Magnetic hollow fibers of covalent organic frameworks (COF) for pollutant degradation and adsorptive removal.

The shaping of covalent organic frameworks (COFs) from non-processible powder forms into applicable architectures with additional functionality remains a challenge. Using pre-electrospun polymer fibers as a sacrificial template, herein, we report a green synthesis of an architecture in the form of COF hollow fibers with an inner layer of peroxidase-like iron oxide nanoparticles as a catalytic material. When compared to peroxidase-like pristine iron oxide nanoparticles, these COF hollow fibers demonstrate higher catalytic breakdown of crystal violet due to their peroxidase-like activity via advanced oxidation process. Furthermore, as a potential adsorbent, hollow COF fibers exhibit significantly effective adsorption capacity and removal efficiency of organic solvent and oil from water. Because of their magnetic nature, COF hollow fibers can be easily recovered and have exhibited high recycling stability for both catalytic dye degradation and organic solvent removal from water.

Development of aptamers for rapid airborne bacteria detection.

Airborne microbes can rapidly spread and cause various infectious diseases worldwide. This necessitates the determination of a fast and highly sensitive detection method. There have been no studies on receptors targeting Citrobacter braakii (C. braakii), a pathogenic bacterium which can exist in the air. In this study, we rapidly isolate an aptamer, a nucleic acid molecule that can specifically bind to C. braakii by centrifugation-based partitioning method (CBPM) reported previously by our groups as omitting the repeated rounds of binding incubation, separation, and amplification that are indispensable for SELEX. The binding affinity and specificity of isolated aptamers are checked using bacteria in liquid culture and recollection solution from aerosolized bacteria. Recollection solutions of the recovered bacteria are obtained by nebulizing, drying, and recapturing with a biosampler. The CB-5 aptamer shows high affinity and specificity for C. braakii (Kd: 16.42 in liquid culture and 26.91 nM in recollection from aerosolized sample). Our results indicate the current protocol can be employed for the rapid development of reliable diagnostic receptors targeting airborne bacteria.

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.

Stabilized and Immobilized Carbonic Anhydrase on Electrospun Nanofibers for Enzymatic CO2 Conversion and Utilization in Expedited Microalgal Growth.

Carbonic anhydrases convert CO2 to bicarbonate at a high turnover rate up to 106 s-1, but their actual applications in CO2 conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO2 to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO2 reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO2 conversion and utilization technologies.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents.

Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while Mag-EPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

Microwave-Assisted Protein Digestion in a Plate Well for Facile Sampling and Rapid Digestion.

Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwave-assisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively. EC/3D can effectively prevent the interference of sampling by accommodating EC in the separated compartment from the sampling hole in the middle. EC/3D in the plate well maintained its protein digestion performance under shaking over 160 days. Microwave irradiation enabled the digestion of bovine serum albumin within 10 min, generating the MALDI-TOF MS results of 75.0% sequence coverage and 61 identified peptides. EC/3D maintained its protein digestion performance under microwave irradiation after 30 times of recycled uses. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable immobilized enzymes and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications, including bioconversion and biosensors.

In vitro gene expression-coupled bacterial cell chip for screening species-specific antimicrobial enzymes.

Targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, bacterial autolysins, and other bacteriolysins are useful antibiotic alternatives due to their exceptional target selectivity, which may be used to lysins rapidly kill target bacteria and their high specificity permit the normal commensal microflora to be left undisturbed. Genetic information of numerous lysins is currently available, but the identification of their antimicrobial function and specificity has been limited because most lysins are often poorly expressed and exhibit low solubilities. Here, we report the development of bacterial cell chip for rapidly accessing the function of diverse genes that are suggestive of encoding lysins. This approach can be used to evaluate rapidly the species-specific antimicrobial activity of diverse lysins synthesized from in vitro transcription and translation (TNT) of plasmid DNA. In addition, new potent lysins can be assessed that are not expressed in hosts and display low solubility. As a result of evaluating the species-specific antimicrobial function of 11 (un)known lysins with an in vitro TNT-coupled bacterial cell chip, a potent recombinant lysin against Staphylococcus strains, SA1, was identified. The SA1 was highly potent against not only S. aureus, but also both lysostaphin-resistant S. simulans and S. epidermidis cells. To this end, the SA1 may be applicable to treat both methicillin-resistant S. aureus (MRSA) and lysostaphin-resistant MRSA mutants. Biotechnol. Bioeng. 2017;114: 1648-1657. © 2017 Wiley Periodicals, Inc.

Ethanol-dispersed and antibody-conjugated polymer nanofibers for the selective capture and 3-dimensional culture of EpCAM-positive cells.

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells.

One-pot enzymatic conversion of carbon dioxide and utilization for improved microbial growth.

