Adsorption Of Trypsin Research Articles

Exploring Nano‐Protein Corona Dynamics: Tracing the Hard‐to‐Soft Corona Transition with Trypsin and Graphene Oxide in a Silver Nanocomposite Model

AbstractIn this work, the adsorption of Trypsin on synthesized Silver (Ag), Graphene Oxide: Silver (GO: Ag) nanocomposite (2:1), and Graphene Oxide (GO) nanoparticles (NPs) and, their effect on the conformation of Trypsin was studied by various spectroscopic and microscopic techniques to understand the transition of nano Protein Corona (PC) from “hard” to “soft.” The results showed that the adsorption of Trypsin on synthesized NPs followed the Freundlich adsorption isotherm and pseudo‐second‐order kinetics. The conformational changes that occurred in Trypsin upon interaction with synthesized NPs were investigated by using Circular Dichroism (CD), Fluorescence, and Fourier Transform Infrared (FTIR) spectroscopy. The morphological investigation of nano PC using High‐Resolution Transmission Electron Microscopy (HR‐TEM) and Atomic Force Microscopy (AFM) indicated the formation of hard, semi‐soft, and soft nano PC in the presence of Ag, GO: Ag, and GO NPs, respectively. The negative value of the thermodynamic parameters ΔG, ΔH, and ΔS in all three cases suggests the reaction to be exothermic and spontaneous. The van der Waals interaction and hydrogen bonding are the major forces responsible for binding Trypsin on the surface of Ag, GO: Ag, and GO. The catalytic efficiency of trypsin in the absence and presence of NPs was examined by performing the protein assay showing the highest catalytic activity of pure Trypsin compared with trypsin‐NPs constructs. Our findings provide useful information for the applications of GO‐based nanocomposites for various biological applications.

Humic acid-based biomass-derived magnetic nanocomposites modified by a deep eutectic solvent for the efficient adsorption of trypsin

Application of DES-modified humic acid-based biomass-derived magnetic nanocomposites for the MSPE of Tryp.

Adsorption of trypsin onto pH sensitive poly-N-vinylpyrrolidone-co-poly-4-vinylpyridine and its magnetic based hydrogel: Sorption isotherms and thermodynamic parameters

Adsorption of trypsin onto pH sensitive poly-N-vinylpyrrolidone-co-poly-4-vinylpyridine and its magnetic based hydrogel: Sorption isotherms and thermodynamic parameters

Protein corona formed on the TiO2 nanoparticles promotes the hydrolysis of collagen in simulated gastrointestinal fluids

Titanium dioxide nanoparticles (TiO2 NPs) could interact with proteins in foods and gastrointestinal fluids, thus changing the properties and functions of proteins. Many research articles have only studied the interactions between TiO2 NPs and digestive enzymes or the gastrointestinal fate of TiO2 NPs without considering the effect of food matrix. Therefore, the interactions between TiO2 NPs and trypsin and its effects on the digestion of collagen in vitro were investigated. Results showed that the functionalization of amino acids (Asp, and Ser) significantly harmed the adsorption of trypsin by TiO2 NPs. The fluorescence quenching and isothermal titration calorimetry confirmed that the hydrogen bond and van der Waals force played an essential role in the interactions between TiO2 NPs and trypsin. The β-sheet content of trypsin bound to TiO2 NPs decreased from 52.40% to 50.73% while the random coil content increased from 20.20% to 22.63%. Binding sites were identified by molecular docking. Additionally, the digestibility of collagen was improved in vitro in the presence of TiO2 NPs analyzed by SDS-PAGE, hydroxyproline quantification, and the antioxidation of digested products. Hydroxyproline concentration increased by nearly 25% and ABTS radical scavenging rate increased by 50%. This study provides a new insight into the impact of nanoparticles on gastrointestinal digestion.

Excellent performance separation of trypsin by novel ternary magnetic composite adsorbent based on betaine-urea- glycerol natural deep eutectic solvent modified MnFe2O4-MWCNTs

