Immobilization Of Papain Research Articles

Laser-induced multi-doped graphene extended-gate field-effect transistor sensor for enhanced detection of cystatin C

In this study, we amplified the capabilities of laser-induced graphene (LIG) by developing a multi-doped LIG extended-gated field-effect transistor (EG-FET) sensor. This sensor integrates a multi-doped LIG EG electrode array as a disposable sensing component with a standard MOSFET for reusable transduction. The multi-doped LIG was synthesized using a dual-approach: initially, by using a MnCl2-doped polyimide (MnCl2-PI) film through precursor compounding, and subsequently, by employing a CO2 laser to respectively in situ generate MnO2 nanoparticles and gold nanoparticles (Au NPs) via direct laser conversion. By incorporating the resultant multi-doped LIG (Au NPs/MnO2/LIG) as the EG electrode, we boosted its electrical efficiency and provided ideal sites for the papain immobilization. This facilitated the selective binding of protein complexes with cystatin C (Cys C), allowing for precise measurement. Notably, the sensor exhibited a robust linear correlation across a concentration range from 50 ag/μL to 0.25 ng/μL and achieved a detection limit of 50 ag/μL. These advancements not only address traditional limitations of LIG applications but also highlight the potential of LIG-based EG-FET portable devices for accurate and early screening of chronic kidney disease (CKD).

Enhanced stability of papain at extreme pHs and temperatures by its immobilization on cellulose nanocrystals coated with polydopamine

In this investigation, a nano-support containing cellulose nanocrystals – polydopamine (CP) was functionalized with glutaraldehyde (G) and utilized to immobilize papain. The properties of the supports and stabilized enzyme were characterized by TEM, SEM, DLS, Zeta-potential, FTIR, and EDS analyses. The optimal temperature of both papain forms was 60 °C, while the optimal pH value of the stabilized papain shifted from 7 to 8 after immobilization. The immobilized enzyme preparations showed higher stability at 80 and 90 °C and under acidic and alkaline conditions compared to the free enzyme. CPG-papain stability was higher than that of CP-papain. The Km values for free and immobilized papain on CP and CPG were 3.92, 3, and 2.74 mM respectively. Kcat values were 9.05, 15.18, and 11.59 min−1 and catalytic efficiency values were 2.3, 5.05, and 4.23 mM−1.min−1. Additionally, the potential advantages of using a silver nanohydrid support were assessed. Although silver nanoparticles conveyed some catalytic activity to the final biocatalysts, they also caused a decrease in stability and activity of the immobilized papain. Therefore, the non-hybrid supports CP and CPG were more suitable candidates for the immobilization of papain than the hybrid supports.

Immobilization of papain onto nitrogen graphene quantum dots and its use as an efficient catalyst for facile MW-assisted synthesis of polyhydroquinolines

Immobilization of papain onto nitrogen graphene quantum dots and its use as an efficient catalyst for facile MW-assisted synthesis of polyhydroquinolines

Immobilization of Papain in Chitosan Membranes as a Potential Alternative for Skin Wounds.

Papain (an enzyme from the latex of Carica papaya) is an interesting natural bioactive macromolecule used as therapeutic alternative for wound healing due to debridement action in devitalized or necrotic tissues. However, its use in high doses can induce potential skin irritation and side effects. In this study, experiments explored the ability of chitosan membrane to immobilize papain, consequently improving enzymatic activity and controlling enzyme release. Papain-loading capacity was tested via experiments of force microscopy (AFM), scanning electron microscopy (SEM-FEG), and X-ray diffraction analyses. Fourier transform infrared spectroscopy and thermal analyses assessed the enzyme interactions with the copolymer. The investigation of the feasibility of membranes included pH on the surface, elasticity, and breaking strength measurements. The surface wettability and swelling capacity of different formulations revealed the best formulation for in vitro papain release experiments. The membranes had a transparent, rough, crystalline characteristic, which was homogeneous with the membrane within the neutrality. The immobilization of papain in the chitosan membrane resulted in a decrease in the vibration band characteristic of pure papain, suggesting a displacement in the vibration bands in the FTIR spectrum. The presence of papain decreased hydrophobicity on the surface of the membrane and disturbed the membrane's ability to swell. Chitosan membranes containing papain 2.5% (0.04 g) and 5.0% (0.08 g) preserved feasible properties and improved the enzymatic activity compared (0.87 ± 0.12 AU/mg and 1.59 ± 0.10 AU/mg) with a free papain sample (0.0042 ± 0.001 AU/mg). Concentrations of over 10% (0.16 g) led to phase separation into membranes. Chitosan membranes exhibited a slow papain release behavior adjusted via the Higushi model. The experimental achievements suggest a novel and promising method for the enhancement of papain. The results indicate the potential for prolonged bioactivity for use on wounds.

