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In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid's apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.

Alginate is a natural polysaccharide resource abundant in brown algae and it can be cleaved into alginate oligosaccharides by alginate lyase. Alginate lyases and the bioactive alginate oligosaccharides have been applied in diverse fields such as pharmaceutical therapy and nutraceutical supplementation. Immobilized enzymes greatly facilitate their industrial application owing to their reusability, stability, and tunability. In this study, magnetic Fe 3 O 4 nanoparticles were synthesized and used to immobilize an exolytic alginate lyase AlgL17 that was characterized previously. The immobilized AlgL17 demonstrated enhanced thermal and pH tolerance, extended storage stability, and moderate reusability. The mass spectrum indicated the specific activity of the immobilized AlgL17 to release alginate oligosaccharides (AOS) from alginate polysaccharide. The produced AOS exhibited their antioxidant and antiapoptotic activities in H 2 O 2 -stressed human umbilical vein endothelial cells by upregulation of reactive oxygen species scavenging activities and attenuation of the caspase-mediated apoptosis pathway., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Composite polycaprolactone-chitosan material was produced by an electrospinning method and used as a support for immobilization of tyrosinase by mixed ionic interactions and hydrogen bonds formation. The morphology of the fibers and enzyme deposition were confirmed by SEM images. Further, multivariate polynomial regression was used to model the experimental data and to determine optimal conditions for immobilization process, which were found to be pH 7, temperature 25 °C and 16 h process duration. Under these conditions, novel type of biocatalytic system was produced with immobilization yield of 93% and expressed activity of 95%. Furthermore, as prepared system was applied in batch experiments related to biodegradation of bisphenol A under various remediation conditions. It was found that over 80% of the pollutant was removed after 120 min of the process, in the temperature range 15-45 °C and pH 6-9, using solutions at concentration up to 3 mg/L. Experimental data collected proved that the stability and reusability of the tyrosinase were significantly improved upon immobilization: the immobilized biomolecule retained around 90% of its initial activity after 30 days of storage, and was still capable to remove over 80% of bisphenol A even after 10 repeated uses. By contrast, free enzyme was able to remove over 80% of bisphenol A at pH 7-8 and temperature range 15-35 °C, and retained less than 60% of its initial activity after 30 days of storage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metal-organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH 2 , or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes.

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe 3 O 4 @Chitosan@Xylanase in the harsh temperature condition of relevant industries.

The organization of enzymes into different subcellular compartments is essential for correct cell function. Protein-based cages are a relatively recently discovered subclass of structurally dynamic cellular compartments that can be mimicked in the laboratory to encapsulate enzymes. These synthetic structures can then be used to improve our understanding of natural protein-based cages, or as nanoreactors in industrial catalysis, metabolic engineering, and medicine. Since the function of natural protein-based cages is related to their three-dimensional structure, it is important to determine this at the highest possible resolution if viable nanoreactors are to be engineered. Cryo-electron microscopy (cryo-EM) is ideal for undertaking such analyses within a feasible time frame and at near-native conditions. This review describes how three-dimensional cryo-EM is used in this field and discusses its advantages. An overview is also given of the nanoreactors produced so far, their structure, function, and applications.

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu 3 (PO 4 ) 2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu 3 (PO 4 ) 2 /CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu 3 (PO 4 ) 2 /Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ee s ) reached 98% in only 10 min, and the corresponding Cu 3 (PO 4 ) 2 /CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Multienzymes, such as the protein metazoan fatty acid synthase (FAS), are giant and highly dynamic molecular machines for critical biosynthetic processes. The molecular architecture of FAS was elucidated by static high-resolution crystallographic analysis, while electron microscopy revealed large-scale conformational variability in FAS with some correlation to functional states in catalysis. However, little is known about time scales of conformational dynamics, the trajectory of motions in individual FAS molecules, and the extent of coupling between catalysis and structural changes. Here, we present an experimental single-molecule approach to film immobilized or selectively tethered FAS in solution at different viewing angles and high spatiotemporal resolution using high-speed atomic force microscopy. Mobility of individual regions of the multienzyme is recognized in video sequences, and correlation of shape features implies a convergence of temporal resolution and velocity of FAS dynamics. Conformational variety can be identified and grouped by reference-free 2D class averaging, enabling the tracking of conformational transitions in movies. The approach presented here is suited for comprehensive studies of the dynamics of FAS and other multienzymes in aqueous solution at the single-molecule level.