The effective trypsin purification methods should be established since trypsin plays a crucial role in biosome. In this work, a novel ternary magnetic composite adsorbent (MnFe2O4-MWCNTs@B–U-G) with the features of strong specific selectivity, good adsorption effect, simple and efficient separation process, no secondary pollution brought in was prepared by integrating the superior physicochemical properties of ternary based natural deep eutectic solvent, multi-walled carbon nanotubes and MnFe2O4. The property, composition and microtopography structure of MnFe2O4-MWCNTs@B–U-G were characterized in detail. Combined with magnetic solid-phase extraction, MnFe2O4-MWCNTs@B–U-G was utilized to adsorb trypsin. Response surface methodology experiment was prepared under Box-Behnken design to optimize the adsorption conditions and the results showed that the practical maximum adsorption capacity for trypsin was 1020.1 mg g−1. Besides, the adsorption isotherms, adsorption kinetics, regeneration studies and method validation studies were investigated systematically to evaluate the established adsorption separation system. Mechanism exploration proved that electrostatic interaction, hydrogen bonding interaction and chelation interaction were the dominant forces for the high-performance adsorption of trypsin. The activity of trypsin after elution had been analyzed by UV–vis spectrophotometer and CD spectrometer with three methods, which illustrated that the enzyme activity, conformation and secondary structure of trypsin did not change significantly during the adsorption-desorption process. In addition, the proposed method was successful and practical applicability to isolation trypsin from crude bovine pancreas. As a result, due to the superiority of the MnFe2O4-MWCNTs@B–U-G, the proposed method not only exhibites high-performance adsorption of trypsin, but also provides a green and sustainable potential value in the adsorption of biomacromolecule.

Immobilization of Trypsin from the Subphase to the Langmuir Monolayer of Fluorocarbon-Modified Single-Walled Carbon Nanotube and Its Activity Maintenance.

Single-walled carbon nanotubes (SWCNTs) synthesized by the improved arc discharge method were modified with a fluorocarbon chain, and the maintenance of trypsin activity upon adsorption from the subphase to the interfacial monolayer of SWCNTs was evaluated. The adsorption of trypsin on the fluorocarbon-modified SWCNT monolayer was confirmed by morphological and spectroscopic evaluations. Fiber morphology studies revealed that the fluorocarbon-modified SWCNT monolayer was covered by trypsin, and a trypsin-derived amide band was detected in the infrared spectra of the multilayers. After adsorption onto the fluorocarbon-modified SWCNT film, the ability of trypsin to cleave the fluorescent casein chain was maintained even at 160 °C. Furthermore, circular dichroism (CD) spectra showed that the second-order structure of the activity of trypsin adsorbed on the fluorocarbon-modified SWCNT was maintained up to nearly 200 °C. At 200 °C, the enhancement of emission intensity by casein chain cleavage was negligible, and the CD signal resulting from the negative Cotton effect was completely altered at 250 °C.

The interaction of graphene oxide-silver nanoparticles with trypsin: Insights from adsorption behaviors, conformational structure and enzymatic activity investigations

In this work, we synthesized graphene oxide-silver nanoparticles (GO-AgNPs) hybrids by one-pot method. Since there are relatively few reports on whether GO-AgNPs bind and change the structure and function of trypsin, A variety of methods were employed to systematically characterize the molecular interaction between GO-AgNPs and trypsin. Results exhibited that GO-AgNPs bound with trypsin to form a ground state complex. GO-AgNPs had higher adsorption capacity for trypsin compared with single GO. Langmuir-Blodgett assembly method was used to confirm that AgNPs did not interfere with the adsorption of trypsin by GO. The secondary structure and the microenvironment of amino acid residues of trypsin were altered after interacting with GO-AgNPs. In addition, GO-AgNPs can enhance the activity of trypsin and promote the hydrolysis of bovine serum protein (BSA) by trypsin. These findings provide important support for the application of GO-based nanocomposites in the efficient immobilization of enzymes.

Different electrically charged proteins result in diverse transport behaviors of plastic particles with different surface charge in quartz sand

The influence of proteins on the transport and deposition behaviors of microplastics (MPs) in quartz sand was examined at both low (5 mM) and high ionic strength (25 mM) in NaCl solutions at pH 6. Carboxylate- and amine-modified polystyrene latex microspheres with size of 200 nm were employed as negatively (CMPs) and positively surface charged MPs (AMPs), respectively, while bovine serum albumin (BSA) and bovine trypsin were utilized as representative negatively and positively charged proteins, respectively. The results showed that for two examined protein concentrations (both 1 and 10 mg/L TOC) under both ionic strength conditions, the presence of BSA increased the transport of both CMPs and AMPs, while the presence of trypsin decreased the transport of CMPs yet increased the transport of AMPs in porous media. The mechanisms driving to the changed transport of MPs induced by two types of proteins were found to be different. Particularly, steric interaction induced by BSA corona adsorbed onto CMPs surface as well as the repel effects resulted from BSA suspending in solutions were found to contribute to the enhanced CMPs transport with BSA copresent in suspensions. The increased sizes and the decreased electrostatic repulsion of CMPs due to the adsorption of trypsin onto CMPs, together with the addition of extra deposition sites due to the adsorption of trypsin onto quartz sand drove to the decreased CMPs transport with trypsin copresent in suspensions. The increased electrostatic repulsion due to the adsorption of BSA onto AMPs surfaces caused the enhanced AMPs transport with BSA in solutions. While, the decreased electrostatic attraction of AMPs due to the adsorption of trypsin onto AMPs, as well as the competition of deposition sites due to the adsorption of trypsin onto quartz sand contributed to the increased AMPs transport with trypsin copresent in suspensions. The results showed that the presence of different types of proteins would induce different transport behaviors of microplastics with different surface charge in porous media. Since proteins are widely present in aquatic systems, to more accurately predict the fate and transport of MPs in natural environments, the effects and mechanisms of proteins on the transport of MPs should be considered.

