for pharmaceuticals degradation.

Enzymatic membrane reactors equipped with multifunctional biocatalytic membranes are promising and sustainable alternatives for removal of micropollutants, including steroid estrogens, under mild conditions. Thus, in this study an effort was made to produce novel multifunctional biocatalytic polyelectrolyte multilayer membranes via polyelectrolyte layer-by-layer assembly with laccase enzyme immobilized between or into polyelectrolyte layers. In this study, multifunctional biocatalytic membranes are considered as systems composed of commercially available filtration membrane modified by polyelectrolytes and immobilized enzymes, which are produced for complex treatment of water pollutants. The multifunctionality of the proposed systems is related to the fact that these membranes are capable of micropollutants removal via simultaneous catalytic conversion, membrane adsorption and membrane rejection making remediation process more complex, however, also more efficient. Briefly, cationic poly-l-lysine and polyethylenimine as well as anionic poly(sodium 4-styrenesulfonate) polyelectrolytes were deposited onto NP010 nanofiltration and UFX5 ultrafiltration membranes to produce systems for removal of 17α-ethynylestradiol. Images from scanning electron microscopy confirm effective enzyme deposition, whereas results of zeta potential measurements indicate introduction of positive charge onto the membranes. Based on preliminary results, four membranes with over 70%, activity retention produced using polyethylenimine in internal and entrapped mode, were selected for degradation tests. Systems based on UFX5 membrane allowed over 60% 17α-ethynylestradiol removal within 100min, whereas NP010-based systems removed over 75% of estrogen within 150min. Further, around 80% removal of 17α-ethynylestradiol was possible from the solutions at concentration up to 0.1mg/L at pH ranging from 4 to 6 and at the pressure up to 3bar, indicating high activity of the immobilized laccase over wide range of process conditions. Produced systems exhibited also great long-term stability followed by limited enzyme elution from the membrane. Finally, removal of over 70% and 60% of 17α-ethynylestradiol, respectively by NP010 and UFX5 systems after 8 cycles of repeated use indicate high reusability potential of the systems and suggest their practical application in removal of micropollutants, including estrogens.

The application of bioremediation in wastewater treatment plants for microplastics removal: a practical perspective.

Wastewater treatment plants (WWTPs) play the role of intercepting microplastics in the environment and provide a platform for bioremediation to remove microplastics. Despite, this opportunity has not been adequately studied. This paper shows the potential ways microplastics-targeted bioremediation could be incorporated into wastewater treatment through the review of relevant literature on bioaugmentation of watertreatment processes for pollutants removal. Having reviewed more than 90 papers in this area, it highlights that bioremediation in WWTPs can be employed through bioaugmentation of secondary biological treatment systems, particularly the aerobic conventional activated sludge, sequencing batch reactor, membrane bioreactor and rotating biological contactor. The efficiency of microplastics removal, however, is influenced by the types and forms of microorganisms used, the polymer types and the incubation time (100% for polycaprolactone with Streptomyces thermoviolaceus and 0.76% for low-density polyethylene with Acinetobacter iwoffii). Bioaugmentation of anaerobic system, though possible, is constrained by comparatively less anaerobic microplastics-degrading microorganisms identified. In tertiary system, bioremediation through biological activated carbon and biological aerated filter can be accomplished and enzymatic membrane reactor can be added to the system for deployment of biocatalysts. During sludge treatment, bioaugmentation and addition of enzymes to composting and anaerobic digestion are potential ways to enhance microplastics breakdown. Limitations of bioremediation in wastewater treatment include longer degradation time of microplastics, incomplete biodegradation, variable efficiency, specific microbial activities and uncertainty in colonization. This paper provides important insight into the practical applications of bioremediation in wastewater treatment for microplastics removal.