Laccase from Pycnoporus sanguineus CS43 was successfully immobilized onto Immobead-150 and Eupergit-C by covalent binding and by entrapment in LentiKats. The highest immobilization was onto Immobead-150 (97.1±1.2%) compared to the other supports, LentiKats (89±1.1%) and Eupergit-C (83.2±1.4%). All three immobilized enzyme systems showed increased thermostability and better mechanical properties than free laccase. Moreover, after 5 cycles of reuse of these systems, 90% of initial laccase activity was retained. Immobead-150 and LentiKats systems exhibited the highest efficiencies in removal of m-cresol under the combined actions of biodegradation and adsorption, while laccase entrapped in LentiKats showed a high ability for degradation of m-cresol within 24h. In addition, the typical Michaelis-Menten enzymatic model effectively described the kinetic profile of m-cresol degradation by the enzyme entrapped in LentiKats. Based on the results obtained in the present study, it can be established that the immobilized biocatalysts developed here possess significant potential for wastewater treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Biocatalytic pulp fibers were prepared using surface functionalization of bleached kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase (G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry, and dynamic light scattering. According to standard assays, the G* content on the resulting fibers (FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal- and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in the present case, as the introduction of amino groups allowed the immobilization of larger amount of enzymes and rendered more efficient systems. Using the approach described on this paper, several advanced materials from wood pulp fibers and new bioprocesses might be developed by selecting the correct enzyme for the target applications.

Industrial use of Novozym 435 in synthesis of structured lipids and biodiesel via alcoholysis is limited by mass transfer effects of the glycerides through immobilized enzymes and its low operational stability under operation conditions. To better understand this, differently modified Novozym 435 preparations, differing in their surface nature and in their interactions with reactants, have been compared in the alcoholysis of Camelina sativa oil. The three modifications performed have been carried out under conditions where all exposed groups of the enzyme have been modified. These modifications were: 2,4,6-trinitrobenzensulfonic acid (Novo-TNBS), ethylendiamine (Novo-EDA) and polyethylenimine (Novo-PEI). Changes in their operational performance are analyzed in terms of changes detected by scan electron microscopy in the support morphology. The hydrophobic nature of the TNBS accelerates the reaction rate; t-ButOH co-solvent swells the macroporous acrylic particles of Lewatit VP OC 1600 in all biocatalysts, except in the case of Novo-PEI. This co-solvent only increases the maximal conversions obtained at 24h using the modified biocatalysts. t-ButOH reduces enzyme inactivation by alcohol and water. In a co-solvent system, these four biocatalysts remain fully active after 14 consecutive reaction cycles of 24h, but only Novo-TNBS yields maximal conversion before cycle 5. Some deposits on biocatalyst particles could be appreciated during reuses, and TNBS derivatization diminishes the accumulation of product deposits on the catalyst surface. Most particles of commercial Novozym(®) 435 are broken after operation for 14 reaction cycles. The broken particles are fully active, but they cause problems of blockage in filtration operations and column reactors. The three derivatizations studied make the matrix particles more resistant to rupture., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10(th) cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37°C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, Km and Vmax were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (Km=15g/L, Vmax=719U/mg versus Km=12.6g/L and Vmax=787U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (Km=18g/L, Vmax=685U/mg). The values for the anchored enzyme-glass were Km=19g/L, Vmax=763U/mg., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Enzymatic biofuel cells have many great features as a small power source for medical, environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers (PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the maximum power density output of 37.4 μW/cm(2), while the power density output of 3.1 μW/cm(2) was shown without mediators. Under room temperature and 4°C for 28 days, enzymatic biofuel cells maintained 54 and 70% of its initial power density, respectively., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this study, a mat/membrane consisting of overlaid PVA/PA6-Cu(II) composite nanofibers was prepared via the electrospinning technique followed by coordination/chelation with Cu(II) ions; an enzyme of catalase (CAT) was then immobilized onto the PVA/PA6-Cu(II) nanofibrous membrane. The amount of immobilized catalase reached a high value of 64 ± 4.6 mg/g, while the kinetic parameters (Vmax and Km) of enzyme were 3774 μmol/mg·min and 41.13 mM, respectively. Furthermore, the thermal stability and storage stability of immobilized catalase were improved significantly. Thereafter, a plug-flow type of immobilized enzyme membrane reactor (IEMR) was assembled from the PVA/PA6-Cu(II)-CAT membrane. With the increase of operational pressure from 0.02 to 0.2 MPa, the flux value of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/m(2)·min, whereas the conversion ratio of H2O2 decreased slightly from 92 ± 2.5% to 87 ± 2.1%. After 5 repeating cycles, the production capacity of IEMR was merely decreased from 0.144 ± 0.006 to 0.102 ± 0.004 mol/m(2)·min. These results indicated that the assembled IEMR possessed high productivity and excellent reusability, suggesting that the IEMR based on electrospun PVA/PA6-Cu(II) nanofibrous membrane might have great potential for various applications, particularly those related to environmental protection.