Chitosan flakes for papain immobilization: improving the stability and reusability of the enzyme

Chitosan flakes for papain immobilization: improving the stability and reusability of the enzyme

Chitosan flakes for papain immobilization: improving the stability and reusability of the enzyme [pdf

Chitosan flakes for papain immobilization: improving the stability and reusability of the enzyme [pdf

Hollow spherical vaterite calcium carbonate prepared by spray-bubble template method for immobilization of papain

Calcium carbonate (CaCO3) possesses unique advantages as a carrier , however, some critical issues need to be addressed, such as low productivity, irregular structures, and weak adsorption ability. Herein, hollow spherical vaterite CaCO3 (HSVC) was synthesized in an improved spray apparatus with high mass transfer using carbon dioxide (CO2) as a template and was in turn applied for papain immobilization. Results revealed that HSVC can be prepared cleanly and efficiently with a regular spherical morphology having a surface area of 17.03 m2·g−1 and immobilization amount of 1068 mg·g−1 for papain. Interestingly, the pH stability, temperature stability and reusability of papain were dramatically improved after immobilization by HSVC. Molecular dynamics simulation (MDS) disclosed that the formation of CO32– and Ca2+ ion pairs was the key factor for the preparation of HSVC and the binding energy (BE) between amino acid molecules and vaterite (110) facilitated the ultra-high adsorption of papain. The decrease in BE of system after immobilization facilitated high catalytic capacity of the immobilized papain. The prepared HSVC exhibits promising prospects in enzymes immobilization based applications.

The Relationship Between the Cross-Linker on Chitosan-Coated Magnetic Nanoparticles and the Properties of Immobilized Papain.

The immobilized enzymes' properties can be affected by cross-linkers on the surface of supports. To study how cross-linkers alter enzymes function, chitosan-coated magnetic nanoparticles (CMNPs) with immobilized papain were prepared using glutaraldehyde and or genipin, and then, the properties of the nanoparticles and the immobilized enzymes were assessed. The Scanning Electron Microscope (SEM), Fourier Transform Infrared (FTIR), and X-Ray Diffraction (XRD) results showed that the CMNPs were prepared and papain molecules were immobilized on CMNPs by glutaraldehyde (CMNP-Glu-Papain) or by genipin (CMNP-Gen-Papain). Also, the results associated with enzymes activity indicated that the immobilization by glutaraldehyde and genipin increased the pH optimum of papain from 7 to 7.5 and 9, respectively. The kinetic results indicated that the immobilization by genipin slightly affects the enzyme affinity to the substrate. The stability results showed that CMNP-Gen-Papain has more thermal stability than CMNP-Glu-Papain and papain immobilization on CMNPs by genipin leads to stabilization of the enzyme in the presence of polar solvents, probably due to the more hydroxyl groups on CMNPs activated by genipin. In conclusion, this study suggests that there is a relationship between the types of cross-linker on the surface of supports, and the mechanism of action, kinetic parameters, and the stability of immobilized papain.