We developed a process for one-pot CO2 conversion and utilization based on simple conversion of CO2 to bicarbonate at ambient temperature with no energy input, by using the cross-linking-based composites of carboxylated polyaniline nanofibers (cPANFs) and carbonic anhydrase. Carbonic anhydrase was immobilized on cPANFs via the approach of magnetically separable enzyme precipitate coatings (Mag-EPC), which consists of covalent enzyme attachment, enzyme precipitation, and cross-linking with amine-functionalized magnetic nanoparticles. Mag-EPC showed a half-life of 236 days under shaking, even resistance to 70% ethanol sterilization, and recyclability via facile magnetic separation. For one-pot CO2 conversion and utilization, Mag-EPC was used to accelerate the growth of microalga by supplying bicarbonate from CO2, representing 1.8-fold increase of cell concentration when compared to the control sample. After two repeated uses via simple magnetic separation, the cell concentration with Mag-EPC was maintained as high as the first cycle. This one-pot CO2 conversion and utilization is an alternative as well as complementary process to adsorption-based CO2 capture and storage as an environmentally friendly approach, demanding no energy input based on the effective action of the stabilized enzyme system.

Biocatalytic carbon capture via reversible reaction cycle catalyzed by isocitrate dehydrogenase.

The practice of carbon capture and storage (CCS) requires efficient capture and separation of carbon dioxide from its gaseous mixtures such as flue gas, followed by releasing it as a pure gas which can be subsequently compressed and injected into underground storage sites. This has been mostly achieved via reversible thermochemical reactions which are generally energy-intensive. The current work examines a biocatalytic approach for carbon capture using an NADP(H)-dependent isocitrate dehydrogenase (ICDH) which catalyzes reversibly carboxylation and decarboxylation reactions. Different from chemical carbon capture processes that rely on thermal energy to realize purification of carbon dioxide, the biocatalytic strategy utilizes pH to leverage the reaction equilibrium, thereby realizing energy-efficient carbon capture under ambient conditions. Results showed that over 25 mol of carbon dioxide could be captured and purified from its gas mixture for each gram of ICDH applied for each carboxylation/decarboxylation reaction cycle by varying pH between 6 and 9. This work demonstrates the promising potentials of pH-sensitive biocatalysis as a green-chemistry route for carbon capture.

Highly stabilized lipase in polyaniline nanofibers for surfactant-mediated esterification of ibuprofen.

Lipase (LP) from Candida rugosa was immobilized and stabilized in polyaniline nanofibers (PANFs) via a three-step process of enzyme adsorption, precipitation, and cross-linking, which generates the final immobilization called "EAPC". The activity of EAPC was 5.1 and 5.9 times higher than those of LP immobilizations via enzyme adsorption (EA) and enzyme adsorption/cross-linking (EAC), respectively. After incubation in an aqueous buffer under shaking (200 rpm) for 84 days, EAPC maintained 74% of its initial activity, while EA and EAC retained 11 and 24% of their initial activities, respectively. Highly stable and active EAPC was employed for the resolution of racemic ibuprofen via esterification of S-(+)-ibuprofen with 1-propanol in isooctane. The addition of 100 mM dioctyl sulfosuccinate (AOT) into the reaction medium increased the esterification activity by 61-fold, which can be explained by the better dispersion of EAPC in isooctane. EAPC showed 42% conversion in the esterification of racemic ibuprofen after 102 h, whereas EA and EAC showed only 1.2 and 1.4% conversion in the same condition, respectively. The EAPC approach increases both loading and stability of LP, and the combination of EAPC with the surfactant addition can be employed for efficient enzymatic reactions in organic solvents.

Effective antifouling using quorum-quenching acylase stabilized in magnetically-separable mesoporous silica.

Highly effective antifouling was achieved by immobilizing and stabilizing an acylase, disrupting bacterial cell-to-cell communication, in the form of cross-linked enzymes in magnetically separable mesoporous silica. This so-called "quorum-quenching" acylase (AC) was adsorbed into spherical mesoporous silica (S-MPS) with magnetic nanoparticles (Mag-S-MPS), and further cross-linked for the preparation of nanoscale enzyme reactors of AC in Mag-S-MPS (NER-AC/Mag-S-MPS). NER-AC effectively stabilized the AC activity under rigorous shaking at 200 rpm for 1 month, while free and adsorbed AC lost more than 90% of their initial activities in the same condition within 1 and 10 days, respectively. When applied to the membrane filtration for advanced water treatment, NER-AC efficiently alleviated the biofilm maturation of Pseudomonas aeruginosa PAO1 on the membrane surface, thereby enhancing the filtration performance by preventing membrane fouling. Highly stable and magnetically separable NER-AC, as an effective and sustainable antifouling material, has a great potential to be used in the membrane filtration for water reclamation.

Nanoimmobilization of marine epoxide hydrolase of Mugil cephalus for repetitive enantioselective resolution of racemic styrene oxide in aqueous buffer.

