Noncovalent Immobilization Research Articles

SORPTION OF BISMUTON (III) IONS FROM AQUEOUS SOLUTIONS BY SILICAS MODIFIED BY ORGANIC REAGENTS

In current paper, adsorbents based on silica L 40/100 modified with 4-(2-pyridylazo)resorcinol, 1-(2-pyrylazo)naphthol‑2, pyrocatechol violet and sodium N, N‑diethyldithiocarbamate were obtained. Adsorbents were modified by non-covalent immobilization of organic analytical reagents on the silica surface. It is noted that the preparation of such adsorbents is fast and easy to manufacture. The conditions of Bismuth (III) adsorption from dilute aqueous solutions by the proposed adsorbents are studied and optimized. The optimal values of the medium acidity for the effective Bismuth (III) preconcentration by the proposed adsorbents based on silica modified with organic analytical reagents have been established. It is shown that the use of modified silicas allows efficient (95–98%) removal of Bismuth (III) from dilute aqueous solutions. Under optimal adsorption conditions, the capacity of modified adsorbents is determined. It is established that the modification of silica leads to a significant (2–3 times) increase in the capacity of the obtained sorbents by Bismuth (III) in comparison with the unmodified silica, which is associated with the processes of complexation on the surface. The desorption of Bismuth (III) from the surface of unmodified and modified silicas by solutions of mineral acids has been studied. It is shown that Bi(III) is quantitatively desorbed from the surface of unmodified silicas by solutions of sulfuric and nitric acids, and in the case of modified silicas the degree of desorption is small and does not exceed 35%. The data obtained can then be used to develop a test system for determination of Bismuth (III) via corresponding colorimetric scales or for quantitative solid phase extraction and adsorption-spectroscopic quantification of Bismuth (III) in some real samples.

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Pyranine (HPTS) is a remarkably interesting pH‐sensitive dye that has been used for plenty of applications. Its high quantum yield and extremely sensitive ratiometric fluorescence against pH change makes it a very favorable for pH‐sensing applications and the development of pH nano‐/microsensors. However, its strong negative charge and lack of easily modifiable functional groups makes it difficult to use with charged substrates such as silica. This study reports a methodology for noncovalent HPTS immobilization on silica microparticles that considers the retention of pH sensitivity as well as the long‐term stability of the pH microsensors. The study emphasizes the importance of surface charge for governing the sensitivity of the immobilized HPTS dye molecules on silica microparticles. The importance of the immobilization methodology, which preserves the sensitivity and stability of the microsensors, is also assessed.

Non-covalent and covalent immobilization of papain onto Ti3C2 MXene nanosheets

Papain was immobilized onto Ti3C2 MXene nanosheets by physical adsorption and physical adsorption combined with covalent crosslinking with glutaraldehyde. Ti3C2 MXene nanosheets were prepared by hydrofluoric acid etching method. The resulting products were well characterized by SEM, BET, XRD, FTIR, XPS. The optimized immobilization conditions are pH 6.5, immobilization time of 20 h, immobilization temperature of 10℃, and 10 mL 2 mg mL−1 papain, the amount of papain immobilized was 156 mg g−1, the activity of the immobilized papain determined was 1701 U∙g−1. The immobilized papain exhibited enhanced pH and temperature endurances, immobilized papain also showed improved storage stability (39.25 % and 65.57 % after 20 days of storage at 4 °C). papain reusability was significantly improved after immobilization and it retained more than 50 % of its initial activity after 5 repeated cycles. Interestingly, the results of immobilized enzymes demonstrated that the immobilization of enzymes on Ti3C2 MXene is feasible. Such approach could be transferred to other support systems for anchoring enzyme.

Noncovalent microarrays from synthetic amino-terminating glycans: Implications in expanding glycan microarray diversity and platform comparison.

Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward β-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.

