Covalent Immobilization Of Trypsin Research Articles

Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde

Magnetic nanoparticles were coated with polyvinyl alcohol and activated with glutaraldehyde for trypsin immobilization. The prepared magnetic nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, thermal gravimetric analysis, zeta potential meter and vibrating sample magnetometer. Free and immobilized trypsin showed optimum activity at pH 6.0, 30 °C and pH 7.0, 40 °C, respectively. Immobilized trypsin was more stable than the free enzyme at 40 °C. After immobilization, Km of the immobilized trypsin increased, however, Vmax value was almost the same with free trypsin. According to the results, the immobilized trypsin retained 50 % of its initial activity, whereas free trypsin retained 19 % of its initial activity after 12-days at 4 °C. Immobilized trypsin sustained 56 % of its initial activity after eight times of successive reuse. The performance of the immobilized trypsin was evaluated by digestion of cytochrome c. The peptide fragments in digest solution were determined by using MALDI-TOF mass spectrometry. Immobilized trypsin showed effective proteolytic activity in shorter time (15 min) than free trypsin (24 h). Hence, immobilized trypsin on the polyvinyl alcohol coated magnetic nanoparticles could be promising biocatalyst for large-scale proteomics studies and practical applications.

Extracción purificación y caracterización de inhibidores de tripsina provenientes de semillas andinas

El presente trabajo determinó las mejores condiciones para inmovilizar covalentemente tripsina en una matriz de sepharosa, con el fin de aplicarla en la purificación de inhibidores de la misma proteasa. Los mayores valores de retención de actividad funcional y de actividad enzimática inmovilizada se obtuvieron en una matriz de Sepharosa 6B-CL a temperatura ambiente, a un pH de 10,5, con una carga enzimática de 25 mg/mL de matriz, en un tiempo mínimo de inmovilización de 12 h, para alcanzar un preparado inmovilizado estable. Posteriormente, se seleccionaron los inhibidores de tripsina más activos a través de la comparación de extractos provenientes de semillas de amaranto (Amaranthus caudatus L.), arveja (Pisum sativum), lupino o “chocho” (Lupinus mutabilis), fréjol (Phaseolus vulgaris) y sangorache (Amaranthus hybridus L.) que fueron purificados parcialmente mediante ultrafiltración centrífuga, tratamiento calórico y precipitación con TCA. El permeado y retenido de sangorache fueron los extractos inhibidores más activos y se purificaron selectivamente, por separado, mediante cromatografía de afinidad en la matriz de tripsina-glioxil-sepharosa 6B-CL. Su caracterización cinética determinó la presencia de dos inhibidores; el inhibidor I que correspondió a una inhibición competitiva y el inhibidor II, a una de tipo mixta.

Covalent immobilization of trypsin onto modified magnetite nanoparticles and its application for casein digestion

The immobilization method consists of the production magnetite nanoparticles (Fe3O4) by solvothermal treatment of FeCl3 and sodium acetate (NaAc) in the presence of ethylene glycol. Subsequently, the surface of magnetite nanoparticles was modified with a well-known polyphenol tannic acid. Trypsin was covalently immobilized on the tannic acid modified magnetite nanoparticles after exposing the modified nanoparticles to pH 9.4. Then, tryptic digestion of casein by free and immobilized trypsin was carried out for 13h and 1h, respectively. TGA curves, FTIR spectra, and magnetization curves demonstrated the decent amount of trypsin immobilization without compromising the enzyme activity. Digestion efficiency of casein was investigated using liquid chromatography–mass spectrometry (LC–MS/MS) technique. LC–MS chromatograms confirmed the efficient digestion of casein by immobilized trypsin compared to free trypsin owing to prevention of autohydrolysis. Also, the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis confirmed the satisfactory digestion of casein by immobilized trypsin.

Covalent biofunctionalization of chitosan nanofibers with trypsin for high enzyme stability

The electrospun chitosan nanofibers provide excellent material for immobilized proteolytic enzymes, and are biocompatible, nontoxic and hydrophilic matrices with large specific area. This paper deals with an application of electrospun chitosan nanofibers and optimizing conditions for their biofunctionalization by model proteolytic enzyme trypsin. Nanofibers from chitosan were prepared using NanospiderTM technology and covalent immobilization of trypsin followed. Three immobilization techniques preserving biocompatibility and utilizing amine and/or hydroxyl groups of chitosan were optimized and compared to simple adsorption to achieve maximum proteolytic activity per cm2 of the functionalized chitosan nanofibers (Tryp-NF). Significant differences were observed. Trypsin immobilized by the carbodiimide one-step protocol demonstrated the highest activity of the three procedures, ranging from 132 to 210IU/cm2 (i.e., 548–874IU/mg of nanofibers), depending on the initial amount of trypsin used. Long-term storage stability together with high reusability of Tryp-NF confirmed advantages of the immobilized enzyme. Tryp-NF showed no cytotoxicity toward growth of HeLa cells. The in vivo tests for irritation and skin sensitization demonstrated no undesirable skin reactions.