The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions.

The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136h of incubation at 50°C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this paper, we present a simple but efficient biomimetic method to encapsulate laccase on mesoporous silica-modified electrospun (ES) ultrafine fibers. Because of the mild immobilization conditions (room temperature, aqueous condition), the encapsulated laccase retained a high activity of 94%. Because of the protection from the silica layer, the laccase worked efficiently at 60 °C and retained a long-term activity in the presence of proteinase K. After recycling for 10 times the laccase still preserved 96% of its original reactivity. More remarkably, the immobilized laccase on fibers could completely recover its activity after thermal denature, while the free laccase permanently lost the activity. We also demonstrated that the laccase on silica-coated fibers exhibited an enhanced decolorization capability of Brilliant Blue KN-R (BBKN-R) as compared to the free laccase, showing its great potential for industrial applications.

Nanomaterials in biological solutions are known to interact with proteins and have been documented to affect protein function, such as enzyme activity. Understanding the interactions of nanoparticles with biological components at the molecular level will allow for rational designs of nanomaterials for use in medical technologies. Here we present the first detailed molecular mechanics model of functionalized gold nanoparticle (NP) interacting with an enzyme (L-lactate dehydrogenase (LDH) enzyme). Molecular dynamics (MD) simulations of the response of LDH to the NP binding demonstrate that although atomic motions (dynamics) of the main chain exhibit only a minor response to the binding, the dynamics of side chains are significantly constrained in all four active sites that predict alteration in kinetic properties of the enzyme. It is also demonstrated that the 5 nm gold NPs cause a decrease in the maximal velocity of the enzyme reaction (V(max)) and a trend towards a reduced affinity (increased K(m)) for the β-NAD binding site, while pyruvate enzyme kinetics (K(m) and V(max)) are not significantly altered in the presence of the gold NPs. These results demonstrate that modeling of NP:protein interactions can be used to understand alterations in protein function., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactor-binding N domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. p97 plays crucial roles in a variety of cellular processes such as the ubiquitin-proteasome pathway, the endoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP are linked to neurodegenerative diseases. The key mechanism of p97 in these various functions has been proposed to be the disassembly of protein complexes. To understand the molecular mechanism of p97, we studied the conformational changes of hexameric CDC-48.1, a Caenorhabditis elegans p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates ~23 ± 8° clockwise and resets relative to the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500-600 nm) were synthesized by a modified sol-gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100-150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A simple method is used to covalently encapsulate enzymes in silica nanoparticles. The encapsulation is highlighted by the high enzyme loading and porous channels that provide efficient diffusion for small substrate and product molecules while preventing protease degradation.

Transglutaminase (TGase) was cross-linked with glutaraldehyde, and cross-linked crystalline transglutaminase was immobilized on a polypropylene microporous membrane by UV-induced grafting. Immobilized enzyme activity were calculated to be 0.128 U/cm² polypropylene microporous membrane. The microstructure and enzyme characteristics of free, cross-linked and immobilized transglutaminase were compared. The optimum temperature of free transglutaminase was determined to be approximately 40 °C, while cross-linking and immobilization resulted in an increase to approximately 45 °C and 50 °C. At 60 °C, immobilized, cross-linked and free transglutaminase retained 91.7 ± 1.20%, 63.2 ± 1.05% and 37.9 ± 0.98% maximum activity, respectively. The optimum pH was unaffected by the state of transglutaminase. However, the thermal and pH stabilities of cross-linked and immobilized transglutaminase were shown to increase.

