Efficient Enzyme Immobilization Research Articles

Study of Molecular Conformation and Activity-Related Properties of Lipase Immobilized onto Core-Shell Structured Polyacrylic Acid-Coated Magnetic Silica Nanocomposite Particles.

A facile approach for the preparation of core-shell structured poly(acrylic acid) (PAA)-coated Fe3O4 cluster@SiO2 nanocomposite particles as the support materials for the lipase immobilization is reported. Low- or high-molecular-weight (1800 and 100,000, respectively) PAA molecules were covalently attached onto the surface of amine-functionalized magnetic silica nanoacomposite particles. The successful preparation of particles were verified by scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential measurement, and Fourier-transform infrared (FTIR) techniques. Once lipase is covalently immobilized onto the particles with an average diameter of 210 ± 50 nm, resulting from high binding sites concentrations on the low- and high-molecular-weight PAA-coated particles, high lipase immobilization efficiencies (86.2% and 89.9%, respectively), and loading capacities (786 and 816 mg g(-1), respectively) are obtained. Results from circular dichroism (CD) analysis and catalytic activity tests reveal an increase in the β-sheet content of lipase molecules upon immobilization, along with an enhancement in their activities and stabilities. The lipases immobilized onto the low- and high-molecular-weight PAA-coated particles show maximum activities at 55 and 50 °C, respectively, which are ∼28% and ∼15% higher than that of the free lipase at its own optimum temperature (40 °C), respectively. The immobilized lipases exhibit excellent performance at broader temperature and pH ranges and high thermal and storage stabilities, as well as superior reusability. These prepared magnetic nanocomposite particles can be offered as suitable support materials for efficient immobilization of enzymes and improvement of the immobilized enzymes properties.

Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: application to levansucrase for fructooligosaccharide synthesis

Vinyl sulfone-silica was efficient to covalently immobilize levansucrase at neutral pH. LEV-VS showed altered selectivity towards FOS and good operational stability.

Facile and controllable synthesis of triplex Au@Ag–Pt@infinite coordination polymer core–shell nanoparticles for highly efficient immobilization of enzymes and enhanced electrochemical biosensing activity

Well-dispersed Au@Ag–Pt@infinite coordination polymer core–shell nanoparticles (Au@Ag–Pt@ICPs NPs) are presented as effective host matrixes for biosensing of enzymes.

Nitrated carbon nanoblisters for high-performance glucose dehydrogenase bioanodes.

Recently, many strategies are being explored for efficiently wiring glucose dehydrogenase (GDh) enzymes capable of glucose (fuel) oxidation. For instance, the use of GDh NAD(+)-dependent for glucose oxidation is of great interest in biofuel cell technology because the enzyme are unaffected by the presence of molecular oxygen commonly present in electrolyte. Here we present the fabrication of flexible carbon fibers modified with nitrated carbon nanoblisters and their application as high-performance GDh bioanodes. These bioelectrodes could electro-oxidize glucose at -360 mV (vs. Ag/AgClsat) in the presence of a molecular oxygen saturated electrolyte with current densities higher than 1.0 mAcm(-2) at 0.0 V. It is corroborated by open circuit potential, where a potential stabilization occurs at -150 mV in a long term stability current-transient experiment. This value is in agreement with the quasi-steady current obtained at very low scan rate (0.1 mVs(-1)), where the onset potential for glucose oxidation is -180 mV. X-ray photoelectron spectroscopy and scanning electron microscopy revealed that the nitrated blisters and edge-like carbon structures, enabling highly efficient enzyme immobilization and low overpotential for electron transfer, allowing for glucose oxidation with potential values close to the thermodynamic cofactor.

Facile Method To Prepare Microcapsules Inspired by Polyphenol Chemistry for Efficient Enzyme Immobilization.

In this study, a method inspired by polyphenol chemistry is developed for the facile preparation of microcapsules under mild conditions. Specifically, the preparation process includes four steps: formation of the sacrificial template, generation of the polyphenol coating on the template surface, cross-linking of the polyphenol coating by cationic polymers, and removal of the template. Tannic acid (TA) is chosen as a representative polyphenol coating precursor for the preparation of microcapsules. The strong interfacial affinity of TA contributes to the formation of polyphenol coating through oxidative oligomerization, while the high reactivity of TA is in charge of reacting/cross-linking with cationic polymer polyethylenimine (PEI) through Schiff base/Michael addition reaction. The chemical/topological structures of the resultant microcapsules are simultaneously characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), etc. The wall thickness of the microcapsules could be tailored from 257±20 nm to 486±46 nm through changing the TA concentration. The microcapsules are then utilized for encapsulating glucose oxidase (GOD), and the immobilized enzyme exhibits desired catalytic activity and enhanced pH and thermal stabilities. Owing to the structural diversity and functional versatility of polyphenols, this study may offer a facile and generic method to prepare microcapsules and other kinds of functional porous materials.