Time-dependent study of graphene oxide-trypsin adsorption interface and visualization of nano-protein corona

Understanding of interactions of nanomaterials with biomolecules (especially proteins) is of great importance to the area of nanobiotechnology. Graphene and its derivative such as graphene oxide (GO), are two-dimensional (2-D) nanomaterials with remarkable physical and chemical properties and have been broadly explored in biotechnology and biomedical application. Here, we have reported the nature of adsorption of trypsin on the GO surface, considering its biomedical implications. A simple incubation of trypsin on GO surface exhibits varying resistance to autolysis. The structural morphology of trypsin on the GO surface was studied by using atomic force microscopy (AFM), circular dichroism (CD), fluorescence, and total internal reflection fluorescence (TIRF) microscopies. Results suggest that the trypsin follows the Freundlich Isotherm. By the Langmuir model, the maximum adsorption capacity was found to be 100 mg/g. From protein assay results we have concluded that the native trypsin exhibits the highest catalytic efficiency (33.97*104 L mol−1 min−1) in comparison to other Trp-GO constructs. We have further visualized morphological change on GO-trypsin interface throughout the adsorption process by taking samples at definite time intervals, which suggests that the interaction of trypsin with GO is an example of the soft corona. Our findings may be implicated in enzyme engineering as well as enzyme-based bio-sensing applications.

Interactions of Surface-Functionalized Starch Nanoparticles with Pepsin and Trypsin in Simulated Gastrointestinal Fluids.

Nanoparticles (NPs) can form a protein corona (PC) with proteins in biological fluids. We examined whether starch nanoparticles (SNPs) form a PC and interact with digestive enzymes in simulated gastric and intestinal fluids. We investigated the adsorption of pepsin and trypsin on unmodified, carboxyl-, and amino-modified SNPs (SNPs, COOH-SNPs, and NH2-SNPs, respectively). Quartz crystal microbalance data showed that a tight and irreversible pepsin corona formed on the NH2-SNPs, pepsin had little or no binding to the SNPs and COOH-SNPs, and trypsin had weak binding to all three kinds of NPs. Dynamic light scattering data showed that pepsin significantly increased the size of the NH2-SNPs from 120 ± 2.6 to 203 ± 12.2 nm and decreased their surface potential from 23.2 ± 1.0 to 12.7 ± 0.2 mV. NH2-SNPs could induce the fluorescence quenching of pepsin and change its secondary structures without affecting its activity.

Gold nanorods–trypsin biocorona: a novel nano composite for in vitro cytotoxic activity towards MCF-7 and A-549 cancer cells

In the present work, we have synthesized cetyltrimethyl ammonium bromide (CTAB) capped gold nanorods (Au NRs) to evaluate apparent binding affinities for the adsorption of trypsin (TRP).

Multiscale Molecular Dynamics Simulation of Multiple Protein Adsorption on Gold Nanoparticles.

A multiscale molecular dynamics simulation study has been carried out in order to provide in-depth information on the adsorption of hemoglobin, myoglobin, and trypsin over citrate-capped AuNPs of 15 nm diameter. In particular, determinants for single proteins adsorption and simultaneous adsorption of the three types of proteins considered have been studied by Coarse-Grained and Meso-Scale molecular simulations, respectively. The results, discussed in the light of the controversial experimental data reported in the current experimental literature, have provided a detailed description of the (i) recognition process, (ii) number of proteins involved in the early stages of corona formation, (iii) protein competition for AuNP adsorption, (iv) interaction modalities between AuNP and protein binding sites, and (v) protein structural preservation and alteration.