Sol-gel-derived aerogels are three-dimensional, nanoscale materials that combine large surface areas and high porosities. These traits make them useful for any rate-critical chemical process, particularly sensing or electrochemical applications, once physical or chemical moieties are incorporated into the gels to add their functionality into the ultraporous scaffold. Incorporating biomolecules into aerogels has been challenging due to the inability of most biomolecules to remain structurally intact within the gels during the necessary supercritical fluid processing. However, the heme protein cytochrome c (cyt. c) forms self-organized superstructures around gold (or silver) nanoparticles in buffer that can be encapsulated within silica and processed to form aerogels in which cyt. c retains its characteristic visible absorption. The gold (or silver) nanoparticle-nucleated superstructures protect the majority of the protein from the harsh physicochemical conditions necessary to form an aerogel. The Au∼cyt. c superstructures exhibit rapid gas-phase recognition of nitric oxide (NO) within the aerogel matrix, as facilitated by the high-quality pore structure of the aerogel, and remain viable for weeks at room temperature.

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

The proteasome is the essential prime protease in all eukaryotes. The large, multisubunit, modular, and multifunctional enzyme is responsible for the majority of regulated intracellular protein degradation. It constitutes a part of the multienzyme ubiquitin-proteasome pathway, which is broadly implicated in recognition, tagging, and cleavage of proteins. The name "proteasome" refers to several types of protein assemblies sharing a common catalytic core particle. Additional protein modules attach to the core, regulate its activities, and broaden its functional capabilities. The structure of proteasomes has been studied extensively with multiple methods. The crystal structure of the core particle was solved for several species. However, only a single structure of the core particle decorated with PA26 activator has been determined. NMR spectroscopy was successfully applied to probe a much -simpler, archaebacterial type of the core particle. In turn, electron microscopy was very effective in exploring the spatial arrangement of many classes of assemblies. Still, the makeup of higher-order -complexes is not well established. Besides, the crystal structure provided very limited information on proteasome molecular dynamics. Atomic force microscopy (AFM) is an ideal technique to address questions that are unanswered by other approaches. For example, AFM is perfectly suited to study allosteric regulation of proteasome, the role of protein dynamics in enzymatic catalysis, and the spatial organization of modules and subunits in assemblies. Here, we present a method that probes the conformational diversity and dynamics of yeast core particle using the oscillating mode AFM in liquid. We are taking advantage of the observation that the tube-shaped core particle is equipped with a swinging gate leading to the catalytic chamber. We demonstrate how to identify distinct gate conformations in AFM images and how to characterize the gate dynamics controlled with ligands and disturbed by mutations.

Papain having the characteristics of metal binding ability is immobilized on alginate bead. Design Expert Software (Version 7.1.6) uses Response Surface Methodology (RSM) for statistical designing of operating condition for immobilization of papain on alginate bead considering concentration of papain, concentration of sodium alginate, concentration of calcium chloride and pH as numeric factors and Specific Enzymatic Activity (SEA) of immobilized papain sample as response. Immobilization using 25.96 g/L papain, 20 g/L sodium alginate and 20 g/L calcium chloride at pH 7 gives the desired product as indicated by ANOVA (Analysis of Variance). Three parameters viz., initial concentration of mercury (II), amount of AIP and pH are varied in a systematic manner. Maximum 98.88% removal of mercury (II) has been achieved within 8 min when simulated aqueous solution of mercury (II) with initial concentration of 10mg/L has been contacted with 5 g of AIP at pH 9 and at 35 degrees C in a batch contactor. A mathematical model has been developed and the value of equilibrium constant for binding of mercury (II) with AIP has been found to be 126797.3., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

We report the immobilization of a model enzyme, papain, within silica matrices by combining vesiclization of poly-l-lysine-b-polyglycine block copolypeptides with following silica mineralization. Our novel strategy utilizes block polypeptide vesicles to induce the condensation of orthosilicic acid while trapping an enzyme within and between vesicles. The polypeptide mediated silica-immobilized enzyme exhibits enhanced pH and thermal stability and reusability, comparing with the free and vesicle encapsulated enzyme. The enhanced enzymatic activity in the immobilized enzyme is due to the confinement of the enzyme in the polypeptide mediated silica matrices. Kinetic analysis shows that the enzyme functionality is determined by the structure and property of silica/polypeptide matrices. The proposed novel strategy provides an alternative route for the synthesis of a broad range of functional bionanocomposites entrapped within silica nanostructures.