Designing Hydrophobically Modified Polysaccharide Derivatives for Highly Efficient Enzyme Immobilization.

In this contribution, a hydrophobically modified polysaccharide derivative is synthesized in an eco-friendly solvent water by conjugation of benzylamine with the backbone of the biopolymer. Owing to the presence of aromatic moieties, the resulting water-soluble polysaccharide derivative self-assembles spontaneously and selectively from solution on the surface of nanometric thin films and sheets of polystyrene (PS). The synthetic polymer modified in this way bears a biocompatible nanolayer suitable for the immobilization of horseradish peroxidase (HRP), a heme-containing metalloenzyme often employed in biocatalysis and biosensors. Besides the detailed characterization of the polysaccharide derivative, a quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) are used to investigate the binding efficiency and interaction of HRP with the tailored polysaccharide interfaces. Subsequent enzyme activity tests reveal details of the interaction of HRP with the solid support. The novel polysaccharide derivative and its use as a material for the selective modification of PS lead to a beneficial, hydrophilic environment for HRP, resulting in high enzymatic activities and a stable immobilization of the enzyme for biocatalytic and analytic purposes.

Interfacial Polymerization of Dopamine in a Pickering Emulsion: Synthesis of Cross-Linkable Colloidosomes and Enzyme Immobilization at Oil/Water Interfaces.

Colloidosomes are promising carriers for immobilizing enzyme for catalytic purposes in aqueous/organic media. However, they often suffer from one or more problems regarding catalytic performance, stability, and recyclability. Here, we report a novel approach for the synthesis of cross-linkable colloidosomes by the selective polymerization of dopamine at oil/water interfaces in a Pickering emulsion. An efficient enzyme immobilization method was further developed by covalently bonding enzymes to the polydopamine (PDA) layer along with the formation of such colloidosomes with lipase as a model enzyme. In this enzyme system, the PDA layer served as a cross-linking layer and enzyme support for simultaneously enhancing the colloidosomes' stability and improving surface availability of the enzymes for catalytic reaction. It was found that the specific activity of lipases immobilized on the colloidosome shells was 8 and 1.4 times higher than that of free lipase and encapsulated lipase positioned in the aqueous cores of colloidosomes, respectively. Moreover, the immobilized lipases demonstrated excellent operational stability and recyclability, retaining 86.6% of enzyme activity after 15 cycles. It is therefore reasonable to expect that this novel approach for enzyme immobilization has great potential to serve as an important technique for the construction of biocatalytic systems.

Low-temperature crosslinking of proteins using non-toxic citric acid in neutral aqueous medium: Mechanism and kinetic study

For the first time, mechanism and kinetics of citric acid crosslinking of protein in neutral aqueous condition under low temperature are reported. Crosslinking was a conventional method to improve properties of protein products to replace petroleum-derived polymers. However, current crosslinking methods were toxic, expensive or required harsh conditions. Non-toxic carboxylic acid could effectively enhance wet performance properties of multiple protein products under mild conditions, which were critical for preserving the activities during immobilization of enzymes. In this study, more than one carboxyl groups in one citric acid molecule were found capable of reacting with protein in aqueous medium at pH 6.8 under 50 and 75°C, verifying occurrence of crosslinking. The pseudo second-order reaction had activation energy as low as 24.956kJmol−1, indicating its easy occurrence. Mild crosslinking using citric acid might provide alternative to accelerate low-cost and efficient enzyme immobilization as well as industrial utilization of protein-based products.

A novel nitrite biosensor based on the direct electrochemistry of hemoglobin immobilized on MXene-Ti3C2

Two-dimensional layered Ti3C2-based material (MXene-Ti3C2), which is a new graphene-like nanomaterials, has been synthesized and then used to immobilize hemoglobin (Hb) to fabricate a mediator-free biosensor. The morphology and structure of MXene-Ti3C2 were characterized by scanning electron microscopy and X-ray diffraction. Spectroscopic and electrochemical results revealed that MXene-Ti3C2 is an excellent immobilization matrix with biocompatibility for redox protein, affording good protein bioactivity and stability. Due to the large surface area and the high conductivity of MXene-Ti3C2, the direct electron transfer of Hb is facilitated and the prepared biosensors displayed good performance for the detection of nitrite with a wide linear range of 0.5–11800μM, as well as an extremely low detection limit of 0.12μM. This work suggests that MXene-Ti3C2 is a potential candidate for efficient enzyme immobilization and promising in environmental analysis.