Papain immobilization on interconnected-porous chitosan macroparticles: Application in controllable hydrolysis of egg white for foamability improvement

Herein, we proposed an interconnected-porous chitosan macroparticles (IPCMs) immobilized papain to exactly control the enzymatic hydrolysis degree of egg white (EW) via a non-heating technology in order to improve its foamability. The SEM images showed that the sodium carbonate (Na2CO3) as gas porogen with varying concentration was able to improve the porosity structure of surface layer of interconnected-porous chitosan macroparticles (IPCMs). The activity determination results indicated that the IPCMs prepared with 0.4 M Na2CO3 hold the highest activity immobilized of papain. In comparison with free enzyme, immobilized papain had a higher range of pH and temperature adaptability, as well as the thermal stability. With a limited hydrolysis, the immobilized papain obtained through condition optimization could enhance the foaming capability (FC) and foaming stability (FS) of EW to 646.67% and 71.62%, respectively. The Fourier transform infrared spectroscopy (FTIR), particle size, zeta potential and surface hydrophobicity were used to analyze the molecular structure change of EW after hydrolysis. Importantly, the immobilized papain could control the enzymatic reaction processing via simple separation, ensuring the optimal hydrolysis degree of EW for foamability enhancement, it also prevented the decrease in foamability during storage due to continuous enzymatic hydrolysis.

Novel Immobilized Biocatalysts Based on Cysteine Proteases Bound to 2-(4-Acetamido-2-sulfanilamide) Chitosan and Research on Their Structural Features.

Briefly, 2-(4-Acetamido-2-sulfanilamide) chitosan, which is a chitosan water-soluble derivative, with molecular weights of 200, 350, and 600 kDa, was successfully synthesized. The immobilization of ficin, papain, and bromelain was carried out by complexation with these polymers. The interaction mechanism of 2-(4-acetamido-2-sulfanilamide) chitosan with bromelain, ficin, and papain was studied using FTIR spectroscopy. It was found that the hydroxy, thionyl, and amino groups of 2-(4-acetamido-2-sulfanilamide) chitosan were involved in the complexation process. Molecular docking research showed that all amino acid residues of the active site of papain formed hydrogen bonds with the immobilization matrix, while only two catalytically valuable amino acid residues took part in the H-bond formation for bromelain and ficin. The spectral and in silico data were in good agreement with the catalytic activity evaluation data. Immobilized papain was more active compared to the other immobilized proteases. Moreover, the total and specific proteolytic activity of papain immobilized on the carrier with a molecular weight of 350 kDa were higher compared to the native one due to the hyperactivation. The optimal ratio of protein content (mg × g −1 of carrier), total activity (U × mL−1 of solution), and specific activity (U × mg−1 of protein) was determined for the enzymes immobilized on 2-(4-acetamido-2-sulfanilamide) chitosan with a molecular weight of 350 kDa.

Chitosan Graft Copolymers with N-Vinylimidazole as Promising Matrices for Immobilization of Bromelain, Ficin, and Papain.

This work aims to synthesize graft copolymers of chitosan and N-vinylimidazole (VI) with different compositions to be used as matrices for the immobilization of cysteine proteases—bromelain, ficin, and papain. The copolymers are synthesized by free radical solution copolymerization with a potassium persulfate-sodium metabisulfite blend initiator. The copolymers have a relatively high frequency of grafting and yields. All the synthesized graft copolymers are water-soluble, and their solutions are characterized by DLS and laser Doppler microelectrophoresis. The copolymers are self-assembled in aqueous solutions, and they have a cationic nature and pH-sensitivity correlating to the VI content. The FTIR data demonstrate that synthesized graft copolymers conjugate cysteine proteases. The synthesized copolymer adsorbs more enzyme macromolecules compared to non-modified chitosan with the same molecular weight. The proteolytic activity of the immobilized enzymes is increased up to 100% compared to native ones. The immobilized ficin retains up to 97% of the initial activity after a one-day incubation, the immobilized bromelain retains 69% of activity after a 3-day incubation, and the immobilized papain retains 57% of the initial activity after a 7-day incubation. Therefore, the synthesized copolymers can be used as matrices for the immobilization of bromelain, ficin, and papain.