Objectives: A method is described for construction of an amperometric triglyceride (TG) biosensor using PVA membrane bound enzymes., Design and Methods: A mixture of commercial lipase, glycerol kinase, glycerol-3-phosphate oxidase and horseradish peroxidase was co-immobilized onto polyvinyl alcohol (PVA) membrane through glutaraldehyde coupling. The biosensor measures current when polarized at 0.4 V., Results: The sensor showed optimum response within 2 s at pH 7.0 and 25 degrees C. The current (mA) was in proportion to concentration of TG in the range 0.56-2.25 mM. The minimum detection limit of the method was 0.21 mM. The analytic recovery of added TG was 94.3%. Within batch and between batch coefficients of variations (CV) were <5.85% and <4.13%, respectively. A good correlation (r=0.99) was found. Among various serum substances tested, only cholesterol caused slight stimulation (20%)., Conclusions: An amperometric method was developed for determination of TG employing this enzyme electrode., (Copyright 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E((0.3))), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3U/mg protein, which is 0.46 times less than that of the free lipase (35.6U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 degrees C respectively. Kinetic parameters V(max) and K(m) were also determined for the immobilized lipase. It was observed that there is an increase of the K(m) value (7.54mM) and a decrease of the V(max) value (145.0U/mg-protein) comparing with that of the free lipase.

In this work, lipase from Arthrobacter sp. was immobilized by sol-gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, gamma-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS=1/1 (mol/mol), water/silane molar ratio (R value)=20 and lipase loading=0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form.

Cellulase is an enzymatic complex which synergically promotes the degradation of cellulose to glucose. The adsorption behavior of cellulase from Trichoderma reesei onto Si wafers or amino-terminated surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM) as a function of temperature. Upon increasing temperature from (24 +/- 1) to (60 +/- 1) degrees C, adsorption of cellulase became faster and more pronounced and the mean roughness of cellulase adsorbed layers increased. In the case of cellulase adsorbed onto Si wafers, Arrhenius's plot allowed us to estimate the adsorption energy as 24.2 kJ mol(-1). The hydrolytic activity of free cellulase and cellulase immobilized onto Si wafers was tested using cellulose dispersions as substrates. The incubation temperature ranged from (37 +/- 1) to (60 +/- 1) degrees C. The highest efficiency was observed at (60 +/- 1) degrees C. The amount of glucose produced by free cellulase was approximately 20% higher than that obtained from immobilized cellulase. However, immobilizing cellulase onto Si wafers proved to be advantageous because they could be reused six times while retaining their original activity level. Such an effect was attributed to surface hydration, which prevents enzyme denaturation. The hydrolytic activity of cellulase immobilized onto amino-terminated surfaces was slightly lower than that observed for cellulase adsorbed onto Si wafers, and reuse was not possible.

DPN (Dip-Pen nanolithography) is one kind of widely used technique to create nanoscopic patterns of many different materials. FoF1-ATPase is nano scale rotary molecular motor, and it would be an ideal motor or energy providing device in micro/nano system. In this paper, we used DPN technique to create nanoarrays of F0F1-ATPase within chromatophore on gold surface. The feature size of our F0F1-ATPase patterns was 270 nm in average, and there were no more than 20 F0F1-ATPases in each dot. The activity of patterned F0F1-ATPase was demonstrated by its ATP synthesis, which was indicated by the fluorescence change of labeled F1300. The patterned F0F1-ATPase nanoarrays can be further used as biosensor, or power providing system. And precisely patterning F0F1-ATPase with desired size, position and biological activity will accelerate its application in many basic and application research fields.

