Enzyme Loading Capacity Research Articles

Affinity Immobilization of a Bacterial Prolidase onto Metal-Ion-Chelated Magnetic Nanoparticles for the Hydrolysis of Organophosphorus Compounds.

In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His6-tagged Escherichia coli prolidase (His6-EcPepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni2+-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His6-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 °C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl p-nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His6-EcPepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His6-EcPepQ-mediated biocatalysis.

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%).

Enhancing Enzyme Immobilization on Carbon Nanotubes via Metal-Organic Frameworks for Large-Substrate Biocatalysis.

Biocatalysis of large-sized substrates finds wide applications. Immobilizing the involved enzymes on solid supports improves biocatalysis yet faces challenges such as enzyme structural perturbation, leaching, and low cost-efficiencies, depending on immobilization strategies/matrices. Carbon nanotubes (CNTs) are attractive matrices but challenged by enzyme leaching (physical adsorption) or perturbation (covalent linking). Zeolitic imidazolate frameworks (ZIFs) overcome these issues. However, our recent study [ J. Am. Chem. Soc., 2018, 140, 16032-16036] showed reduced cost-efficiency as enzymes trapped below the ZIF surfaces cannot participate in biocatalysis; the enzyme-ZIF composites are also unstable under acidic conditions. In this work, we demonstrate the feasibility of using ZIFs to immobilize enzymes on CNT surfaces on two model enzymes, T4 lysozyme and amylase, both of which showed negligible leaching and retained catalytic activity under neutral and acidic conditions. To better understand the behavior of enzymes on CNTs and CNT-ZIF, we characterized enzyme orientation on both matrices using site-directed spin-labeling (SDSL)-electron paramagnetic resonance (EPR), which is immune to the complexities caused by CNT and ZIF background signals and enzyme-matrix interactions. Our structural investigations showed enhanced enzyme exposure to the solvent compared to enzymes in ZIFs alone; orientation of enzymes in matrices itself is directly related to substrate accessibility and, therefore, essential for understanding and improving catalytic efficiency. To the best of our knowledge, this is the first time ZIFs and one-pot synthesis are employed to anchor large-substrate enzymes on CNT surfaces for biocatalysis. This is also the first report of enzyme orientation on the CNT surface and upon trapping in CNT-ZIF composites. Our results are essential for guiding the rational design of CNT-ZIF combinations to improve enzyme stabilization, loading capacity, and catalytic efficiency.

Influence of silica architecture on the catalytic activity of immobilized glucose oxidase

Immobilizing enzymes on solid support materials is of great interest for many applications as it improves enzyme lifetime and enables its recycling. Synthetic mesoporous silica materials are widely used as supports for enzyme immobilization due to their high enzyme-loading capacity and their excellent mass transport properties through interconnected channels of mesopores. Much less is known about the enzyme support properties of naturally occurring mesoporous silica materials. These are readily available through the growth of diatoms, which biosynthesize hierarchically porous silica microshells with pore diameters ranging from 10 to 1000 nm. Here the authors investigated the performance as enzyme support materials of biosilica from three diatom species in comparison to synthetic silica materials. Using glucose oxidase (GOx) as a model enzyme, the authors determined the enzyme-loading capacities and the specific catalytic activities of the GOx-bearing (bio)silicas both under suspension conditions and in a flow-through setting. Remarkably, the specific activity of GOx was strongly dependent on the type of diatom biosilica material and in the best case was as high as on synthetic mesoporous silica. Porosity analysis of the materials points to an important role of large mesopores (diameters >20 nm) for supporting a high specific activity of (bio)silica-immobilized GOx.

Hairy Particles with Immobilized Enzymes: Impact of Particle Topology on the Catalytic Activity.