Homogeneous and noncovalent immobilization of NHC-Cu catalyzed azide-alkyne cycloaddition reaction

Abstract A series of new pyrene tagged nitrogen heterocyclic carbene copper (NHC-Cu) molecular complexes were synthesized and characterized by 1H- and 13C-NMR, high resolution ESI-mass spectrometry (HR-MS). The corresponding NHC-Cu complex was immobilized onto the surface of multi-walled carbon nanotubes (MWNTs) by using π-π stacking interaction, the noncovalent immobilization catalytic material MWNTs@NHC-Cu were characterized by FT-IR, XRD, XPS, thermogravimetry (TGA) and TEM. The new NHC-Cu complexes were tested both in the homogeneous phase and once immobilized onto multi-walled carbon nanotubes support for the azide-alkyne cycloaddition reaction. Both were active in the two-component click cycloaddition reaction of terminal alkynes and organic azides and three-component cycloaddition reaction that employs various terminal alkynes, NaN3 and organic halides with the advantage of excellent yields, low catalyst dosage, short reaction time. The MWNTs@NHC-Cu catalyst could be separated from the reaction system at the end of the reaction and reused in another catalytic cycle, but with loss of product yield.

Magnetically Responsive PA6 Microparticles with Immobilized Laccase Show High Catalytic Efficiency in the Enzymatic Treatment of Catechol

Herewith we report the first attempt towards non-covalent immobilization of Trametes versicolor laccase on neat and magnetically responsive highly porous polyamide 6 (PA6) microparticles and their application for catechol oxidation. Four polyamide supports, namely neat PA6 and such carrying Fe, phosphate-coated Fe and Fe3O4 cores were synthesized in suspension by activated anionic ring-opening polymerization (AAROP) of ε-caprolactam (ECL). Enzyme adsorption efficiency up to 92% was achieved in the immobilization process. All empty supports and PA6 laccase complexes were characterized by spectral and synchrotron WAXS/SAXS analyses. The activity of the immobilized laccase was evaluated using 2,2’-Azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) and compared to the native enzyme. The PA6 laccase conjugates displayed up to 105% relative activity at room temperature, pH 4, 40 °C and 20 mM ionic strength (citrate buffer). The kinetic parameters of the ABTS oxidation were also determined. The reusability of the immobilized laccase-conjugates was proven for five consecutive oxidation cycles of catechol.

Development of MALDI MS peptide array for thrombin inhibitor screening

The development of in situ methods for the analysis and visualization of enzyme activity is of paramount importance in drug discovery, research, and development. In this work, the functionalized and array patterned indium tin oxide (ITO) glass slides were fabricated by non-covalent immobilization of amphipathic phospholipid-tagged peptides encompassing the thrombin cleavage site on steric acid-modified ITO slides. The fabricated peptide arrays provide 60 spots per slide, and are compatible with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) measurement, free matrix peak interference, and tolerance to repeated aqueous washing. The peptide arrays were used for the investigation of thrombin activity and screening for its potential inhibitors. The thrombin activity and its Michaelis-Menten constant (Km) for immobilized peptide substrate was determined using developed MALDI MS peptide array. To investigate the applicability and effectiveness of peptide arrays, the anti-thrombin activity of grape seed proanthocyanidins with different degrees of polymerization (DP) was monitored and visualized. MALDI MS imaging results showed that the fractions of proanthocyanidins with the mean DP of 4.61–6.82 had good thrombin inhibitory activity and their half-maximal inhibitory concentration (IC50) were below 10 μg/mL. Therefore, the developed peptide array is a reliable platform for the discovery of natural thrombin inhibitors.

Controlled density glycodendron microarrays for studying carbohydrate-lectin interactions.

Glycodendron microarrays with defined valency have been constructed by on-chip synthesis on hydrophobic indium tin oxide (ITO) coated glass slides and employed in lectin-carbohydrate binding studies with several plant and human lectins. Glycodendrons presenting sugar epitopes at different valencies were prepared by spotwise strain-promoted azide-alkyne cycloaddition (SPAAC) between immobilised cyclooctyne dendrons and azide functionalised glycans. The non-covalent immobilisation of dendrons on the ITO surface by hydrophobic interaction allowed us to study dendron surface density and SPAAC conversion rate by in situ MALDI-TOF MS analysis. By diluting the dendron surface density we could study how the carbohydrate-lectin interactions became exclusively dependant on the valency of the immobilised glycodendron.