Enzymatic cascade reaction in simple-coacervates

The design of enzymatic droplet-sized reactors constitutes an important challenge with many potential applications such as medical diagnostics, water purification, bioengineering, or food industry. Coacervates, which are all-aqueous droplets, afford a simple model for the investigation of enzymatic cascade reaction since the reactions occur in all-aqueous media, which preserve the enzymes integrity. However, the question relative to how the sequestration and the proximity of enzymes within the coacervates might affect their activity remains open. Herein, we report the construction of enzymatic reactors exploiting the simple coacervation of ampholyte polymer chains, stabilized with agar. We demonstrate that these coacervates have the ability to sequester enzymes such as glucose oxidase and catalase and preserve their catalytic activity. The study is carried out by analyzing the color variation induced by the reduction of resazurin. Usually, phenoxazine molecules acting as electron acceptors are used to characterize glucose oxidase activity. Resazurin (pink) undergoes a first reduction to resorufin (salmon) and then to dihydroresorufin (transparent) in presence of glucose oxidase and glucose. We have observed that resorufin is partially regenerated in the presence of catalase, which demonstrates the enzymatic cascade reaction. Studying this enzymatic cascade reaction within coacervates as reactors provide new insights into the role of the proximity, confinement towards enzymatic activity.

On-flow enzymatic inhibitor screening: The emerging success of liquid chromatography-based assays

Enzymes are targets commonly explored in screening assays aiming to discover new leads in the drug development process. Among the diverse assay models to identify new enzymatic inhibitors, on-flow assays based on liquid chromatography (LC) can be highlighted. In these approaches, the ligand-enzyme interaction can be examined by monitoring the catalytic activity or the affinity/retention. Most applications use the biological target immobilized in solid supports resulting in the acquisition of an immobilized enzymatic reactor (IMER). Coupling IMERs to LC or mass spectrometry (MS) systems allows monitoring enzyme activity online and studying binding events between target and ligands. On-flow screening assays present many advantages for the hit-to-lead process, such as the possibility of system automation, reusability, and high stability. This review covers articles from the last decade that combine the use of varied immobilization methods on different solid supports and several equipment setups in on-flow systems, emphasizing the performance and capacity of recognizing and identifying biologically active compounds in various matrices.

Flow-Through Acetylcholinesterase Sensor with Replaceable Enzyme Reactor.

Fast and reliable determination of enzyme inhibitors are of great importance in environmental monitoring and biomedicine because of the high biological activity and toxicity of such species and the necessity of their reliable assessment in many media. In this work, a flow-through biosensor has been developed and produced by 3D printing from poly(lactic acid). Acetylcholinesterase from an electric eel was immobilized on the inner walls of the reactor cell. The concentration of thiocholine formed in enzymatic hydrolysis of the substrate was monitored amperometrically with a screen-printed carbon electrode modified with carbon black particles, pillar[5]arene, electropolymerized Methylene blue and thionine. In the presence of thiocholine, the cathodic current at −0.25 V decreased because of an alternative chemical reaction of the macrocycle. The conditions of enzyme immobilization and signal measurements were optimized and the performance of the biosensor was assessed in the determination of reversible (donepezil, berberine) and irreversible (carbofuran) inhibitors. In the optimal conditions, the flow-through biosensor made it possible to determine 1.0 nM–1.0 μM donepezil, 1.0 μM–1.0 mM berberine and 10 nM to 0.1 μM carbofuran. The AChE biosensor was tested on spiked samples of artificial urine for drugs and peanuts for carbofuran. Possible interference of the sample components was eliminated by dilution of the samples with phosphate buffer. Easy mounting, low cost of replaceable parts of the cell and satisfactory analytical and metrological characteristics made the biosensor a promising future application as a point-of-care or point-of-demand device outside of a chemical laboratory.

Fabrication of Polymer Membrane Enzyme Reactors with a Dual-Responsive Feature for Cascade Enzyme Reaction Regulation.