Preparation of Magnetic Chitosan Beads as Carriers for Papain Immobilization

Papain, a well‐known cysteine protease, is predominantly found and extracted from papaya fruit. Its broad hydrolyzing activity and stability at different temperature and pH, make it a widely used enzyme in food, and pharmaceutical industries. For example, it is used in various food processing procedures and in the production of bioactive peptides. However, under extreme industrial conditions, the activity of this and other enzymes, are significantly decreased. This loss of enzyme activity could be overcome by immobilizing the enzymes on a solid support. The immobilization of enzymes also allows them to be reused. Chitosan is a non‐toxic, biodegradable polymer derived from the deacetylation of chitin, the second most abundant polysaccharide in nature. Chitosan offers multiple functional groups for binding enzymes and other molecules and has myriad enzyme immobilization applications on a wide variety of materials, including nanoparticles, nanocomposites, and nanofibers. In this study, we have prepared mechanically stable magnetic chitosan beads as carriers for immobilized papain. Chitosan beads were prepared using a phase inversion technique. Chitosan (2.0 gm) was dissolved in 50mL diluted acetic acid and mixed thoroughly with 0.3M FeCl3 solution. The homogenous mixture was placed in a syringe and extruded dropwise into a beaker with 0.5M sodium hydroxide with contestant stirring. The resulting beads were stirred for one hour and washed with water. The washed beads were incubated with 0.5% glutaraldehyde solution for 10 hours with constant shaking. This step was carried out to stabilize the beads by crosslinking the chitosan beads while also creating additional free aldehyde groups for papain binding. Following washing, the beads were immersed in 20mL of 10% ammonium hydroxide and 0.2M FeCl2 solution and stirred for 2 hours to create magnetic chitosan beads. The beads were separated using an external magnet and washed thoroughly. The beads were stored in 50mM phosphate buffer (pH 7.5). For immobilization of papain, the beads were incubated at room temperature in 0.1% papain solution for 20 hours with constant shaking. The immobilized papain was washed and stored in buffer. The enzyme activity of free and immobilized papain was determined by measuring the acid soluble peptides generated by hydrolyzed casein. The thermal stability studies shows that the immobilized papain retained 93% activity after 60 minutes incubation at 80oC while the soluble enzyme retained only 40% of its initial activity. There was no loss of immobilized papain activity after six weeks of storage at 4oC. In addition, a significant amount of immobilized papain activity is retained after multiple usage. These results show that attachment of papain on the magnetic chitosan beads could broaden its application in industrial use. Moreover, immobilization on magnetic chitosan beads can impede losses in activity of other industrially important enzymes.

Immobilization of papain on nitrogen-doped graphene quantum dots improves the enzymatic properties and makes it a biosensor for cystatin C

In recent years enzymes had many applications, such as catalysts in organic synthesis and producing biosensors. In this study, nitrogen-doped graphene quantum dots (NGQDs) were functionalized with glutaraldehyde (NGQD-Glu) and used as a support for the immobilization of papain (NGQD-Glu-Papain). Immobilization of papain on NGQDs has considerably improved papain stability. So, with incubation of the enzymes at 70 °C, immobilized papain maintained 80% of its activity for 150 min while the free enzyme's residual activity was 55% after 50 min. The stability results in the presence of organic solvents showed that the immobilization increased papain stability, especially in polar solvents. The activity results showed that the Km value decreases from 2.02 g L−1 to 1.20 g L−1 after immobilization, and the Vmax value decreases from 932.1 μmol mg−1 min−1 to 62.5 μmol mg−1 min−1. The fluorimetric studies showed that the NGQD-Glu-Papain is specific sensitive to cystatin C at concentrations ranging from 0.05 ng L−1 to 50 ng L−1 with a 0.05 ng L−1 of limit for detection. In conclusion, NGQDs can be suitable for enzyme stabilization and biosensor production.

Acyl-Modified Water-Soluble Chitosan Derivatives as Carriers for Adsorption Immobilization of Papain

Acyl-Modified Water-Soluble Chitosan Derivatives as Carriers for Adsorption Immobilization of Papain

Site-specific immobilization of papain on DDI-modified polystyrene beads for the oligo(γ-ethyl-L-glutamate) synthesis