Enzymes are described as ideal green biocatalysts because they are highly specific and selective. However, their practical application is hampered because of the low stability and missing reusability of free enzymes. One method to overcome these problems is the immobilization of enzymes onto carriers. Although numerous publications discuss different immobilization strategies, optimization of these carriers for the highest enzyme activity and loading capacity, enzyme selectivity, reusability, and reactor system configuration still remains a challenging task. In this contribution, we aim to address the role of the core-shell particle design with respect to their geometry as well as the polymer shell thickness on the immobilization of biomolecules. We discovered that spherical particles with a core diameter of 200 nm and intermediate shell thickness as well as platelet-like particles exhibited excellent results with a maximum immobilization yield of laccase from Trametes versicolor of up to 92% and an activity on the carrier material of 5.722 U/(g particle). Especially, the platelet-like particles offered a scalable and convenient alternative for the immobilization of laccase. Circular dichroism measurements proved that the secondary structure of the enzyme is not impaired by immobilization onto all kinds of carrier particles. Moreover, the immobilized laccase was successfully used for the decolorization of Cibacron blue P-3R in up to 18 cycles. Finally, particle separation was achieved via citrate-induced flocculation within 10 min. This detailed study contributes to the understanding of rational design of catalytically active hybrid materials and their effective performance at interfaces for applications in textile industry and environmental technologies.

The study of titanium dioxide modification by glutaraldehyde and its application of immobilized penicillin acylase

In this work, titanium dioxide particle was firstly modified with glutaraldehyde, and modification conditions, i.e. particle size (d), active temperature (Ta), modification temperature (Tm), modification time (tm), pH value, aldehyde ethanol ratio (Ra) and ethanol ratio (Re), were studied and optimized adopting grafting rate (Gr) as the optimizing index, the ultraviolet-visible spectrophotometry as characterization method, and the obtained optimal modification conditions were d = 3.3 μm, Ta = 120 °C, Tm = 45 °C, tm = 30 h, pH = 6.0, Ra = 1:10 and Re = 100%. Under the optimal conditions, Gr of glutaraldehyde arrived at 50%. Thereafter, a series of modified titanium dioxide with Gr in the range of [8%, 46%] and with Gr = 30% obtained from different modification conditions were adopted to study the immobilization of penicillin G acylase (PGA), during this process, the ultraviolet-visible spectrophotometry of 6-aminopenicillanic acid was conscripted as characterization method, the enzyme loading capacity (ELC), the enzyme activity (EA) and enzyme activity retaining ratio (EAR) were explored, and results documented with the increase of Gr, ELC, EA and EAR increased. As Gr kept as a similar level, ELC, EA and EAR also showed a similar value. Finally, the structure of titanium dioxide, glutaraldehyde modified titanium dioxide and PGA immobilized titanium dioxide was characterized by the flourier transform infrared spectroscopy (FTIR), and results exhibited aimed samples were obtained successfully.

Spectroscopic and Electrochemical Features of Glucose Oxidase Incorporation into Polyaniline-Cobaltous Oxalate Nano-complex

Nano-complex as enzyme carrier was prepared via hybridization of nano-scale cobaltous oxalate with conductive polymer: polyaniline. Spectrometric means and electrochemical technique were proposed to explore the influence of interaction between nano-composite and redox protein on structural features, dynamics of electron shuttle and catalysis of immobilized enzyme. Results indicated that collaboration of π–π stacking effect as well as the complexation of metal ions in nano-composite with cofactors in enzyme molecule could lead to the firm linkage of glucose oxidase to the matrix of nano-composite. The ligation would cause orientated attachment of enzyme molecules onto the surface of nano-complex. Synergistic effect would not only enhance the enzyme loading capacity of carrier but also facilitate the direct electrochemistry of tethered enzyme molecules with intact structure and configuration of cofactor. However the complexation would cripple the enzymatic catalysis in electro-oxidation of glucose in spite of high affinity to glucose for glucose oxidase integration into nano-composite. Coordination of metal ions within nano-composite with cofactor of glucose oxidase (GOx) would lead to formation of composite with crippled emission fluorescence and hinder the electro-oxidation of glucose.