Enzymes are known to drive biological processes efficiently and uniquely. However, the integration of multienzymes and immobilization technology to improve the stability of enzymes and boost the catalytic efficiency in cascade reactions by controlling the biocatalysis process in complex conditions via an external stimulus is still facing a significant challenge. Herein, we demonstrate a concept for cascade enzyme reaction regulation by controlling the catalytic efficiency step by step based on a dual-stimuli-responsive porous polymer membrane enzyme reactor (DPMER). Poly(styrene-maleicanhydride-acrylic acid-4-[(4-methacryloyloxy)phenylazo] benzoic acid) was designed for the fabrication of the DPMER with glutaminase and alanine aminotransferase as the model modified enzymes. Under UV light irradiation and by regulating the solution pH, the cascade enzyme reaction was carried out step by step. The polymer membrane surface demonstrated a configuration change while improving the enzymolysis efficiency of the DPMER. The enzymatic kinetics of the DPMER was investigated by a chiral capillary electrophoresis technique, and the cascade enzyme reaction regulation capability was evaluated. Under pH of 4.9 and 365 nm UV irradiation, the poly(4-[(4-methacryloyloxy)phenylazo] benzoic acid) and poly(acrylic acid) moieties changed from a "stretched state" to a "curled state" to form surface nanopores, which embedded the enzymes into the surface nanopores while causing the spatial confinement effect and enhancing the enzymolysis efficiency of the DPMER by 9.9-fold in comparison with the free enzymes. This concept provided a potential platform for cascade enzyme reaction regulation and highlighted the perspective of stimuli-responsive polymers.

Controllable Enzyme Immobilization via Simple and Quantitative Adsorption of Dendronized Polymer-Enzyme Conjugates Inside a Silica Monolith for Enzymatic Flow-Through Reactor Applications.

Although many different methods are known for the immobilization of enzymes on solid supports for use in flow-through applications as enzyme reactors, the reproducible immobilization of predetermined amounts of catalytically active enzyme molecules remains challenging. This challenge was tackled using a macro- and mesoporous silica monolith as a support and dendronized polymer–enzyme conjugates. The conjugates were first prepared in an aqueous solution by covalently linking enzyme molecules and either horseradish peroxidase (HRP) or bovine carbonic anhydrase (BCA) along the chains of a water-soluble second-generation dendronized polymer using an established procedure. The obtained conjugates are stable biohybrid structures in which the linking unit between the dendronized polymer and each enzyme molecule is a bisaryl hydrazone (BAH) bond. Quantitative and reproducible enzyme immobilization inside the monolith is possible by simply adding a defined volume of a conjugate solution of a defined enzyme concentration to a dry monolith piece of the desired size. In that way, (i) the entire volume of the conjugate solution is taken up by the monolith piece due to capillary forces and (ii) all conjugates of the added conjugate solution remain stably adsorbed (immobilized) noncovalently without detectable leakage from the monolith piece. The observed flow-through activity of the resulting enzyme reactors was directly proportional to the amount of conjugate used for the reactor preparation. With conjugate solutions consisting of defined amounts of both types of conjugates, the controlled coimmobilization of the two enzymes, namely, BCA and HRP, was shown to be possible in a simple way. Different stability tests of the enzyme reactors were carried out. Finally, the enzyme reactors were applied to the catalysis of a two-enzyme cascade reaction in two types of enzymatic flow-through reactor systems with either coimmobilized or sequentially immobilized BCA and HRP. Depending on the composition of the substrate solution that was pumped through the two types of enzyme reactor systems, the coimmobilized enzymes performed significantly better than the sequentially immobilized ones. This difference, however, is not due to a molecular proximity effect with regard to the enzymes but rather originates from the kinetic features of the cascade reaction used. Overall, the method developed for the controllable and reproducible immobilization of enzymes in the macro- and mesoporous silica monolith offers many possibilities for systematic investigations of immobilized enzymes in enzymatic flow-through reactors, potentially for any type of enzyme.

Transient response of immobilized enzyme reactors - the effects of reactor type and shape of core-shell bio-catalytic pellets

Transient response of immobilized enzyme reactors - the effects of reactor type and shape of core-shell bio-catalytic pellets

Nanoplatforms with donor-acceptor Stenhouse adduct molecular switch for enzymatic reactions remotely controlled with near-infrared light