In this paper, a reusable heterogeneous catalyst of papain was prepared and used to catalyze the polymerization for oligo-glutamate. In silico, the 9,9-bis(2,3-epoxypropyl) fluorenyl group (DDI) carried on polystyrene beads (PS-PEG-DDI) showed site-specific binding for sites which are far away the active site of papain. The immobilization of papain on PS-PEG-DDI reached the optimal value of 126 ± 2.5 mg/g beads and 1726.2 ± 34 U/g beads at the papain/beads ratio of 1:5 in phosphate buffer (pH 8.0, 200 mM) at 45 °C for 4 h. The storage stability, pH stability, thermal stability, solvent tolerance and resistance to metal ions of papain were enhanced by the immobilization. The oligomerization of L-glutamic acid diethyl ester hydrochloride via PS-PEG-DDI immobilized papain in phosphate buffer (pH 8, 200 mM) at 45 °C demonstrated the yield of oligo(γ-ethyl-L-glutamate) reached 78.6 ± 1.2% under the conditions of 2 U/ml of papain, 60 mg/ml L-glutamic acid diethyl ester hydrochloride for 4 h. The immobilization increased the yield by about 40%. The analysis by mass spectrometry (MS) and 1H NMR showed an average degree of polymerization (DPavg) of product was approximately 7–8, identical to that produced using free papain. The biocatalytic activity of the PS-PEG-DDI immobilized papain remained stable after 5 repeated uses, all giving a yield of over 70%. The reduced activation energy of the reaction catalyzed by the PS-PEG-DDI immobilized papain suggests a more favorable active conformation was probably achieved following immobilization.

Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies.

The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography–mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.

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.

Molecular Docking and Antibacterial Studies of Pyranopyrazole Derivatives Synthesized Using [Pap-Glu@Chi] Biocatalyst Through a Greener Approach

In the present work, papain enzyme was immobilized on a polymer support chitosan through a linkage of glutaraldehyde to form the [Pap-Glu@Chi] biocatalyst through the covalent bonding method. The immobilization of papain on Chitosan was confirmed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) which revealed the change in morphology of chitosan due to immobilization of papain through glutaraldehyde. Thus prepared heterogeneous biocatalyst was used for the four-component synthesis of pyranopyrazole derivatives by the reaction of substituted aromatic aldehydes, malononitrile, ethylacetoacetate and hydrazine hydrate affording excellent yields within short reaction times at moderate temperature. The catalyst exhibited significant activity up to six cycles. Synthesized compounds were checked for their molecular docking activity using AutoDock Vina against the bacterial protein receptors (PDB code 2VF5 and 1BAG) obtained from the RCSB Protein Data Bank. Significant binding energy values obtained from docking studies revealed good interaction between synthesized compounds and bacterial protein receptor. Synthesized compounds were tested against human pathogenic bacterial strains Escherichia coli, Salmonella typhi, Bacillus subtilis, Pseudomonas aeruginosa along with Gentamicine as a reference antibiotic using disk diffusion method. The compounds were found to exhibit moderate-to-high antibacterial activity.

The quest for reusability: The facile and stable immobilization of papain on cysteine functionalized iron oxide nanoparticles activated glass surface

The study displays the immobilization of papain on cysteine functionalized iron oxide nanoparticle coated glass beads. Glass beads were treated with (3-mercaptopropyl) trimethoxysilane and iron oxide nanoparticles capped with cysteine, to create a layer of cysteine on the surface of glass bead. This functionalized glass bead surface was further used to immobilize papain through glutaraldehyde treatment. The average size of cysteine capped iron oxide nanoparticles were found to be 50 nm. The binding of cysteine through the iron oxide nanoparticles was validated by Fourier transform infra-red spectroscopy. The activity of enzyme was found to be stable at variable temperature and pH conditions. The covalently immobilized enzyme on the glass bead sustained high enzyme activity and could be reused for 5 times without losing its activity. The immobilized papain retained 81% of its initial activity after 5 consecutive cycle. The storage stability analysis of the immobilized system revealed that it is stable for 6 months without loss in its activity. An average of 1.8 mg papain was successfully immobilized per gram of glass beads. In this case cysteine emerges as a new and effective medium for immobilizing biomolecules as it provides high efficiency towards immobilization.

Papain immobilization on heterofunctional membrane bacterial cellulose as a potential strategy for the debridement of skin wounds

We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3–7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.