The work is focused on the development of microspheres based on the combination of two polysaccharides; chitosan and alginic acid with the aim to allocate, hold, release and protect environmentally sensible molecules. The microspheres were prepared using a solvent-free, low cost and scalable approach and two enzymes; trypsin and protease from Aspergillus Oryzae have been used as a model to evaluate the microspheres peculiarities. The proteins were encapsulated during the microspheres preparation. The relationship between the polysaccharides weight ratio and the morphology, stability and ability of the carrier to allocate the enzymes has been evaluated. The enzymatic activity and the release kinetics were assessed in different conditions to assess the impact of the external environment. Obtained results demonstrate the efficacy of the prepared microspheres to preserve the activity of relevant bioactive compounds which are highly relevant in food, cosmetic and pharmaceutic, but the application is limited due to their high sensibility.
- MeSH
- Aspergillus oryzae enzymology MeSH
- NIH 3T3 Cells MeSH
- Chitosan chemistry toxicity MeSH
- Enzymes, Immobilized chemistry metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Alginic Acid chemistry toxicity MeSH
- Humans MeSH
- Microspheres * MeSH
- Mice MeSH
- Materials Testing MeSH
- Capsules MeSH
- Trypsin chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Bacterial cellulose (BC) produced by Komagataeibacter sucrofermentans was magnetically modified using perchloric acid stabilized magnetic fluid. Magnetic bacterial cellulose (MBC) was used as a carrier for the immobilization of affinity ligands, enzymes and cells. MBC with immobilized reactive copper phthalocyanine dye was an efficient adsorbent for crystal violet removal; the maximum adsorption capacity was 388mg/g. Kinetic and thermodynamic parameters were also determined. Model biocatalysts, namely bovine pancreas trypsin and Saccharomyces cerevisiae cells were immobilized on MBC using several strategies including adsorption with subsequent cross-linking with glutaraldehyde and covalent binding on previously activated MBC using sodium periodate or 1,4-butanediol diglycidyl ether. Immobilized yeast cells retained approximately 90% of their initial activity after 6 repeated cycles of sucrose solution hydrolysis. Trypsin covalently bound after MBC periodate activation was very stable during operational stability testing; it could be repeatedly used for ten cycles of low molecular weight substrate hydrolysis without loss of its initial activity.
- MeSH
- Acetobacteraceae chemistry MeSH
- Polysaccharides, Bacterial chemistry MeSH
- Cellulose chemistry MeSH
- Enzymes, Immobilized chemistry MeSH
- Gentian Violet chemistry MeSH
- Cells, Immobilized cytology MeSH
- Indoles chemistry MeSH
- Magnetics MeSH
- Drug Carriers chemistry MeSH
- Organometallic Compounds chemistry MeSH
- Saccharomyces cerevisiae cytology MeSH
- Cattle MeSH
- Trypsin chemistry MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.
Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.
- MeSH
- Adhesiveness MeSH
- Adsorption MeSH
- Alzheimer Disease diagnosis MeSH
- Amino Acid Motifs MeSH
- Biomarkers chemistry MeSH
- Chymotrypsin chemistry MeSH
- Epitopes chemistry MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Magnetics MeSH
- Antibodies, Monoclonal chemistry MeSH
- tau Proteins chemistry MeSH
- Proteolysis MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Thiazoles chemistry MeSH
- Trypsin chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.
- MeSH
- Adsorption MeSH
- Glycosylation MeSH
- Capillary Electrochromatography instrumentation methods MeSH
- Humans MeSH
- Nanoparticles chemistry MeSH
- Peptides analysis MeSH
- Proteins chemistry MeSH
- Trypsin chemistry MeSH
- Gold chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.
- MeSH
- Biocatalysis MeSH
- Chromatography, Affinity methods MeSH
- Immobilized Proteins chemistry MeSH
- Immunoglobulin G chemistry MeSH
- Polymethacrylic Acids chemical synthesis MeSH
- Humans MeSH
- Magnets MeSH
- Microspheres MeSH
- Microscopy, Electron, Scanning MeSH
- Polyhydroxyethyl Methacrylate chemical synthesis MeSH
- Polymerization MeSH
- Spectrophotometry, Infrared MeSH
- Trypsin chemistry MeSH
- Ferric Compounds chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Nearly monodispersed superparamagnetic maghemite nanoparticles (15-20nm) were prepared by a one-step thermal decomposition of iron(II) acetate in air at 400 degrees C. The presented synthetic route is simple, cost effective and allows to prepare the high-quality superparamagnetic particles in a large scale. The as-prepared particles were exploited for the development of magnetic nanocomposites with the possible applicability in medicine and biochemistry. For the purposes of the MRI diagnostics, the maghemite particles were simply dispersed in the bentonite matrix. The resulting nanocomposite represents very effective and cheap oral negative contrast agent for MRI of the gastrointestinal tract and reveals excellent contrast properties, fully comparable with those obtained for commercial contrast material. The results of the clinical research of this maghemite-bentonite contrast agent for imaging of the small bowel are discussed. For biochemical applications, the primary functionalization of the prepared maghemite nanoparticles with chitosan was performed. In this way, a highly efficient magnetic carrier for protein immobilization was obtained as demonstrated by conjugating thermostable raffinose-modified trypsin (RMT) using glutaraldehyde. The covalent conjugation resulted in a further increase in trypsin thermostability (T(50)=61 degrees C) and elimination of its autolysis. Consequently, the immobilization of RMT allowed fast in-solution digestion of proteins and their identification by MALDI-TOF mass spectrometry.
We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.
- MeSH
- Bioreactors MeSH
- Time Factors MeSH
- Chromatography, Affinity methods instrumentation MeSH
- Enzymes, Immobilized chemistry MeSH
- Financing, Organized MeSH
- Humans MeSH
- Ligands MeSH
- Magnetics MeSH
- Metalloendopeptidases chemistry MeSH
- Nanoparticles chemistry MeSH
- Neurotensin analysis MeSH
- Silicon Dioxide MeSH
- Peptides analysis chemistry MeSH
- Proteomics MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Serine Endopeptidases chemistry MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Trypsin chemistry isolation & purification MeSH
- Check Tag
- Humans MeSH
Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.
- MeSH
- Coated Materials, Biocompatible MeSH
- Electrophoresis, Capillary methods MeSH
- Electrophoresis, Microchip MeSH
- Electrolytes MeSH
- Enzymes, Immobilized chemical synthesis classification MeSH
- Financing, Organized MeSH
- Spectrometry, Mass, Electrospray Ionization methods MeSH
- Microchemistry MeSH
- Online Systems MeSH
- Pepsin A metabolism MeSH
- Proteins analysis chemistry MeSH
- Sequence Analysis, Protein methods MeSH
- Sensitivity and Specificity MeSH
- Feasibility Studies MeSH
- Trypsin metabolism MeSH
- MeSH
- Electrophoresis methods instrumentation utilization MeSH
- Enzymes, Immobilized analysis MeSH
- Spectrometry, Mass, Electrospray Ionization methods instrumentation MeSH
- Peptide Mapping methods instrumentation utilization MeSH
- Proteins analysis MeSH
- Trypsin analysis MeSH
- Protein Binding genetics MeSH