Enzymes have been widely used in biomedical applications owing to their excellent biocatalysis functions. However, the precise control of enzymatic reactions remains a challenge in vivo. Here, a nanoplatform functionalized with upconversion nanoparticles (UCNPs) as the core and a donor-acceptor Stenhouse adduct as a molecular switch (MS) was designed for near-infrared (NIR)-controlled enzymatic reactions. Under NIR irradiation, the UCNPs could emit bright green light to isomerize the MS and then transform the permeability of the MS-gated polymer layer, allowing small molecules to penetrate the polymer layer. Consequently, the contact between the enzyme and substrate could be regulated to control the enzymatic reaction remotely. As the photoisomerism of MS is reversible and the enzyme activity is not changed, the enzyme reactor based on this nanoplatform is also reversible, which can achieve precise temporal and spatial control of the enzyme reaction. Glucose oxidase (GOX), lactate oxidase (LOX), urate oxidase (UOX), and luciferase were used to assess the controllability of the enzymatic reactions. Both the substrates in and out of the nanoplatform were satisfactorily controlled, indicating that the gated nanoplatform equipped with valve-like characteristics could realize remotely controlled enzymatic reactions upon NIR irradiation.

Spatial confinement of multi-enzyme for cascade catalysis in cell-inspired all-aqueous multicompartmental microcapsules

Biocatalytic reaction networks in eukaryotic cells is realized by the immobilized and compartmental multi-enzymatic system. Inspired by the spatial localization of natural cells, multiple enzymes were confined within the multicompartmental microcapsules, which were created using a gas-shearing method coupled with surface-triggered in situ gelation strategy. Heterogeneous multicompartmental (two-, three-, four-, six-, or eight-faced) core particles, due to their capacity for positional assembly, were encapsuled in alginate hydrogel shells. The generated microcapsules integrate logic network to access complex digital design through a three-step convergent enzymatic cascade reaction as a model, and the capsules with high stability, recyclability and cytocompatibility are ideal enzymatic reactor systems to be used for biomimetic biocatalysis process.

Integrated microsphere-packed bed enzymatic membrane reactor for enhanced bioconversion efficiency and stability: A proof-of-concept study

Fabricating high-performance enzyme reactors is requested for achieving efficient and stable bioconversions, but remains challenging, because few of them can possess high enzyme loading, sufficient mixing, and efficient mass transfer at the same time. Herein, we propose to develop a novel enzymatic packed bed membrane reactor (EPBMR) by integrating the advantages of both packed bed reactor (PBR) and enzymatic membrane reactor (EMR). A prototype study is conducted with the simplified enzyme-loaded microsphere-ultrafiltration EMR model (Mic-UF EMR). Invertase and dextranase are used in this work to produce glucose and oligodextran by hydrolysis of sucrose and dextran, respectively. Specifically, the use of microspheres can enlarge the contact area between enzymes and substrates and mitigate membrane fouling induced by free enzymes. Thus, Free&Mic-UF EMR (with both free and immobilized enzymes) exhibits a higher sucrose conversion rate (84%) than the EMR with free invertase (34%) and a negligible decline in sucrose conversion for 36 h continuous operation. Membrane fouling is ameliorated by alkaline cleaning and implementation of covalent bonding strategy. In addition, commercial resins with larger sizes are employed to replace konjac glucomannan microspheres (KGM) which reduce the pressure drop of EMR. Finally, by selecting the UF membrane with proper molecular weight cut-off (MWCO), the dextranase-based Mic-UF EMR system successfully produces oligodextran with desired molecular weight (Mw) and narrow Mw distribution. The outcome of this work not only offers a novel enzyme reactor construction strategy but also provides guidance for regulating the performance of EMR.

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis.

Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.

Deacidification of high-acid rice bran oil by the tandem continuous-flow enzymatic reactors

Rice bran oil (RBO) contains a variety of nutrients, but the high acid values largely hinder its processing into edible oil. Thus, the tandem continuous-flow reactors are proposed and developed for the enzymatic deacidification of RBO and simultaneous production of functional oils. The results indicate that the Candida antarctica lipase B (CALB) immobilized on the hydrophobic ordered mesoporous silicon (OMS-C18) increased 6.6 times of the catalytic activity and improved at least 20 ℃ of temperature tolerance compared to the commercial Novozym 435. The tandem continuous-flow enzymatic reactors removed 91.4% of free fatty acid and increased 9 and 12 times of phytosterol ester and diacylglycerol in RBO, respectively. Moreover, the retention rate of γ-oryzanol was at least 40% higher than that obtained by traditional alkali refining. This study provides an effective and sustainable method to continuously convert the low-value RBO into value-added products, which brings huge potential to cleaner industrial production.