Peptide-mediated surface coatings for the release of wound-healing cytokines.

Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg-Gly-Asp (RGD) are commonly used to enhance cell-surface interactions, peptide-mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel-derived surface coating as a prospective cytokine delivery system using covalently bound heparin mimetics, receptor-derived chemokine-binding peptides, and heparin-binding peptides (HBP). The latter enabled non-covalent immobilization of heparin on the surface followed by chemokine binding and release, whereas the former allowed direct non-covalent chemokine immobilization. The peptide displayed excellent binding to custom-made polystyrene 96-well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells, especially for the non-covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin-mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics.

Silk Fibroin-Based Materials for Catalyst Immobilization.

Silk fibroin is a widely and commercially available natural protein derived from silkworm cocoons. Thanks to its unique amino acid composition and structure, which lead to localized nanoscale pockets with limited but sufficient hydration for protein interaction and stabilization, silk fibroin has been studied in the field of enzyme immobilization. Results of these studies have demonstrated that silk fibroin offers an important platform for covalent and noncovalent immobilization of enzymes through serving as a stabilization matrix/support with high retention of the biological activity of the enzymes of interest. In the hope of providing suggestions for potential future research directions, this review has been written to briefly introduce and summarize key advances in silk fibroin-based materials for immobilization of both enzymes/biocatalysts (including alkaline phosphatase, β-glucosidase, glucose oxidase, lipase, urease, uricase, horseradish peroxidase, catalase, xanthine oxidase, tyrosinase, acetylcholinesterase, neutral protease, α-chymotrypsin, amylase, organophosphorus hydrolase, β-galactosidase, carbonic anhydrase, laccase, zymolyase, phenylalanine ammonia-lyase, thymidine kinase, and several others) and non-enzymatic catalysts (such as Au, Pd, Fe, α-Fe2O3, Fe3O4, TiO2, Pt, ZnO, CuO, Cu2O, Mn3O4, and MnO2).

Three-phase partitioning and immobilization of Bacillus methylotrophicus Y37 cellulase on organo-bentonite and its kinetic and thermodynamic properties

AbstractIn this study, for the first time Bacillus methylotrophicus Y37 cellulase was purified and recovered in a single step by three-phase partitioning (TPP). The optimal purification parameters for TPP were 40% ammonium sulfate saturation (m/v) with a 1.0:1.0 (v/v) ratio of crude extract:t-butanol, which gave 5.8-fold purification with 155% recovery of cellulase. Non-covalent immobilization of the partitioned cellulase was performed using bentonite as a support material. The activity observed in the 20th experiment was 100%. The optimal pH values and temperatures determined for the free enzyme and the immobilized enzyme were 5.0 and 6.0 and 45°C and 50°C, respectively. The Arrhenius activation energy (Ea) of the immobilized enzyme was lower than that of the free enzyme, whereas the Michaelis–Menten constant (Km) and maximum velocity (Vm) of the immobilized enzyme increased. The turnover number (kcat) and the catalytic performance (kcat/Km) demonstrated the improved catalytic properties of the immobilized enzyme compared to the free enzyme. Immobilization of cellulase is thermodynamically preferred.

Immobilization of β-glucuronidase: biocatalysis of glycyrrhizin to 18β-glycyrrhetinic acid and in-silico lead finding

Abstract: The non-covalent immobilization of ?-glucuronidase enzyme obtained from Rhizopus oryzae was carried out by entrapment in natural fiber (papaya and coconut). The bioconversion capability of immobilized enzyme was analyzed based on conversion of glycyrrhizin to 18?-glycyrrhetinic acid under different conditions. The hydrolytic activity of the ?-glucuronidase enzyme was highly depended on the microbial source and matrix, in which enzyme was immobilized. R. oryzae ?-glucuronidase immobilized in papaya fibers produced the highest GA content (13.170 µg/mL) at 10 h of reaction. However R. oryzae ?-glucuronidase immobilized in coconut fibers produced the highest GA content (21.425 µg/mL) at 15 h of reaction. Online Molinspiration software was used to predict drug like molecular properties of the 18?-glycyrrhetinic acid, and software suggested that the compounds had potential of becoming the orally active molecules. Therefore, in silico studies were conducted on proposed 18?-glycyrrhetinic acid to select the best possible drug candidates based on drug properties and bioactivity score of the compounds.