The engineering and immobilization of penicillin G acylase onto thermo‐sensitive tri‐block copolymer system

In this work, the relationships between catalytic performances of penicillin G acylase (PGA) and the molar ratio of carrier, thermo‐sensitive tri‐block polymer, poly (N,N‐diethylacrylamide‐b‐ β‐hydroxyethyl methacrylate‐b‐glycidyl methacrylate) (PDEA‐b‐PHEMA‐b‐PGMA) were studied firstly, and result documented the optimal molar ratio was nDEA:nHEMA:nGMA = 100:47:24, which presented a suitable lower critical solution temperature (LCST) of 35°C and the activity retention ratio of 80.62% (±0.50%). Based on the suitable carrier, immobilization conditions were investigated and optimized. When pH of solution, concentration of PGA, immobilized time, and immobilization temperature were 8.0, 1/10 (m/v), 16 hours, and 36°C, respectively, enzyme loading capacity (L), enzyme activity (Ea), and activity retention ratio (Ar) of PGA arrived at the highest value of 21 223 U, 16 199 U/g, and 93.50% (±0.50%), respectively. Besides, the response rate (Rr) of immobilized PGA was the same as free PGA, the reusable stability (Rs) was 77.00% (±1.00%) after using for 11 times, which indicated that the carrier has better compatibility with L, Ar, Rs, and Rr.

Integrated Microcentrifuge Carbon Entrapped Glucose Oxidase Poly (N-Isopropylacrylamide) (pNIPAm) Microgels for Glucose Amperometric Detection

This study demonstrates a miniaturized integrated glucose biosensor based on a carbon microbeads entrapped by glucose oxidase (GOx) immobilized on poly (N-isopropylacrylamide) (pNIPAm) microgels. Determined by the Lowry protein assay, the pNIPAm microgel possesses a high enzyme loading capacity of 31 mg/g. The pNIPAm GOx loaded on the microgel was found to maintain a high activity of approximately 0.140 U determined using the 4-aminoantipyrine colorimetric method. The integrated microelectrochemical cell was constructed using a microcentrifuge vial housing packed with (1:1, w/w) carbon entrapped by pNIPAm GOx microgels, which played the dual role of the microbioreactor and the working electrode. The microcentrifuge vial cover was used as a miniaturized reference electrode and an auxiliary electrode holder. The device can work as biosensor, effectively converting glucose to H2O2, with subsequent amperometric detection at an applied potential of −0.4 V. The microelectrochemical biosensor was used to detect glucose in wide linear range from 30 µM to 8.0 mM, a low detection limit of 10 µM, a good linear regression coefficient (R2) of 0.994, and a calibration sensitivity of 0.0388 µA/mM. The surface coverage of active GOx, electron transfer rate constant (ks), and Michaelis–Menten constant (KMapp) of the immobilized GOx were 4.0 × 10−11 mol/cm2, 5.4 s−1, and 0.086 mM, respectively. To demonstrate the applicability and robustness of the biosensor for analysis of high sample matrix environment, glucose was analyzed in root beer. The microelectrochemical device was demonstrated for analysis of small sample (<50 µL), while affording high precision and fast signal measurement (≤5 s).

Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase

Rapid, low-cost and efficient assays for penicillinase activity and inhibition are of vital importance for therapeutics and diagnostics of bacterial resistance to antibiotics. Herein we report a novel approach for on-line enzyme assays for penicillinase utilizing capillary electrophoresis-integrated immobilized enzyme reactors (CE-IMERs). The CE-IMERs are fabricated based on penicillinase-mediated alginate hydrogelation, allowing single-step in-situ encapsulation of enzymes without any additional manipulation process. We show that the fabricated CE-IMERs have high enzyme loading capacity with approximately 61.8% of the original penicillinase in the sol mixture being encapsulated in the “egg-box” hydrogel matrix. Excellent intraday and interday stability and batch-to-batch reproducibility are proved, indicating the reliability of our method for accurate on-line enzyme assays for penicillinase. Enzymatic activities and inhibition of immobilized penicillinase are analyzed, the results of which are in good agreement with those using free enzymes. The proposed method is successfully used for determination of penicillin in pork samples, indicating the potential applications for analysis of complicated real samples.