Flowing on-line preparation of deglycosylation, labeling and purification for N-glycan analysis

The current in-solution analysis of N-glycans suffers from several disadvantages including tedious de-glycosylation time and multi-step pre-treatment procedures. Here, an ultra-simple flowing on-line analysis of labeled N-glycans for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for eliminating the deficiencies. This on-line analysis consisted of an immobilized enzyme reactor (IMER) of PNGase F for efficient release of N-glycans, labeling of released N-glycans and following purification of derivatives on microfluidic chip. Notably, efficient preparations for all type of N-glycans were completed within ∼30 min. To our best knowledge, this is the first time to integrated the whole preparation of N-glycan deglycosylation, labeling and purification only by a simple fluidic flow with our developed device. Good reproducibility and stability were achieved with the relative standard deviation (RSD) less than 10%. Furthermore, the glycome studies with human serum revealed a good adaptability for biological samples. Our work provides an efficient N-glycomic strategy that can be applied to further multilayered clinical analysis.

Experimental and modeling of tetracycline degradation in water in a flow-through enzymatic monolithic reactor.

In this work, the laccase from Trametes versicolor was immobilized in highly porous silica monoliths (0.6-cm diameter, 0.5-cm length). These monoliths feature a unique homogeneous network of interconnected macropores (20 μm) with mesopores (20 nm) in the skeleton and a high specific surface area (330 m2/g). The enzymatic monoliths were applied to degrade tetracycline (TC) in model aqueous solutions (20 ppm). For this purpose, a tubular flow-through reactor (FTR) configuration with recycling was built. The TC degradation was improved with oxygen saturation, presence of degradation products, and recirculation rate. The TC depletion reaches 50% in the FTR and 90% in a stirred tank reactor (CSTR) using crushed monoliths. These results indicate the importance of maintaining a high co-substrate concentration near active sites. A model coupling mass transfers with a Michaelis-Menten kinetics was applied to simulate the TC degradation in real wastewaters at actual TC concentration (2.8 10-4 ppm). Simulation results show that industrial scale FTR reactor should be suitable to degrade 90% of TC in 5 h at a flow rate of 1 mL/min in a single passage flow configuration. Nevertheless, the process could certainly be further optimized in terms of laccase activity, oxygen supply near active sites, and contact time.

Production of 4-Ethyl Malate through Position-Specific Hydrolysis of Photobacterium lipolyticum M37 Lipase.

Microbial lipases are used widely in the synthesis of various compounds due to their substrate specificity and position specificity. 4-Ethyl malate (4-EM) made from diethyl malate (DEM) is an important starting material used to make argon fluoride (ArF) photoresist. We tested several microbial lipases and found that Photobacterium lipolyticum M37 lipase position-specifically hydrolyzed DEM to produce 4-EM. We purified the reaction product through silica gel chromatography and confirmed that it was 4-EM through nuclear magnetic resonance analysis. To mass-produce 4-EM, DEM hydrolysis reaction was performed using an enzyme reactor system that could automatically control the temperature and pH. Effects of temperature and pH on the reaction process were investigated. As a result, 50°C and pH 4.0 were confirmed as optimal reaction conditions, meaning that M37 was specifically an acid lipase. When the substrate concentration was increased to 6% corresponding to 0.32 M, the reaction yield reached almost 100%. When the substrate concentration was further increased to 12%, the reaction yield was 81%. This enzyme reactor system and position-specific M37 lipase can be used to mass-produce 4-EM, which is required to synthesize ArF photoresist.