FTIR-ATR study for adsorption of trypsin in aqueous environment on bare and TiO2 coated ZnSe surfaces

Undesired adsorption of proteins brings big troubles to marine structures. The settled proteins change the physical and chemical properties of the surfaces, which allow marine fouling organisms to settle down on the structures. Therefore, to understand the adsorption mechanism of proteins is very helpful to find an environment-friendly solution against biofouling. Many approaches have been developed to study protein adsorption, but most of them are insufficient to give the chemical interaction information between proteins and surfaces. Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR) is an efficient, fast and non-destructive method for in situ surface measurement, which greatly minimizes the interference of water to infrared spectra, because of the very small depth of penetration of the evanescent wave. In this paper, an in situ FTIR-ATR technology was used to investigate the adsorption process of trypsin on a bare ZnSe surface and on a TiO2 coated ZnSe surface, and the effect of calcium cation strength and ultraviolet light irradiation on the secondary structure of trypsin were also evaluated. FTIR spectra of trypsin showed that Amide I band red shift and Amide II band blue shift in aqueous environment on both surfaces compared with the dry trypsin powder, and the addition of calcium cations further changed the Amide bands position, which indicated that the change of the secondary structure could be interfered by the environment. The hydrogen bond formation between water and trypsin, the interaction between surface and trypsin, the interaction between hydrated calcium cations and trypsin, are major factors to change the secondary structure of trypsin, and UV light irradiation also showed its influence for the secondary structure.

Chemically modified alginate bead matrix for efficient adsorptive recovery of trypsin from fresh bovine pancreas.

The adsorption of commercial trypsin (Try) onto epichlorohydrin cross-linked alginate-guar gum matrix has been studied at equilibrium in batch and in fixed bed column. Experiments were conducted to study the effect of ionic strength, temperature and to obtain a thermodynamic characterization of the adsorption process. The resulting adsorption isotherm fitted the Hill equation. Experimental breakthrough curve profiles were compared with the theoretical breakthrough profiles obtained from the mathematical model, bed depth service time. At pH 5.0, 1.0 g hydrated matrix adsorbed 480.0 milligram of Try per gram of dried bed. The desorption process showed 80% of Try recovery in 50 mM phosphate buffer, pH 7.00-500 mM NaCl-20% propylene glycol. The obtained results were applied to an adsorption/washing/desorption process with fresh pancreas homogenate yielded 20% of recovery and 5.7 purification factor of Try. The matrix remained functional until the fifth cycle of repeated batch enzyme adsorption. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

Different electrically charged proteins result in diverse bacterial transport behaviors in porous media

The influence of proteins on bacterial transport and deposition behaviors in quartz sand was examined in both NaCl (10 and 25 mM) and CaCl2 solutions (1.2 and 5 mM). Bovine Serum Albumin (BSA) and bovine trypsin were used to represent negatively and positively charged proteins in natural aquatic systems, respectively. The presence of negatively charged BSA in suspensions increased the transport and decreased bacterial deposition in quartz sand, regardless of the ionic strength and ion types. Whereas, positively charged trypsin inhibited the transport and enhanced bacterial deposition under all experimental conditions. The potential mechanisms controlling the changes of bacterial transport behaviors varied for different charged proteins. The steric repulsion resulting from BSA adsorption onto both bacteria and quartz sand was found to play a dominant role in the transport and deposition of bacteria in porous media with BSA copresent in suspension. BSA adsorption onto bacterial surfaces and competition for deposition sites onto sand surfaces (adsorption of quartz sand surfaces) contributed to the increased cell transport with BSA in suspension. In contrast, the attractive patch-charged interaction induced by the adsorption of trypsin onto both bacteria and quartz sand had great contribution to the decreased bacterial transport in porous media with trypsin copresent in suspension. The increase in bacteria size, and the adsorption of trypsin onto cell surfaces (resulting in less negative cell surface charge) and quartz sand surfaces (providing extra deposition sites) were found to be the main contributors to the decreased transport and increased deposition of bacteria in quartz sand with trypsin in suspension.