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

ABSTRACTPoly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g−1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (Vmax) and Michaelis constant (Km) of immobilized enzyme were found to be 0.74 μM s−1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43343.

A bioactive film based on cashew gum polysaccharide for wound dressing applications

This work presents the development of a new bioactive material for wound therapeutics which may play a dual role of modulate metallo proteinases activity while prevents infection blocking out pathogenic microorganisms and foreign materials. A CGP/PVA film was activated by covalent immobilization of trypsin. Results from biocompatibility test revealed that PDL fibroblasts grown on the surface of CGP/PVA and the high amount of viable cells proved absence of cytotoxicity. Trypsin immobilized onto CGP/PVA film remained 100% active after 28 days stored dried at room temperature. In addition, CGP/PVA-trypsin film could be used for 9 cycles of storage/use without loss of activity. After immobilization, trypsin retained its collagenolytic activity, indicating this material as a promising material for wound dressing applications.

Covalent immobilization of trypsin on glutaraldehyde-activated silica for protein fragmentation

Trypsin was immobilized by covalent binding to glutaraldehyde-activated silica with and without a spacer arm; 1,6-diaminohexane and polyethyleneglycol as well. The addition of polyethyleneglycol (PEG) to the immobilization media increased the activity of immobilized trypsin in organic solvents, whilst free trypsin activity disappeared under the same conditions. Thermal, pH, storage, and operational stabilities of the free and immobilized enzyme were found to be better than the free enzyme. Furthermore, use of immobilized enzyme for protein fragmentation was achieved by solid-phase, on-line, protein digestion in organic solvents. Reaction times were reduced to a few minutes and the sample handling was minimized.

Ammonia Plasma Treated Polyethylene Films for Adsorption or Covalent Immobilization of Trypsin: Quantitative Correlation between X-ray Photoelectron Spectroscopy Data and Enzyme Activity

The ammonia plasma process was used for generating reactive groups, particularly primary amine functions on the surface of polyethylene (PE) films, to immobilize the enzyme trypsin. The attachment of the enzyme was achieved by directly applying an aqueous solution of trypsin to the plasma-activated surface or by using glutaraldehyde as a chemical linker. In both cases, the utilization of sodium cyanoborohydride efficiently stabilized the immobilization. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and enzymatic activity measurements. Active trypsin was successfully immobilized on the surface with a mean activity of 0.09 ± 0.02 U/cm(2). The study of the stability of the immobilized enzyme during repetitive assays showed that some activity could be maintained during several months. An original quantitative correlation between the immobilized enzyme activity and the XPS signal intensity of the S 2p electrons present in the sulfur-containing amino acid residues was evidenced.

IMPROVEMENT OF PURIFICATION OF TRYPSIN INHIBITOR FROM WILD SOYBEAN (GLYCINE SOJA SIEB. & ZUCC.) USING CHITOSAN RESIN-IMMOBILIZED TRYPSIN

Chitosan resin-trypsin was prepared for one-step affinity purification of trypsin inhibitor (TI) from a wild-type soybean (Glycine soja Sieb. & Zucc.). The influences of several parameters (cross-linking, chemical modifying and activating agent) on the chitosan resin-trypsin activity were investigated. The results showed that the chitosan resin-trypsin composite demonstrated considerable trypsin activity at levels of, 3.6% glutaraldehyde (cross-linking agent), 0.06 mol/L NaBH4 (chemical modifying agent) and 15% epichlorohydrin (activating agent). Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65C) and wide pH range (5.0–9.0), compared with free trypsin (45C, pH 6.0–8.0). With chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk trypsin inhibitor from a wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The affinity chromatography purification method described for TI was 279-fold with a 58.3% recovery rate of TI, much improved over established procedures. PRACTICAL APPLICATIONS The use of chitosan resin as a support for enzymes in affinity chromatography has advantages over other materials: low cost, good stability and versatility. The covalent immobilization of trypsin on chitosan resin permits easy recovery and reuse of the enzyme, and produces a quick and effective tool to isolate the Bowman-Birk trypsin inhibitor form wild soybean. This method could be a competitive choice for large-scale purification of this functional molecule.