Immobilization of glycerol dehydrogenase and NADH oxidase for enzymatic synthesis of 1,3‐dihydroxyacetone with in situ cofactor regeneration

AbstractBACKGROUNDNicotinamide cofactor‐dependent oxidoreductases have been widely applied to the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these oxidoreductase‐catalyzed reactions. Herein, enzymatic production of 1, 3‐dihydroxyacetone (DHA) via immobilized enzymes with in situ cofactor regeneration was reported.RESULTSGlycerol dehydrogenase (GDH) and NADH oxidase (Nox) were immobilized on two carriers: macroporous resin and epoxy functionalized magnetic nanoparticles (EFMN). Higher enzyme loading capacity and activity recovery were achieved on EFMN. A high immobilization yield of 90% and &gt;70% recovery activity were obtained for both enzymes using EFMN as carriers. Besides, both immobilized enzymes showed enhanced pH stability and temperature tolerance. Co‐immobilized and mixed immobilized enzymes were evaluated for synthesis of DHA, and mixed immobilized enzymes showed higher catalytic rate. The mixed reaction system also was investigated in view of various operational factors. Under the optimal conditions, a high DHA concentration with mixed immobilized enzymes system can reach up to 3.5 mmol L–1, which was about four times higher than that without cofactor regeneration system.CONCLUSIONThe immobilized GDH and Nox on magnetic nanoparticles showed enhanced stabilities. A cofactor regeneration system with immobilized enzymes could be utilized for efficient production of DHA from inexpensive substrates. © 2018 Society of Chemical Industry

Assembly of graphene oxide-formate dehydrogenase composites by nickel-coordination with enhanced stability and reusability.

Featuring unique planar structure, large surface area and biocompatibility, graphene oxide (GO) has been widely taken as an ideal scaffold for the immobilization of various enzymes. In this regard, nickel-coordinated graphene oxide composites (GO-Ni) were prepared as novel supporters for the immobilization of formate dehydrogenase. The catalytic activity, stability and morphology were studied. Compared with GO, the enzyme loading capacity of GO-Ni was enhanced by 5.2-fold, besides the immobilized enzyme GO-Ni-FDH exhibited better thermostability, storage stability and reuse stability than GO-FDH. GO-Ni-FDH retained 40.9% of its initial activity after 3h at 60°C, and retained 31.4% of its initial relative activity after 20 days' storage at 4°C. After eight times usages, GO-Ni-FDH maintained 63.8% of its initial activity. Mechanism insights of the multiple interactions of enzyme with the GO-Ni were studied, considering coordination bonds, hydrogen bonds, electrostatic forces, coordination bonds, and etc. A practical and simple immobilization strategy by metal ions coordination for multimeric dehydrogenase was developed.

Facile immobilization of Bacillus licheniformis γ-glutamyltranspeptidase onto graphene oxide nanosheets and its application to the biocatalytic synthesis of γ-l-glutamyl peptides