Binding behaviors and kinetics studies on the interaction of silver nanoparticles with trypsin

The interaction of nanoparticles (NPs) with proteins is a topic of high relevance for the medical application of nanomaterials. In this study, a comprehensive investigation was performed to clarify the binding mechanism, adsorption isotherms and kinetics of the interaction between silver nanoparticles (AgNPs) and trypsin. The experimental results indicate that the binding of AgNPs to trypsin seems to be a static quenching mechanism. Thermodynamic analysis reveals that AgNPs binding to trypsin is synergistically driven by enthalpy and entropy, and the major driving forces are hydrophobic and electrostatic interactions. The adsorption of trypsin on AgNPs was analyzed by Langmuir and Freundlich models, suggesting that the equilibrium adsorption data fit well with Freundlich model. The kinetics of adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetic equations. The results indicate that a pseudo-second-order kinetic equation describes better. The conformational change at the secondary structural level of trypsin induced by AgNPs was investigated with the circular dichroism (CD) measurements and no obvious changes in trypsin secondary structural elements are observed. These fundamental works will provide some new insights into the safe and effective application of AgNPs in biological and medical areas.

Adsorption of Trypsin Onto Chitosan/PSf Affinity Membranes: Effects of Physio-chemical Environment

This study aimed to investigate the significance of the physico-chemical environment during trypsin adsorption onto a highly bio-specific affinity membrane. The affinity matrix polysulfone (PSf) membranes were prepared via a simple dry/wet phase inversion technique. Surface modification of PSf membranes was employed using chitosan in order to improve membrane hydrophilicity. Glutaraldehyde and ovomucoid were used as the membrane activator and affinity ligand, respectively. Inspection of membrane morphology was done using scanning electron microscopy. The functional groups on the membrane surface were determined using Fourier-transform infrared spectroscopy equipped with attenuated total reflection (ATR-FTIR). In order to determine the optimum conditions for the maximum adsorption capacity of trypsin, adsorption studies were performed at different pH levels (5, 7, 8, 10, 12), ionic strengths (0.01, 0.05, 0.1, 0.3 and 0.5 M) and initial trypsin concentrations (0.1, 0.3, 0.5, 0.7 and 0.9 mg/ml). The optimum adsorption was obtained with a 0.9 mg/ml initial trypsin solution at pH 7 and an ionic strength of 0.1 M.

Selective adsorption of trypsin using molecularly imprinted polymers prepared with PEG-based hydrogels containing anionic functional monomers

AbstractMolecularly imprinting (MI) hydrogels for selective adsorption of trypsin are reported. The trypsin imprinted hydrogels were prepared using a polyethylene glycol (PEG)-based dimethacrylate as a crosslinker and anionic functional monomers. The hydrogel prepared without any functional monomers showed significantly low ability to adsorb a variety of proteins. We optimized the concentration and the length of PEG units of the crosslinkers to achieve the complete removal of the template molecule and suitable selective adsorption. Additionally, the functional monomers chosen were anionic since the template, trypsin, is a basic protein. The adsorption tests for proteins, done on the prepared MI gels, indicated that the MI gel prepared with sodium allyl sulfonate (AS) as a functional monomer showed much higher selective adsorption for trypsin, even though a mixture of trypsin and cytochrome c was used as the protein solution. The selective adsorption was more effective in a NaCl solution in which the non-specific adsorption by a sulfonate is suppressed, similarly to our findings in a previous study. The MI gel prepared with acrylic acid also showed the selectivity, although the adsorption strength was lower than that of the MI gel containing AS. We believe that the present study constitutes the first approach for the selective adsorption of trypsin using PEG-based hydrogels.

Nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films.

In this study, we investigated how the adsorption properties governed by the nanometer-scale surface morphology of cluster-assembled titanium oxide films influence the catalytic activity of immobilized serine-protease trypsin. We developed an activity assay for the parallel detection of physisorbed enzyme activity and mass density of the adsorbed proteins in microarray format. The method combines a microarray-based technique and advanced quantitative confocal microscopy approaches based on fluorescent labeling of enzymes and covalent labeling of active sites of surface-bound enzymes. The observed diminishing trypsin binding affinity with increasing roughness, as opposed to the steep rise in its saturation uptake, was interpreted as heterogeneous nucleation-driven adsorption of trypsin at the rough nanoporous titania surface. The increase in relative activity of adsorbed trypsin is proportional to the fractional saturation of titania surfaces, expressed as percentage of saturation uptake. In turn, the specific activity, that is, the ratio of active proteins to the absolute number of adsorbed proteins, drops with growing saturation uptake and surface roughness, witnessing a reduction in the accessibility of enzyme active sites. Both geometrical constraints of titania nanopores and the clusterwise adsorption of trypsin were identified as the key factors underpinning the steric hindrance of the immobilized enzyme. These findings are relevant for the optimization of rough nanoporous surfaces as carriers of immobilized enzymes. The proposed activity assay is particularly advantageous in the screening of candidate materials for enzyme immobilization.

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9±2.6mg/g beads. Adsorbed trypsin was eluted by using (0.1M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.