Immobilization of trypsin on sub-micron skeletal polymer monolith

A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-α-benzoyl- l-arginine (BA) which is the digestion product of a substrate N-α-benzoyl- l-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30 °C, which is comparable to 24 h digestion in solution at 37 °C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.

Cotton gauze bandage: a support for protease immobilization for use in biomedical applications

The covalent immobilization of trypsin on sterilized cotton gauze bandage was carried out for future use as an anti-inflammatory agent on wound dressing. The optimal immobilization conditions were determined: the influence of pH, concentration, and volume of the trypsin solution used in the immobilization procedure was studied. The catalytic properties and kinetic parameters, as well as the storage stability of the immobilized and free enzyme were compared. Results showed that the optimal pH and temperature for the immobilized trypsin in the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide were 9.5 and 55 oC, which were higher than those of the free form (7.5 and 45 oC). It was found, at 37 oC and pH 7.0 (approximately, the physiological conditions), that the Km (Michaelis constant) was 3.98 µmol/mL and that Vmax (maximal reaction rate) was 0.719 µmol/(min mg) for the immobilized trypsin, compared to a Km of 2.46 mmol/mL and a Vmax of 2.89 µmol/(min mg) for the free trypsin. The amount of immobilized trypsin was 6 mg/g dried support. After 30 days, no trypsin had liberated from the support. The superior performance of the immobilized trypsin demonstrates its potential application in practice.

The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles

Poly(methyl methacrylate-ethyl acrylate-acrylic acid) latex particles with narrow size distribution and with surface carboxyl groups were produced by soap-free emulsion polymerization, and covalent immobilization of trypsin onto these particles was carried out by using the water-soluble carbodiimide (EDC) as an activating agent under various conditions. Different immobilization methods were employed and the factors affecting the efficiency and activity of the immobilized enzyme, such as the amount of trypsin and EDC, pH and temperature of the immobilization reaction were investigated. Results showed that both relatively high immobilization efficiency and high enzyme activity were achieved when pre-adsorption method was employed. The immobilization efficiency decreased as the trypsin amount increased, and increased as pH and temperature increased. When the EDC amount varied, the immobilization efficiency first increased significantly and then decreased slowly. A maximum of enzyme activity can be obtained at the optimum value of 958.0 mg trypsin/g dried particles and 372.5 mg EDC/g dried particles at 25 °C and pH 5.0. The immobilized trypsin exhibited much higher relative activity than its free counterpart.

Chararacterization of a monolithic immobilized trypsin microreactor with on‐line coupling to ESI‐MS

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.

The Properties of Covalently Immobilized Trypsin on Soap‐Free P(MMA‐EA‐AA) Latex Particles

The covalent immobilization of trypsin onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55 degrees C and 8.5, both of which were higher than that of the free form. It was found that K(m) (Michaelis constant) was 45.7 mg . ml(-1) and V(max) (maximal reaction rate) was 793.0 microg . min(-1) for immobilized trypsin, compared to a K(m) of 30.0 mg . ml(-1) and a V(max) of 5 467.5 microg . min(-1) for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times.

The covalent immobilization of trypsin at the galleries of layered γ-zirconium phosphate

Assorted trypsin immobilization trials were thoroughly conducted and compared to identify an efficient enzyme immobilization at the galleries of amine preintercalated γ-zirconium phosphate. That was, (a) direct trypsin binding by physical adsorption, (b) coupling of enzyme to glutaraldehyde (GA) activated carrier, (c) trypsin immobilization by GA crosslinking, (d) incorporating bovine serum albumin (BSA) as a spacer during the immobilization by covalently coupling to GA activated carrier or by covalent trypsin crosslinking with GA. We reported here that covalent immobilization of trypsin by using GA as a crosslinking agent and BSA as a spacer was superior to other immobilization strategies investigated. In this study, the experimental parameters in each step of the immobilization procedure, such as the concentration of the preintercalation agent, the crosslinking agent, the spacer and trypsin, etc., were investigated to improve the efficiency of trypsin immobilization. A reasonable enzymatic activity recovery at 75.6% and improved thermal stability were achieved. The optimum pH value and optimum temperature of immobilized enzyme were determined.

Design of reactive porous polymer supports for high throughput bioreactors: poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide- co-ethylene dimethacrylate) monoliths.