Silaffin-3-derived pentalysine cluster as a new fusion tag for one-step immobilization and purification of recombinant Bacillus subtilis catalase on bare silica particles

Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.

Facile Synthesis and Characterization of a Novel Tamavidin-Luciferase Reporter Fusion Protein for Universal Signaling Applications.

Despite the avidin/biotin reaction being one of the most ubiquitous noncovalent immobilization and sensing strategies in scientific research, the ability to synthesize useful amounts of biotin-binding fusion constructs is hampered by poor solubility in bacterial expression systems. As such, there are few reports of successful genetic reporter fusions incorporating a biotin-binding partner. To address this, a sensitivity-enhanced, synthetically facile reporter fusion is developed to merge the bioluminescence output of Gaussia luciferase (Gluc) with the recently characterized biotin-binding ability of tamavidin 2 (TA2) for general and universal signaling applications in biological and analytical systems. This fusion construct enables direct bacterial expression of a reporter system incorporating two important functionalities in a 1:1 stoichiometric relationship that can provide detection of discrete events at low concentrations. Using a cold-shock expression system, highly concentrated construct can be obtained from standard culture volumes while retaining essentially native protein activity. To demonstrate feasibility and provide an example application, this fusion construct is then included in a standard target-bridged assay design for the sensitive detection of four miRNA targets.

Pyrene-Based Noncovalent Immobilization of Nitrogenase on Carbon Surfaces.

Early work with viologen-mediated nitrogenase bio-electrochemistry focused on bio-electrocatalysis with the enzyme in solution, but biocatalyst immobilization at electrode surfaces is favorable as it provides a high effective concentration of biocatalyst at the electrode surface. We have used a pyrene-peptide linker as a method for immobilizing nitrogenase on carbon electrode surfaces for nitrogenase bio-electrocatalysis.

Noncovalent Integration of a Bioinspired Ni Catalyst to Graphene Acid for Reversible Electrocatalytic Hydrogen Oxidation.

Efficient heterogeneous catalysis of hydrogen oxidation reaction (HOR) by platinum group metal (PGM)-free catalysts in proton-exchange membrane (PEM) fuel cells represents a significant challenge toward the development of a sustainable hydrogen economy. Here, we show that graphene acid (GA) can be used as an electrode scaffold for the noncovalent immobilization of a bioinspired nickel bis-diphosphine HOR catalyst. The highly functionalized structure of this material and optimization of the electrode-catalyst assembly sets new benchmark electrocatalytic performances for heterogeneous molecular HOR, with current densities above 30 mA cm–2 at 0.4 V versus reversible hydrogen electrode in acidic aqueous conditions and at room temperature. This study also shows the great potential of GA for catalyst loading improvement and porosity management within nanostructured electrodes toward achieving high current densities with a noble-metal free molecular catalyst.

Noncovalent immobilization of Co(ii)porphyrin through axial coordination as an enhanced electrocatalyst on carbon electrodes for oxygen reduction and evolution

Noncovalent immobilization of Co(ii)porphyrin on carbon nanotubes through axial coordination provides significantly enhanced electrochemically catalyzed oxygen reduction and oxygen evolution.

Use of functionalized carbon nanotubes for the development of robust nanobiocatalysts.