Four different silica sol-gel films: methyltrimethoxysilane (MTMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTOS) and 3-glycidoxypropyl-trimethoxysilane (GOPMOS) assembled onto highly oriented pyrolytic graphite (HOPG) were characterized using atomic force microscopy (AFM), due to their use in the development of glucose biosensors. The chemical structure of the oxysilane precursor and the composition of the sol-gel mixture both influenced the roughness, the size and the distribution of pores in the sol-gel films, which is relevant for enzyme encapsulation. The GOPMOS sol-gel film fulfils all the morphological characteristics required for good encapsulation of the enzyme, due to a smooth topography with very dense and uniform distribution of only small, 50 nm diameter, pores at the surface. APTOS and MTMOS sol-gel films developed small pores together with large ones of 300-400 nm that allow the leakage of enzymes, while the TEOS film formed a rough and incomplete network on the electrode, less suitable for enzyme immobilisation. GOPMOS sol-gel film with encapsulated glucose oxidase and poly(neutral red) redox mediator, prepared by in situ electropolymerization, were also morphologically characterized by AFM. The AFM results explain the variation of the stability in time, sensitivity and limit of detection obtained with different oxysilane sol-gel encapsulated glucose oxidase biosensors with redox mediator.

Enzyme/polymer/gold nanoparticle hybrids, called "nanozymes", were prepared and structurally analyzed by dynamic light scattering (DLS), ultraviolet-visible spectroscopy, and zeta-potential and transmission electron microscopy (TEM) measurements, which showed that the nanozyme particles were mainly composed of a single gold nanoparticle, on whose surface the enzyme and polymer were coimmobilized. This kind of structure resulted in the high dispersion stability of the nanozyme under various conditions, accompanied by improved thermal stability of the enzyme.

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.

Candida rugosa lipase was immobilized by covalent binding on hybrid matrix of polysiloxane-polyvinyl alcohol chemically modified with different activating agents as glutaraldehyde, sodium metaperiodate and carbonyldiimidazole. The experimental results suggested that functional activating agents render different interactions between enzyme and support, producing consequently alterations in the optimal reaction conditions. Properties of the immobilized systems were assessed and their performance on hydrolytic and synthetic reactions were evaluated and compared with the free enzyme. In hydrolytic reactions using p-nitrophenyl palmitate as substrate all immobilized systems showed higher thermal stability and optima pH and temperature values in relation to the free lipase. Among the activating compounds, carbonyldiimidazole resulted in a total recovery of activity on the support and the highest thermal stability. For the butyl butyrate synthesis, the best performance (molar conversion of 95% and volumetric productivity of 2.33 g L(-1)h(-1)) was attained with the lipase immobilized on POS-PVA activated with sodium metaperiodate. The properties of the support and immobilized derivatives were also evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopies and chemical composition (FTIR).

A microfluidic immobilised enzyme reactor consisting of a catalytically functionalised microstructure fabricated from silicone rubber material was used for steady-state kinetic characterisation of a thermophilic beta-glycosidase under pressure-driven flow conditions and continuous conversion of lactose by this enzyme at 80 degrees C.

(Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization.

RNA transcripts after transcription elongation with T7 RNA polymerase in vitro were visualized by using atomic force microscopy (AFM). Fragment of pGEMEX linear DNA with the length of 1414 nucleotide pairs carrying promoter and terminator of bacteriophage T7 was used as DNA template for transcription. Immobilized on the mica (AFM substrate) RNA transcripts formed rod-like condensed structures with the length of 122 +/- 10 nm and characteristic aspect ratio ca. 4.5-5. Problems of RNA immobilization onto mica for subsequent visualization by AFM are discussed.

Redox enzymes in bioelectronic devices usually lack direct electrical contact with electrodes, owing to the spatial separation of their redox centers from the conductive surfaces by the protein shells. The reconstitution of apo-enzymes on cofactor-functionalized nanostructures associated with electrodes provides a means to align the biocatalysts on the conductive surface and to electrically contact redox enzymes with electrodes. The reconstitution of apo-enzymes on cofactor-functionalized gold nanoparticles or carbon nanotubes has led to effective electrical communication between the redox proteins and the electrodes. Alternatively, the reconstitution of redox enzymes on molecular wires that enable electron tunneling or dynamic charge shuttling represent supramolecular biocatalytic nanostructures exhibiting electrical contact. The bioelectrocatalytic activities of the electrically wired reconstituted enzymes on electrodes have allowed the development of amperometric biosensors and biofuel cell elements.

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.