Synergistic action of laccase treatment and membrane filtration during removal of azo dyes in an enzymatic membrane reactor upgraded with electrospun fibers

Nowadays, the increasing amounts of dyes present in wastewaters and even water bodies is an emerging global problem. In this work we decided to fabricate new biosystems made of nanofiltration or ultrafiltration membranes combined with laccase entrapped between polystyrene electrospun fibers and apply them for decolorization of aqueous solutions of three azo dyes, C.I. Acid Yellow 23 (AY23), C.I. Direct Blue 71 (DB71) and C.I. Reactive Black 5 (RB5). Besides effective decolorization of the permeate stream, the biosystems also allowed removal of dyes from the retentate stream as a result of enzymatic action. The effect of pH and applied pressure on decolorization efficiencies was investigated, and pH 5 and pressure of 2 bar gave the highest removal efficiencies of 97% for AY23 and 100% for both DB71 and RB5 from permeate solutions while decolorization of retentate for RB5 reached 65% under these conditions. Almost 100% decolorization of all dyes was achieved after three consecutive enzyme membrane cycles. Decolorization was shown to be due to the synergistic action of membrane separation and bioconversion. The biocatalytic action also enabled significant reduction of permeate and retentate toxicity, which is one of the biggest environmental health issues for these types of streams.

Síntesis benigna y sencilla de dímeros de tipo lignano usando peroxidasa cruda de rábano rojo (Raphanus sativus var sativus)

Nowadays, immobilized enzymes are utilized in several food industry applications. Some researches use apparent kinetic parameters in immobilized enzyme reactor systems, which are limited to such case studies. To enhance productivity in a packed bed reactor (PBR), a clear description of all the mechanisms (kinetic, intra-particle diffusive mass transport, fluid-particle convective mass transport, and axial dispersion) affecting the process should be established. The objective of this study was to model the isomerization of glucose in a PBR with calcium alginate beads (CAB), using an approach where the kinetic and diffusional mechanisms are described independently. The convective mass transfer (kL) and axial dispersion (Dz) coefficients were calculated from correlations. Validation was performed comparing predictions against experimental data (R2 = 0.907) of glucose conversion at the reactor’s outlet once steady state was reached. Under the study conditions, in contrast to the effect exerted by diffusive mass transport on fructose specific productivity, the effect of axial dispersion and convective mass transport is negligible. Analyzing different operation parameters via simulation, the particle size had the highest impact on the glucose bioconversion. By reducing the CAB size, the surface area is increased and thus the conversion. It is recommended to test new immobilizing agents or decreasing the CAB size, monitoring that immobilizing support preserves its stability and functionality.

Precipitation-based microscale enzyme reactors coupled with porous and adhesive elastomer for effective bacterial decontamination and membrane antifouling on-demand

Bacterial contamination of water environments can cause various troubles in various areas. As one of potential solutions, we develop enzyme-immobilized elastomer, and demonstrate the uses of enzyme reactions on-demand for effective microbial decontamination and antifouling. Asymmetrically-structured elastomer is prepared by combining two polydimethylsiloxane (PDMS) layers with different degrees of crosslinking: highly-crosslinked and lightly-crosslinked PDMS layers. At the surface of highly-crosslinked PDMS layer, porous structure with average diameter of 842 nm is formed by dissolving pre-packed and entrapped latex beads. Lightly-crosslinked PDMS on the other side, due to its adhesive nature, enables iterative attachments on various materials under either dry or wet condition. Glucose oxidase (GOx) is immobilized by using the pores at the surface of highly-crosslinked PDMS matrix via a ship-in-a-bottle protocol of precipitation-based microscale enzyme reactor (p-MER), which consists of GOx adsorption, precipitation and chemical crosslinking (EAPC). As a result, crosslinked enzyme aggregates (CLEAs) of GOx not only are well entrapped within many pores of highly-crosslinked PDMS layer (ship-in-bottle) but also cover the external surface of matrix, both of which are well connected together. Highly-interconnected network of CLEAs themselves effectively prevents enzyme leaching, which shows the 25% residual activity of GOx under shaking at 200 rpm for 156 days after 48% initial drop of loosely-bound p-MER after 4 days. In presence of glucose, the underwater attachment of biocatalytic elastomer demonstrates the generation of hydrogen peroxide via p-MER-catalyzed glucose oxidation, exhibiting effective biocidal activities against both gram-positive S. aureus and gram-negative E. coli. Adhesion-induced GOx-catalyzed reaction also alleviates the biofouling of membrane, suggesting its extendibility to various engineering systems being suffered by biofouling. This study of biocatalytic elastomer has demonstrated its new opportunities for the facile and on-demand enzyme-catalyzed reactions in various environmental applications, such as bactericidal treatment, water treatment/purification, and pollutant degradation.