This chapter deals with the use of functionalized carbon nanotubes (fCNTs) as supports for the development of nanobiocatalytic systems through the immobilization of enzymes. The surface characteristics, properties and production of carbon nanotubes are described, while an analysis in their biological applications is also presented. The results presenting within the text are giving insights to the effect of carbon nanotubes on the catalytic and structural characteristics of different proteins, such as cytochrome c from equine heart (cyt c) and laccase from Trametes versicolor (TvL), either when they are used as additives in the reaction medium or as supports for protein immobilization. A variety of biochemical and spectroscopic techniques is applied to investigate the interactions between the protein biomolecules and carbon nanotubes. The results showed that the presence of fCNTs enables cyt c to maintain both its secondary structure and heme microenvironment. Non-covalent and covalent immobilization approaches are also described, while the immobilized biocatalysts are characterized with respect to their catalytic and structural characteristics. Immobilized TvL was found to exhibit higher catalytic activity when non-specific binding was used as immobilization procedure (up to 0.85Uμg-1), compared to covalent immobilization (up to 0.7Uμg-1), while the increase of the alkyl chain of the functionalized CNTs also seems to affect the catalytic efficiency of the immobilized enzymes. The nanobiocatalytic systems that are presented here demonstrated exceptional stability (up to 31% of their initial activity is maintained after 24h incubation at 60°C) and reusability (up to 58% remaining activity after 8 successive catalytic cycles) compared to the native enzymes, leading to robust biocatalytic systems appropriate for various applications of biotechnological and industrial interest.

Peptide-Mediated Immobilization on Magnetoferritin for Enzyme Recycling.

Ferritin possess favorable properties because its exterior and interior surface can be applied to generate functional nanomaterials, which make them possible for enzyme immobilization and recycling. Here, we report the noncovalent immobilization of a genetically modified β-glucosidase onto the outer surface of synthetic magnetoferritin through the electrostatic interaction of a heterodimeric coiled-coil protein formed by coils containing lysine residues (K-coils) and coils containing glutamic acid (E-coils). The immobilized enzyme was characterized, and its enzymatic properties were evaluated. Furthermore, reusability of immobilized enzyme was demonstrated in aqueous solution under an applied magnetic field. The results showed that magnetoferritin was successfully prepared and it was an excellent support for enzyme immobilization. After three times usages, the retention rates were 93.75%, 82.5%, and 56.25%, respectively, demonstrating that immobilized enzyme possessed good retention efficiency and could be used as potential carrier for other biomolecules. The strategy of enzyme immobilization developed in this work can be applied, in general, to many other target molecules.

Non-covalent and covalent immobilization of Candida antarctica lipase B on chemically modified multiwalled carbon nanotubes for a green acylation process in supercritical CO2

• Efficient immobilization of Candida antarctica B lipase on modified MWCNTs. • Enzyme loading reaching as high as 21 wt.% for non-covalent immobilization. • Active O-acylation of geraniol into geranyl acetate by CAL-B in supercritical CO 2 . • Reaction regioselectivity unchanged through immobilization. • Development of a fully green enzymatic process. Candida antarctica B lipase (CAL-B) was immobilized on purified and functionalized multiwalled carbon nanotubes (MWCNTs). Both immobilization routes, physical adsorption and covalent bonding, were investigated. MWCNT functionalization by a non-aggressive oxidation by potassium permanganate led to an interesting balance between the hydrophilic and the hydrophobic areas of the MWCNT surface; the former being responsible of the good dispersion of MWCNTs in water and the latter having a favorable affinity with CAL-B. The enzyme loadings reached were significant: around 16 wt. % and 21 wt.% for non-covalent and covalent immobilization, respectively. The enzymatic activity was studied with the reaction of O-acylation of geraniol into geranyl acetate by CAL-B in supercritical CO 2 . Even if a decay in synthesis of geranyl acetate was observed over cycling for both CAL-B@MWCNT catalysts, it was demonstrated that the regioselectivity of CAL-B was unchanged through immobilization on the MWCNT surface for both routes. Interestingly, it was shown that a fully green enzymatic process can be achieved with these prepared CAL-B@MWCNT biocatalyst. Such approach could be transferred to other support/enzyme systems for developing new eco-friendly synthesis processes.