For the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we illustrated a simple and efficient approach to fabricate a biocatalytic system by immobilizing the enzyme onto graphene oxide (GO) nanosheets via both non-covalent (GO-BlGGT) and covalent (GO/GA-BlGGT) bonds. The enzyme-loading capacity for the prepared GO/GA nanomaterial was 3.47 mg/mg support, corresponding to 68.7% recovery of the initial activity. Native and enzyme-bound layered GOs were characterized by X-ray diffraction, followed by Raman and Fouier transform infrared spectroscopy, elemental analysis and thermogram analysis. As compared to the free form of BlGGT, the immobilized enzymes exhibited significantly higher activity, possibly due to the beneficial effect of the layered GO carrier. The kinetic behaviors of GO-BlGGT and GO/GA-BlGGT were mostly consistent with those of free enzyme. The covalently immobilized enzyme had a comparable stability respective to free enzyme during a storage period of 30 days and could be recycled nine times with 45.3% retention of the initial activity. Besides, the biocatalytic synthesis of γ-l-glutamyl-phenylalanine and γ-l-glutamyl-leucine by immobilized enzymes resulted in the product yield of more than 31%. Taken together, these results suggest that the facile strategy is an economical means of depositing bioactive enzymes upon GO nanosheets for BlGGT-mediated biocatalysis.

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (kcat), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed.

Physical and Covalent Immobilization of Lipase onto Amine Groups Bearing Thiol-Ene Photocured Coatings.

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37°C (free lipase) to 50-55°C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70% of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56% of its hydrolytic activity and 67% of its synthetic activity in the same cycle period. In the case of hydrolysis V max values slightly decreased after immobilization. For synthetic assay, the V max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.

Hierarchical Nanoplatforms for High‐Performance Enzyme Biocatalysis under Denaturing Conditions

AbstractThe front cover artwork for Issue 07/2016 is provided by TeamNanoTech (TNT), the Magnetic Materials Group (MMG), and Bioengineering &amp; Sustainable Processes (BIOSUV) at Universidade de Vigo (Spain), and the Institute of Nanoscience of Aragón (INA) at Universidad de Zaragoza (Spain). The image illustrates a novel multifunctional nanoplatform featuring a high enzyme loading capacity and remarkable recycling capabilities as an effective means to circumvent the limitations that hinder the implementation of enzymes in industrial biotechnology (image credit: Miguel Spuch‐Calvar). See the Communication itself at http://dx.doi.org/10.1002/cctc.201501401.

Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.

Covalent immobilization of microbial naringinase using novel thermally stable biopolymer for hydrolysis of naringin.

Naringinase induced from the fermented broth of marine-derived fungus Aspergillus niger was immobilized into grafted gel beads, to obtain biocatalytically active beads. The support for enzyme immobilization was characterized by ART-FTIR and TGA techniques. TGA revealed a significant improvement in the grafted gel’s thermal stability from 200 to 300 °C. Optimization of the enzyme loading capacity increased gradually by 28-fold from 32 U/g gel to 899 U/g gel beads, retaining 99 % of the enzyme immobilization efficiency and 88 % of the immobilization yield. The immobilization process highly improved the enzyme’s thermal stability from 50 to 70 °C, which is favored in food industries, and reusability test retained 100 % of the immobilized enzyme activity after 20 cycles. These results are very useful on the marketing and industrial levels.

Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

Lysozyme-Based Antibacterial Nanomotors.

An effective and rapid bacterial killing nanotechnology strategy based on lysozyme-modified fuel-free nanomotors is demonstrated. The efficient antibacterial property of lysozyme, associated with the cleavage of glycosidic bonds of peptidoglycans present in the bacteria cell wall, has been combined with ultrasound (US)-propelled porous gold nanowire (p-AuNW) motors as biocompatible dynamic bacteria nanofighters. Coupling the antibacterial activity of the enzyme with the rapid movement of these p-AuNWs, along with the corresponding fluid dynamics, promotes enzyme-bacteria interactions and prevents surface aggregation of dead bacteria, resulting in a greatly enhanced bacteria-killing capability. The large active surface area of these nanoporous motors offers a significantly higher enzyme loading capacity compared to nonporous AuNWs, which results in a higher antimicrobial activity against Gram-positive and Gram-negative bacteria. Detailed characterization studies and control experiments provide useful insights into the underlying factors controlling the antibacterial performance of the new dynamic bacteria nanofighters. Rapid and effective killing of the Gram-positive Micrococcus lysodeikticus bacteria (69-84% within 1-5 min) is demonstrated.