Simple and Efficient Immobilization of Extracellular His-Tagged Enzyme Directly from Cell Culture Supernatant As Active and Recyclable Nanobiocatalyst: High-Performance Production of Biodiesel from Waste Grease

A simple method for immobilizing extracellular enzyme without prepurification of the enzyme was developed. Extracellular His-tagged Thermomyces lanuginosus lipase (His-TLL) was immobilized via affinity by direct treatment of core–shell structured iron oxide magnetic nanoparticles containing long-armed nickel-nitrilotriacetic acid surface groups (Ni-NTA-MNPs) with the cell culture supernatant of Pichia pastoris (h-TLL), giving high enzyme loading efficiency, specific enzyme loading, and specific enzyme activity. The nanobiocatalyst His-TLL-MNPs (80 nm) (5 wt % loading) catalyzed the one-pot conversion of waste grease (24 wt % FFA) to biodiesel with 94% yield and showed excellent recyclability. Ni-NTA-MNPs were easily regenerated from the recycled biocatalyst and reusable for enzyme immobilization. The immobilization method was proven to be general by the immobilization of extracellular His-tagged Candida antarctica lipase B (His-CALB) from the cell culture supernatant of P. pastoris (h-CALB).

Electrochemical pretreatment of amino-carbon nanotubes on graphene support as a novel platform for bilirubin oxidase with improved bioelectrocatalytic activity towards oxygen reduction.

The electrochemical conditioning of amino-carbon nanotubes (CNTs) on a graphene support in an alkaline solution is used to produce -NHOH as hydrophilic functional groups for the efficient immobilization of bilirubin oxidase enzyme. The application of the immobilized enzyme for the direct electrocatalytic reduction of O2 is investigated. The onset potential of 0.81 V versus NHE and peak current density of 2.3 mA cm(-2) for rotating modified electrode at 1250 rpm, indicate improved biocatalytic activity of the proposed system for O2 reduction.

Charge-switchable gold nanoparticles for enhanced enzymatic thermostability.

This study illustrates a facile strategy for efficient immobilization of enzymes on a metal nanoparticle surface. The strategy proposed here enables the enzymatic activity to be retained while increasing the enzyme thermostability. It is demonstrated that the use of a zwitterionic amino acid tyrosine as a reducing and capping agent to synthesise gold nanoparticles allows efficient immobilization of phytase enzyme through charge-switchable electrostatic interactions. The detailed kinetic and thermodynamic studies reveal that the proposed enzyme immobilization strategy improves the overall quality of phytase by reducing the activation energy required for substrate hydrolysis and broadening the temperature window in which immobilized enzyme is able to operate. The outcomes of this study indicate that the underlying zwitterionic nature of 20 natural amino acids along with significant variability in their isoelectric points and hydropathy indices as well the ability of some of the amino acids to reduce metal ions is likely to offer significant opportunities for tailoring nano-bio interfaces in a rational manner for a range of biological applications.

Facile preparation of porous magnetic polydopamine microspheres through an inverse replication strategy for efficient enzyme immobilization.

An inverse replication method based on porous CaCO3 templates was developed to fabricate porous magnetic polymer microspheres (PMMSs) composed of biocompatible polydopamine and magnetic Fe3O4 nanoparticles. The preparation procedure involved the synthesis of Fe3O4@CaCO3 templates, infiltration and spontaneous polymerization of dopamine in template pores and finally the mild removal of templates. The particle size, the surface morphology and the pore structure (e.g., average pore size, pore volume, surface area, etc.) of porous PMMSs were facilely tailored by altering the templates. The as-prepared polydopamine microspheres PMMSs were applied to covalently immobilize YADH for catalyzing the conversion of formaldehyde to methanol. In comparison to the enzyme-conjugated PDA-coated Fe3O4 nanoparticles (PMNPs), the immobilized enzyme on porous PMMSs exhibited remarkably enhanced activity (specific activity: 162.3 U mg-1 enzyme vs. 97.6 U mg-1 enzyme; methanol yield: 95.5% vs. 57.1%; initial reaction rate: 0.15% s-1vs. 0.08% s-1), and desirable thermal/pH/recycling/storage stabilities, and particularly, easy separation from the bulk solution by an external magnetic field.

Formulation of robust organic-inorganic hybrid magnetic microcapsules through hard-template mediated method for efficient enzyme immobilization.