Cutinase from Fusarium solani pisi was encapsulated in sol-gel matrices prepared with a combination of alkyl-alkoxysilane precursors of different chain-lengths. The specific activity of cutinase in a model transesterification reaction at fixed water activity in n-hexane was highest for the precursor combination tetramethoxysilane/n-butyltrimetoxysilane (TMOS/BTMS) in a 1:5 ratio, lower and higher chain lengths of the mono-alkylated precursor or decreasing proportions of the latter relative to TMOS leading to lower enzyme activity. Results obtained using combinations of three precursors confirmed the beneficial effect of the presence of BTMS in the preparations. Scanning electron microscopy of the 1:5 TMOS/n-alkylTMS gels showed a direct correlation between the macropore dimensions and the alkyl chain length of the alkylated precursor and revealed that TMOS/n-octylTMS gels suffered extensive pore collapse during the drying process. The specific activity of TMOS/BTMS sol-gel entrapped cutinase was similar to that exhibited by the enzyme immobilized by adsorption on zeolite NaY. However, the incorporation of different additives (zeolites, silica, Biogel, grinded sol-gel, etc.) having in common the capability to react with residual silanol groups of the sol-gel matrix brought about remarkable enhancements of cutinase activity, despite the fact that the global porosity of the gels did not change. The behavior of the gels in supercritical CO 2 (sc-CO 2) paralleled that exhibited in n-hexane, although cutinase activity was ca. one order of magnitude lower (i.e. sol-gel encapsulation did not prevent the deleterious effect of CO 2. The impact that functionalization of some of the additives had on cutinase activity indicates that the enzyme/matrix interactions must play an important role. Some of the best additives from the standpoint of enzyme activity were also the best from the standpoint of its operational stability (ca. 80% retention of enzyme activity at the tenth reutilization cycle). None of the additives that proved effective for cutinase could improve the catalytic activity of sol-gel encapsulated Pseudomonas cepacia lipase.

A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system., (Copyright 2005 Wiley Periodicals, Inc.)

In a study of Assemblase, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.

The feasibility of creating nanometer scale depressions in biological substrates using active enzymes delivered with scanning probe microscopes has been previously demonstrated by us and other groups. Here we present a comprehensive study revealing the dependence of channels dimensions on the parameters of the "writing" process and provide a simple way to precisely control their dimensions. Such nanochannels may be used in nanofluidic biochip applications.

Proteolipid sheets (PLSs) obtained using the vesicle fusion technique on a convenient surface are the base to obtain transmembrane protein biosensors. In this preliminary work, we have screened several physicochemical conditions to optimize the visualization of proteolipid sheets formed between different phospholipid matrices and the membrane protein lactose permease (LacP) by atomic force microscopy (AFM). When LacP was reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, the proteolipid sheets were densely packed with an upper layer that protruded from a background layer. Several lipid protein molar ratios (LPR) were screened. High resolution analysis of the upper layer revealed a quasi-crystalline arrangement formed by small entities that could be attributed to the protein. The approach described here may be suitable for the rational design of biosensors based in other transmembrane proteins.

The objective of this work was to select an efficient methodology for preparing active samples of Candida rugosa lipase immobilized in wood cellulignin, to be applied in hydrolysis and ester reactions. For this purpose, lipase was immobilized in the matrix by physical adsorption (pure cellulignin) and covalent binding (activated cellulignin with glutaraldeyde or carbonyldiimidazole [CDI]) in the presence or absence of polyethylene glycol (PEG) (Molecular mass of 1500 Daltons) as stabilizing agent. The activating agent and the presence of PEG-1500 in the immobilization procedure showed a strong influence on enzyme retention in the support. The values for enzyme retention ranged from 20 to 68%, and the highest yield was obtained when the enzyme was immobilized in cellulignin activated with CDI in the presence of PEG-1500. This immobilized derivative presented high hydrolytic (193.27 microM/[mg.min]) and synthetic (522.92 microM/[g.min]) activities when compared with those obtained by other techniques. The superiority of this immobilized system was confirmed by additional analyses, such as infrared spectroscopy and elemental analysis, which demonstrated an appropriate enzyme fixation and the highest level of protein incorporation in the support. Further information on the immobilized derivative was obtained by assessing the recycle potential in both aqueous and nonaqueous media.

The broad applicability of the cross-linking of enzyme aggregates to the effective immobilisation of enzymes is demonstrated and the influence of many parameters on the properties of the resulting CLEAs is determined. The relative simplicity of the operation ideally lends itself to high-throughput methodologies. The aggregation method was improved up to 100% activity yield for any enzyme. For the first time, the physical structures of CLEAs are elucidated., (Copyright 2004 Wiley Periodicals, Inc.)