We developed two nanoimmobilized biocatalyst systems of thermally unstable Mugil cephalus epoxide hydrolase (McEH) for enantioselective resolution of racemic styrene oxide in aqueous buffer. The recombinant and purified McEH enzyme was immobilized onto magnetic nanoparticles (Mag-NPs) via a two step process of enzyme precipitation and crosslinking. McEH enzyme was also adsorbed, precipitated, and cross-linked in/on polyaniline nanofibers (PANFs). The residual relative activity of free McEH, defined as the ratio of residual activity to the initial activity, was 8% after incubation at 30 degrees C for 80 h while those of McEH immobilized onto Mag-NPs and in/on PANFs were 15% and 33% in the same condition, respectively. McEH immobilizations onto Mag-NPs and in/on PANFs could be reused in seven repetitive batch reactions for enantioselective hydrolysis of racemic styrene oxide to prepare (S)-styrene oxide with 98% enantiomeric excess (ee) while retaining greater than 40-50% of their initial activity.

Single step isolation and activation of primary CD3+ T lymphocytes using alcohol-dispersed electrospun magnetic nanofibers.

Electrospun polymer nanofibers with entrapped magnetic nanoparticles (magnetic NP-NF) represent a novel scaffold substrate that can be functionalized for single-step isolation and activation of specific lymphocyte subsets. Using a surface-embedded T cell receptor ligand/trigger (anti-CD3 monoclonal antibody), we demonstrate, as proof of principle, the use of magnetic NP-NF to specifically isolate, enrich, and activate CD3(+) T cells from a heterogeneous cell mixture, leading to preferential expansion of CD8(+)CD3(+) T cells. The large surface area, adjustable antibody density, and embedded paramagnetic properties of the NP-NF permitted enhanced activation and expansion; its use represents a strategy that is amenable to an efficient selection process for adoptive cellular therapy as well as for the isolation of other cellular subsets for downstream translational applications.

Enzyme precipitate coatings of glucose oxidase onto carbon paper for biofuel cell applications.

Enzymatic biofuel cells (BFC) have a great potential as a small power source, but their practical applications are being hampered by short lifetime and low power density. This study describes the direct immobilization of glucose oxidase (GOx) onto the carbon paper in the form of highly stable and active enzyme precipitation coatings (EPCs), which can improve the lifetime and power density of BFCs. EPCs were fabricated directly onto the carbon paper via a three-step process: covalent attachment (CA), enzyme precipitation, and chemical crosslinking. GOx-immobilized carbon papers via the CA and EPC approaches were used as an enzyme anode and their electrochemical activities were tested under the BFC-operating mode. The BFCs with CA and EPC enzyme anodes produced the maximum power densities of 50 and 250 µW/cm(2) , respectively. The BFC with the EPC enzyme anode showed a stable current density output of >700 µA/cm(2) at 0.18 V under continuous operation for over 45 h. When a maple syrup was used as a fuel under ambient conditions, it also produced a stable current density of >10 µA/cm(2) at 0.18 V for over 25 h. It is anticipated that the direct immobilization of EPC on hierarchical-structured electrodes with a large surface area would further improve the power density of BFCs that can make their applications more feasible.

Selective oxidative degradation of organic pollutants by singlet oxygen-mediated photosensitization: tin porphyrin versus C60 aminofullerene systems.

This study evaluates the potential application of tin porphyrin- and C(60) aminofullerene-derivatized silica (SnP/silica and aminoC(60)/silica) as (1)O(2) generating systems for photochemical degradation of organic pollutants. Photosensitized (1)O(2) production with SnP/silica, which was faster than with aminoC(60)/silica, effectively oxidized a variety of pharmaceuticals. Significant degradation of pharmaceuticals in the presence of the 400-nm UV cutoff filter corroborated visible light activation of both photosensitizers. Whereas the efficacy of aminoC(60)/silica for (1)O(2) production drastically decreased under irradiation with λ > 550 nm, Q-band absorption caused negligible loss of the photosensitizing activity of SnP/silica in the long wavelength region. Faster destruction of phenolates by SnP/silica and aminoC(60)/silica under alkaline pH conditions further implicated (1)O(2) involvement in the oxidative degradation. Direct charge transfer mediated by SnP, which was inferred from nanosecond laser flash photolysis, induced significant degradation of neutral phenols under high power light irradiation. Self-sensitized destruction caused gradual activity loss of SnP/silica in reuse tests unlike aminoC(60)/silica. The kinetic comparison of SnP/silica and TiO(2) photocatalyst in real wastewater effluents showed that photosensitized singlet oxygenation of pharmaceuticals was still efficiently achieved in the presence of background organic matters, while significant interference was observed for photocatalyzed oxidation involving non-selective OH radical.

Carbonic anhydrase for CO2 capture, conversion and utilization.

Carbonic anhydrase (CA) enzymes, catalyzing the CO2 hydration at a high turnover number, can be employed in expediting CO2 capture, conversion and utilization to aid in carbon neutrality. Despite extensive research over the last decade, there remain challenges in CA-related technologies due to poor stability and suboptimal use of CAs. Herein, we discuss recent advances in CA stabilization by protein engineering and enzyme immobilization, and shed light on state-of-the-art of in vitro and in vivo CA-mediated CO2 conversion for improved production of value-added chemicals using CO2 as a feedstock.

Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

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.