A mild and facile method for the construction of robust organic-inorganic hybrid magnetic microcapsules was developed by a hard-template mediated method combined with polydopamine (PDA) and Fe3O4 nanoparticles onto a CaCO3 microparticle template. More specifically, negatively charged Fe3O4 nanoparticles were adsorbed on the surface or into the lumen of porous CaCO3 microparticles through electrostatic interaction and physical absorption. Then, the magnetic sacrificial templates were coated with PDA through the self-polymerization of dopamine to obtain the magnetic PDA-CaCO3 microparticles, which was followed by template removal using EDTA to construct organic-inorganic hybrid magnetic microcapsules. Characterization confirmed that the microcapsules possess a robust hollow structure such that the enzyme inside exists in a free state. The Fe3O4 nanoparticles acted as critical factors in the microcapsules for both recyclable component and tough scaffolds to sustain the microcapsules away from collapsing and folding. Combing the merits of the organic layer and the inorganic component, the microcapsules were applied for the encapsulation of Candida Rugosa Lipase (CRL). The encapsulated CRL was demonstrated to have several advantages, including increased encapsulation efficiency, enzyme activity and long-term storage stability. Hopefully, the as-prepared microbioreactor may provide a facile and generic approach for other biochemical applications.

Surface cell immobilization within perfluoroalkoxy microchannels

Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4h contacting with 10% APTES aqueous solution, 10min treatment with 1% glutaraldehyde and 20min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.

Fabrication of Boehmite/Alginate Hybrid Beads for Efficient Enzyme Immobilization

A novel kind of matrix for enzyme immobilization, the boehmite/alginate hybrid beads, was fabricated under mild conditions in this study. The boehmite particles were first prepared via a mild sol–gel technology and were then entrapped into the inherent network of calcium alginate. The characterization results suggest that the hybridization between alginate and boehmite belongs to a physical blending, in which the electrostatic interaction acts an important role. Yeast alcohol dehydrogenase (YADH), which is of high importance for the transformation of CO2, was encapsulated in the boehmite/alginate hybrid beads. The enzyme was loaded as high as 99.4% and uniformly distributed in these hybrid beads according to the fluorescence experiment. It is worthy of noting that the encapsulated YADH can almost reach “zero leaching” after incubation for 67 h and preserve 86.6% activity after 12-recycle using. This kind of hybrid materials may become a promising matrix for the immobilization of industrial enzymes.

Immobilization and catalytic activity of horseradish peroxidase on molybdenum disulfide nanosheets modified electrode

This communication reports the application of layer-structured MoS2 as matrix for enzyme immobilization and biosensor construction. MoS2 nanosheets were prepared using ultrasonic exfoliation in solvent mixture. Data from UV–vis spectra, scanning electron microscopy and transmission electron microscopy demonstrate that the as-prepared MoS2 nanomaterial has ordered layer-structure. The negative charges carried by the MoS2 nanosheets are then used to immobilize horseradish peroxidase (HRP) via the electrostatic attraction, forming HRP–MoS2 hybrid. FTIR spectra indicate that the immobilized HRP retains its native structure. The HRP–MoS2 hybrid modified glassy carbon electrode shows good electrocatalytic activity toward H2O2 reduction. This work suggests that MoS2 nanosheets are a potential candidate for efficient enzyme immobilization and promising in biosensing and biocatalytic applications.

Development and evaluation of microfluidic device for the determination of organophosphorus pesticide incorporating monolith based immobilized AChE with spectrophotometric detection

A spectrophotometric microfluidic bioreactor system is described for the determination of organophosphorus pesticides. The glass chip was designed and fabricated for in situ monolithic preparation and subsequently acetlycholineserase (AChE) immobilization via a covalent bonding method. The porous polymer monolith was prepared using glycidyl methacrylate, ethylenedimethacrylate and 2,2-dimethoxy-1,2-diphenylethan-1-one in binary porogenic solvents of cyclohexanol and dodecanol. The epoxide groups of monolith were reacted with ethylenediamine and gluteraldehyde to allow immobilization of the enzyme using their amine groups. Organophosphorus pesticides can be determined by measuring their inhibition effect on the enzyme AChE using Ellman's reaction. A linear relationship between the absorbance and percentage inhibitions was obtained over the concentration range of 0.25 to 2.50 mg L−1 paraoxon with a correlation coefficient (r 2) of 0.9974. The limit of detection (LOD) defined as 10% inhibition (I 10) was 0.17 mg L−1 for paraoxon. The relative standard deviations (RSD) of 1.0 mg L−1 paraoxon was 3.73% (n = 5). The proposed µFI system incorporates efficient enzyme immobilization and reduces reagent consumption and waste production and could thus be considered to be more environmentally friendly.

Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37°C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.