Enzymatic bioreactors with both high flow characteristics and mechanical stability based on macroporous poly(2-vinyl-4, 4-dimethylazlactone-co-acrylamide-co-ethylene dimethacrylate) monoliths have been prepared. Covalent immobilization of trypsin on these support is achieved in a single reaction step using the azlactone functional groups. Optimization of hydrophilic/hydrophobic properties of the monolith affords a support that does not shrink in water and leads to immobilized enzyme that shows high activity in the hydrolysis of both low and high molecular weight substrates such as L-benzoyl arginine ethyl ester and casein. The catalytic activity of the monolithic reactor is maintained even at a flow velocity of 180 cm/min, which substantially exceeds those reported in the literature for packed bed reactors.

Surface modified and functionalized polyaniline and polypyrrole films

Surface modified and functionalized polyaniline (PANi) and polypyrrole(PPY) films from ozone treatment, Ar plasma treatment, near-UV-light induced graft copolymerization with acrylic acid(AAc), and covalent immobilization of the enzyme trypsin were characterized by angle-resolved X-ray photoelectron spectroscopy(XPS). Both ozone and Ar plasma treatment result in the oxidation of carbon atoms and cause a significant decrease in the intrinsic oxidation states ([=N-]/[-NH-] ratio) of the base polymers. Surface graft copolymerization with AAc readily gives rise to self-protonated PANi and PPY surface structures. The concentration of surface grafted AAc polymer increases with increasing monomer concentration and near-UV irradiation time used during grafting, but decreases with Ar plasma pretreatment time. Covalent immobilization of trypsin on the AAc polymer grafted PANi and PPY films is facilitated by the presence of water-soluble carbodiimide (WSC). The activity of the immobilized enzyme increases initially with increasing concentration of surface grafted AAc polymer, but becomes saturated at moderate to high AAc polymer concentration.

XPS Characterization of Surface Functionalized Electroactive Polymers

Surface modification and functionalization of polyaniline (PAN) and polypyrrole (PPY) films were achieved by Ar plasma treatment, near-UV-induced graft copolymerization with acrylic acid (AAc) and covalent immobilization of an enzyme (trypsin). The compositions and structures of the modified and functionalized polymer surfaces were characterized by angle-resolved XPS. Argon plasma treatment results in minor oxidations of the carbon atoms but is accompanied by a significant decrease in the intrinsic oxidation state ([=N−]/[−NH−] ratio) of the polymers. Graft copolymerization with AAc readily gives rise to self-protonated PAN and PPY surface structures. The surface concentration of the grafted AAc polymer increases with increasing monomer concentration and near-UV irradiation time during grafting but decreases with increasing plasma pretreatment time. Covalent immobilization of trypsin on the AAc-polymer-grafted PAN and PPY films is facilitated by the use of water-soluble carbodiimide intermediate (WSC). The effective enzyme activity increases initially with increasing surface concentration of the grafted AAc polymer, but becomes saturated at moderate to high AAc polymer concentration.

Surface Modification and Functionalization of Polytetrafluoroethylene Films

Argon plasma-pretreated polytetrafluoroethylene (PTFE) films were subjected to further surface modification by near-UV light-induced graft copolymerization with acrylic acid (AAc), sodium salt of styrenesulfonic acid (NaSS), and N,N-dimethylacrylamide (DMAA). The surface compositions and microstructures of the modified films were characterized by angle-resolved X-ray photoelectron spectroscopy (XPS). A stratified surface microstructure with a significantly higher substrate-to-graft chain ratio in the top surface layer than in the subsurface layer was always obtained for PTFE surface with a substantial amount of the hydrophilic graft. The stratified surface microstructure was consistent with the observed hysteresis in advancing and receding water contact angles. The graft yield increased with Ar plasma pretreatment time and monomer concentration. Covalent immobilization of trypsin on the AAc polymer-grafted PTFE films was facilitated by water-soluble carbodiimide (WSC). The effective enzyme activities increased initially with increasing surface concentration of the grafted AAc polymer but became saturated at a moderate AAc polymer concentration. The immobilized enzyme could still retain close to 30% of its original activity. Solution-coating of the polymeric acid-modified PTFE films with the emeraldine (EM) base of polyaniline readily resulted in an interfacial charge transfer interaction and a semiconductive PTFE surface.

Rapid Tryptic Mapping Using Enzymically Active Mass Spectrometer Probe Tips

A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient.