Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis

In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.     

Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles

Mucor javanicus lipase was effectively immobilized on silica nanoparticles which were prepared by Stöber method. Glycidyl methacrylate (GMA), which bears a reactive epoxide group, was incorporated onto the surface of the nanoparticles and the epoxide groups were directly used for multipoint coupling of the enzyme. We also introduced amine residues by coupling ethylene diamine (EDA) to the epoxide group of GMA. M. javanicus lipase was covalently immobilized onto the amine-activated silica nanoparticles by using glutaraldehyde (GA) or 1,4 phenylene diisothiocyanate (NCS) as a coupling agent. The lipase loading capacities of the EDA-GA and EDA-NCS nanoparticles (81.3 and 60.9 mg g⁻¹, respectively) were much higher than that of the unmodified GMA nanoparticles (18.9 mg g⁻¹). The relative hydrolytic activities in an aqueous medium of the lipases immobilized on EDA-GA and EDA-NCS attached silica nanoparticles (115% and 107%, respectively) were significantly high and almost in the same range with the free enzyme. This may be due to the improvement of the enzyme–substrate interaction by avoiding the potential aggregation of free lipase molecules. The immobilized lipases were also more resistant to temperature inactivation than the free form. This work demonstrates that the size-controlled silica nanoparticles can be efficiently employed as host materials for enzyme immobilization leading to high activity and stability of the immobilized enzymes.     

Enzyme immobilization in a biomimetic silica support

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure. Here we show that butyrylcholinesterase entrapped during the precipitation of silica nanospheres retained all of its activity. Ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme. The mechanical properties of silica nanospheres facilitated application in a flow-through reactor. The use of biosilica for enzyme immobilization combines the excellent support properties of a silica matrix with a benign immobilization method that retains enzyme activity.     

Fluorescent (Au@SiO2)SiC Nanohybrids: Influence of Gold Nanoparticle Diameter and SiC Nanoparticle Surface Density

Gold@silica core–shell nanoparticles were prepared with various gold core diameters (ranging from 20 to 150 nm) and silica thicknesses (ranging from 10 to 30 nm). When the gold diameter is increased, the size dispersion became larger, leading to a broader plasmon band. Then, silicon carbide (SiC) nanoparticles were covalently immobilized onto silica to obtain hybrid (Au@SiO₂) SiC nanoparticles. The absorption properties of these hybrid nanoparticles showed that an excess of SiC nanoparticles in the dispersion can be identified by a strong absorption in the UV region. Compared to SiC reference samples, a blue shift of the fluorescence emission, from 582 to 523 nm, was observed, which was previously attributed to the strong surface modification of SiC when immobilized onto silica. Finally, the influence of several elaboration parameters (gold diameter, silica thickness, SiC concentration) on fluorescence enhancement was investigated. It showed that the highest enhancements were obtained with 10 nm silica thickness, low concentration of SiC nanoparticles, and surprisingly, with a 20-nm gold core diameter. This last result could be attributed to the broad plasmon band of big gold colloids. In this case, SiC emission strongly overlapped gold absorption, leading to possible quenching of SiC fluorescence by energy transfer.     

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH₂ nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5–11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5 % of its initial activity at similar conditions. The immobilized protease showed higher k _cat and K _m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH₂ nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.     

A novel route for immobilization of proteins to silica particles incorporating silaffin domains

In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post‐translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1–R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600–700 μg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP‐R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin‐protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin‐fused proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Immobilization of proline-specific endoprotease on nonporous silica nanoparticles functionalized with amino group

Enzyme immobilization is believed to provide an excellent base for increasing environmental tolerance of enzyme and considerable period of time. In this work, a kind of nonporous silica nanoparticles functionalized with amino group was synthesized to immobilize proline-specific endoprotease (PSEP). PSEP is known to specifically cleave peptides (or esters) at the carboxyl side of proline, thus can prevent the formation of haze and prolong the shelf life of beer. After immobilization, the environmental tolerance (temperature and pH, respectively) was obviously improved, and the immobilized enzyme can retain above 90 % of its original activity after 6 uses. Moreover, the immobilized enzyme can effectively prevent the formation of chill-haze using fresh beer fermentation liquid.     

Covalent immobilization of recombinant <Emphasis Type="Italic">Rhizobium etli</Emphasis> CFN42 xylitol dehydrogenase onto modified silica nanoparticles

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t _1/2 of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K _m of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into l-xylulose. A ∼ 71% conversion and a productivity of 10.7 g h^-1 l^-1 were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to l-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.     

Improved stability of the carbon nanotubes-enzyme bioconjugates by biomimetic silicification

Nano-materials have been applied in many fields due to their excellent characteristics, such as the high surface area-to-volume ratio, excellent physicochemical properties and biological compatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were utilized to prepare MWCNTs-papain bioconjugates and then realized the immobilization of papain. MWCNTs functionalized with carboxyl- and amine- groups on their surface were used as immobilization carriers. The immobilization of papain on the functionalized MWCNTs through physical absorption was examined. The conjugates were denoted as MWCNTs-papain bioconjugates. To improve the stability, the bioconjugates were further coated by silica through the biomimetic silicification process that induced by papain (denoted as silica-coated bioconjugates). The as-prepared MWCNTs-papain bioconjugates and the silica-coated bioconjugates were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The preliminary results showed that the bioconjugates could retain most of the initial activity of papain. Compared to free papain and MWCNTs-papain bioconjugates, the silica-coated bioconjugates exhibited significantly improved thermal, pH and recycling stability. Comparisons of the kinetic parameters between MWCNTs-papain bioconjugates and the silica-coated bioconjugates revealed that the K_m value of the immobilized papain experienced a slight increase after silica coating, which suggested that the silica coating did not significantly hinder papain's access to substrate or release of product.     

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants in wastewater. Laccase of the white-rot fungus Coriolopsis polyzona has been successfully immobilized on fumed silica nanoparticles using a novel method. It consists in the sorption of the enzyme to amino-modified silica nanoparticles and the subsequent covalent cross-linking using glutaraldehyde as a homobifunctional linker. The so-produced nanoparticulate material has been characterized by means of scanning electron microscopy and Brunauer–Emmett–Teller surface area analysis revealing modifications of the surface structure and area during the coupling procedure. Laccase immobilization on spherical nanoparticles produced according to the method of St?ber has been shown to be much less efficient than on fumed silica nanoparticles. Long-term stability assays revealed that the novel developed method allows a drastic stabilization of the enzyme. In real wastewater, 77% of the laccase activity remained on the nanoparticles over 1 month, whereas the activity of free laccase dropped to 2.5%. The activity loss on the nanoparticles resulted from partial inactivation of the immobilized enzymes and additional release into the surrounding solution with subsequent fast inactivation of the free enzymes, since almost no activity was found in the supernatants.     

Immobilization of cellulase on polyamidoamine dendrimer-grafted silica

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups, and is suitable to carry biomolecules due to its affinity and biocompatibility. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation. A novel media was developed through immobilizing cellulase onto the prepared PAMAM-grafted silica by adsorption and crosslinking methods and applied in hydrolysis of carboxymethyl cellulose. The results demonstrate that the enzyme binding capacity and enzymolysis efficiency increased with generations of PAMAM. The properties of the immobilized cellulase-PAMAM-grafted silica were investigated, which possessed high enzymatic activity and exhibited better stability with respect to pH, temperature compared with free enzyme. The optimal immobilization conditions for adsorption and crosslinking method were respectively obtained at 5 and 4 mg ml⁻¹ cellulase for 2 h of immobilization. A high enzymolysis efficiency was achieved by employing pH 4.8 and 5.8 substrate solution at 60 °C for adsorbed and crosslinked cellulase, respectively. After repeated three run cycles, the retained activities were found to be 75% and 82%. The results indicate that the PAMAM has a good performance as a carrier, and can be potentially adapted to support other biomacromolecules.     

One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.     

Using fluorescent nanoparticles and SYBR Green I based two-color flow cytometry to determine Mycobacterium tuberculosis avoiding false positives

A method using an improved two-color flow cytometric analysis by a combination of bioconjugated fluorescent silica nanoparticles and SYBR Green I (FSiNP@SG-FCM) has been developed for detection of pathogenic Mycobacterium tuberculosis. Antibody-conjugated nanoparticles were prepared by oriented immobilization of the anti-M. tuberculosis antibody onto Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate-doped fluorescent silica nanoparticles (RuBpy-doped FSiNPs) through Protein A. M. tuberculosis was specially labeled with antibody-conjugated RuBpy-doped FSiNPs, then stained with a nucleic acid dye SYBR Green I to exclude background detrital particles, followed by multiparameter determination with flow cytometry. With this method, false positives caused by aggregates of nanoparticle-bioconjugates and nonspecific binding of nanoparticle-bioconjugates to background debris could be significantly decreased. This assay allowed for detection of as low as 3.5 x 10(3) and 3.0 x 10(4)cellsml(-1)M. tuberculosis in buffer and spiked urine respectively, with higher sensitivities than the FITC-based conventional flow cytometry. The total assay time including sample pretreatment was within 2h. This proposed FSiNP@SG-FCM method will be promising for rapid detection of M. tuberculosis or other pathogenic bacteria in clinical samples.     

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.     

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.     

Biosurfactants as green stabilizers for the biological synthesis of nanoparticles

Taking into consideration the needs of greener bioprocesses and novel enhancers for synthesis using microbial processes, biosurfactants, and/or biosurfactant producing microbes are emerging as an alternate source for the rapid synthesis of nanoparticles. A microemulsion technique using an oil-water-surfactant mixture was shown to be a promising approach for nanoparticle synthesis. Biosurfactants are natural surfactants derived from microbial origin composed mostly of sugar and fatty acid moieties, they have higher biodegradability, lower toxicity, and excellent biological activities. The biosurfactant mediated process and microbial synthesis of nanoparticles are now emerging as clean, nontoxic, and environmentally acceptable “green chemistry’’ procedures. The biosurfactant-mediated synthesis is superior to the methods of bacterial- or fungal-mediated nanoparticle synthesis, since biosurfactants reduce the formation of aggregates due to the electrostatic forces of attraction and facilitate a uniform morphology of the nanoparticles. In this review, we highlight the biosurfactant mediated synthesis of nanoparticles with relevant details including a greener bioprocess, sources of biosurfactants, and biological synthesized nanoparticles based on the available literature and laboratory findings.     

Biosurfactants as green stabilizers for the biological synthesis of nanoparticles

Taking into consideration the needs of greener bioprocesses and novel enhancers for synthesis using microbial processes, biosurfactants, and/or biosurfactant producing microbes are emerging as an alternate source for the rapid synthesis of nanoparticles. A microemulsion technique using an oil-water-surfactant mixture was shown to be a promising approach for nanoparticle synthesis. Biosurfactants are natural surfactants derived from microbial origin composed mostly of sugar and fatty acid moieties, they have higher biodegradability, lower toxicity, and excellent biological activities. The biosurfactant mediated process and microbial synthesis of nanoparticles are now emerging as clean, nontoxic, and environmentally acceptable "green chemistry" procedures. The biosurfactant-mediated synthesis is superior to the methods of bacterial- or fungal-mediated nanoparticle synthesis, since biosurfactants reduce the formation of aggregates due to the electrostatic forces of attraction and facilitate a uniform morphology of the nanoparticles. In this review, we highlight the biosurfactant mediated synthesis of nanoparticles with relevant details including a greener bioprocess, sources of biosurfactants, and biological synthesized nanoparticles based on the available literature and laboratory findings.     

Immobilization and biocatalytic activity of fungal protease on gold nanoparticle-loaded zeolite microspheres

Gold nanoparticles are excellent biocompatible surfaces for the immobilization of enzymes. However, separation of the gold nanoparticle-enzyme bioconjugate material from the reaction medium is often difficult. In this study, we investigate the assembly of the gold nanoparticles on the surface of the amine-functionalized zeolite microspheres in the formation of zeolite-gold nanoparticle "core-shell" structures and, thereafter, the use of this structure in immobilization of fungal protease. The assembly of gold nanoparticles on the zeolite surface occurs through the amine groups present in 3-aminopropyltrimethoxysilane (3-APTS). The fungal proteases bound to the massive "core-shell" structures were easily separated from the reaction medium by mild centrifugation and exhibited excellent reuse characteristics. The biocatalytic activity of fungal protease in the bioconjugate was marginally enhanced relative to the free enzyme in solution. The bioconjugate material also showed significantly enhanced pH and temperature stability and a shift in the optimum temperature of operation.     

Influence of the conditions of trypsin immobilization onto Spherosil on coupling efficiency

Summary The effect of several parameters (pH, time of reaction, temperature, enzyme concentration) on trypsin immobilization onto glutaraldehyde-activated amine-Spherosil was investigated. This activated support could be stored over long periods of time without any important loss of capacity for trypsin coupling. When increasing the amount of trypsin bound to the carrier, enzymatic activity shows an optimal value, beyond which an augmentation of Spherosil enzyme content results in a lowered activity. The influence of the number of available reactive aldehyde groups on silica was investigated by coupling L-lysine to activated support either prior to or simulataneously with trypsin immobilization. In both cases, the activity of trypsin derivatives is decreased when L-lysine concentration is increased, yet the activity of trypsin derivatives is never equal to zero, even in presence of a large excess of L-lysine. This suggests the presence of two types of reactive groups on the activated support.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.