Enzyme Entrapment Research Articles

Synthesis of photo-responsive chitosan-cinnamate for efficient entrapment of β-galactosidase enzyme

A modified photo-crosslinkable chitosan cinnamate (CS-CN) bio-polymer was synthesized via functionalization of the main active –NH2 and –OH groups of chitosan using cinnamoyl chloride. The chemical structure of the manufactured bio-polymer was elucidated using instrumental techniques including FTIR and NMR spectra. The obtained photo-active CS-CN was cross-linked upon irradiation with the UV light and the performed photo-crosslinking was monitored using UV–vis light spectra. Moreover, XRD and SEM were respectively utilized to investigate both crystalline and morphological structures of the cross-linked bio-polymer. The obtained CS-CN bio-polymer was successfully employed as a polymeric support for β-galactosidase immobilization via entrapment technique. The entrapped β-galactosidase maintains around 80% of its initial activity and displayed a considerable stability against temperature and pH changes in comparison with the native soluble enzyme form. In addition, the kinetic studies showed an expected elevation of Km along with lowering of vo values after immobilization reflecting a lower enzyme-substrate affinity. However, the reusability experiments indicated that the entrapped enzyme maintains approximately 88% of its activity after 8 reuse cycles, which reveal the value of the performed immobilization technique especially in economic terms.

Polyacrylamide beads: Polymer entrapment increases the catalytic efficiency and thermal stability of protease

Immobilization is a unique method for the improvement of product yield. During solid phase catalysis, the chemistry of the synthetic matrix plays an essential role in the performance of the biocatalyst. In the current study, immobilized protease within polyacrylamide macrosphere beads exhibited 76.0% entrapment yield with a remarkable stability of enzyme (33.0%) after 30 days of storage period at 4 °C. The entrapment of free enzyme within polyacrylamide also increased the optimal reaction temperature by 55 °C and provided a broad range of pH optima. Moreover, a significant enhancement in thermal stability was also detected. Polyacrylamide entrapped protease revealed up to 30.93% activity after incubation period of 30.0 min at 70 °C whereas, the free enzyme was completely inactivated at this temperature. Additionally, entrapped protease displayed an efficient recycling capacity and retained approximately 24.0% of its initial activity after eight successive reaction cycles. After entrapment of protease, the anchoring of substrate to the active site of the free protease exhibited change in Km and Vmax values. Therefore, owing to economic feasibility, the polyacrylamide entrapped protease might be a promising candidate for various applications in different industrial sectors.

Immobilization of laccase onto porous polyvinyl alcohol/halloysite hybrid beads for dye removal.

Laccase was immobilized in polyvinyl alcohol beads containing halloysite nanotubes (PVA/HNTs) to improve the stability and reusability of enzyme. The porous structure of PVA/HNTs beads facilitates the entrapment of enzyme and prevents the leaching of immobilized laccase as well. Halloysite nanotubes act as bridge to connect the adjacent pores, facilitating the electron transfer and enhancing the mechanical properties. PVA/HNTs beads have high laccase immobilization capacity (237.02 mg/g) and activity recovery yield (79.15%), indicating it can be used as potential support for laccase immobilization. Compared with free laccase, the immobilized laccase on hybrid beads exhibits enhanced pH tolerance (even at pH 8.0), good thermal stability (57.5% of the initial activity can be maintained at 75 °C), and excellent storage stability (81.17% of enzyme activity could be retained after storage at 4 °C for 5 weeks compared with that for free enzyme of 60%). Also, the removal efficiency for reactive blue can reach as high as 93.41% in the presence of redox mediator 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate), in which adsorption and degradation exist simultaneously. The remarkable pH tolerance, thermal and storage stability, and reuse ability imply potential application of porous PVA/HNTs immobilized enzyme in environmental fields.

Regulating immobilization performance of metal-organic coordination polymers through pre-coordination for biosensing

We propose a method for regulating biomolecules immobilization performance of metal-organic coordination polymers (MOCPs) through pre-coordination for highly sensitive biosensing. 2,5-dimercapto-1,3,4-thiadiazole (DMcT) was used as organic monomers. Firstly, using CuCl2 as the source of metal ions to form short oligomers with DMcT (MOCPsCu), which can regulate the length of ligands through pre-coordination. Then exploiting NaAuCl4 as the source of Au ions to coordinate both short oligomers and biomolecules (MOCPsCu+Au), since Au ions can coordinate with both N and S atoms. Through controlling the concentration of CuCl2, oligomers with desired length could be readily obtained to prepare MOCPsCu+Au framework with controllable porosity and enzyme entrapment efficiency. Thus MOCPsCu+Au offers several advantages including improved mass transfer efficiency and biocatalytic sensitivity than conventional MOCPs using single metal ions. Glucose oxidase (GOx) was used as the representative biomolecule, the entrapment ratio of enzyme in MOCPsCu+Au case reached an extreme value of 100%. These MOCPsCu+Au biocomposites modified electrode also showed greatly enhanced biocatalytic sensitivity (127 μA cm−2 mM−1) and very low detection limit (58 nM), compared with those reported analogues. The new materials/strategy may create new avenue to regulate the performance of ligand-constructed polymers and their composites for entrapment-based applications.

A mild route to entrap papain into cross-linked PEG microparticles via visible light-induced inverse emulsion polymerization

Entrapment of enzymes into a cross-linked network is an effective way to enable their recycling. In order to obtain a satisfied recovery of enzyme activity, a mild encapsulating condition is highly required due to the delicate nature of enzymes. Herein, a facile and mild visible light-induced inverse emulsion polymerization technique was developed for in situ entrapment of enzyme. In this method, poly (ethylene glycol) diacrylate (PEGDA) was dissolved in phosphate buffer saline and used as disperse phase, while continuous phase was composed of liquid paraffin, photoinitiators (isopropylthioxanthone and ethyl 4-dimethylaminobenzoate) and emulsifier (Span 80 and Tween 80). Under the irradiation of visible light, PEGDA could be cross-linkedly polymerized and formed microparticles with diameter ranged from 0.75 to 6.5 μm. When the glutaraldehyde cross-linked papain was added into disperse phase, it could be in situ entrapped into the microparticles after the visible light-induced inverse emulsion polymerization. The immobilized papain exhibited higher activity in a wide range of temperature and pH than free papain. Moreover, the immobilized papain could maintain 60% of its initial activity even after ten cycles of usage. This simple and mild strategy to in situ entrapment of enzymes has potential application in fields such as biocatalyst, biosensor and drug delivery.

A novel enzyme based biosensor for catechol detection in water samples using artificial neural network

Biosensors could be used as digital devices to measure the sample infield. Consequently, computational programming along with experimental achievements are required.In this study, a novel biosensor/artificial neural network (ANN) integrated system was developed. Poly (3,4-ethylenedioxy-thiophene)(PEDOT), graphene oxide nano-sheets (GONs) and laccase (Lac) were used to construct a biosensor. The simple and one-pot process was accomplished by electropolymerizing 3,4-ethylenedioxy-thiophene (EDOT) along with GONs and Lac as dopants on glassy carbon electrode. Scanning electron microscopy (SEM) and electrochemical characterization were conducted to confirm successful enzyme entrapment. The modified electrode was employed to detect and measure catechol. The reaction of catechol and the prepared electrode was controlled by adsorption. Linear responses of the biosensor were over two ranges, 0.036–0.35μM and 0.35–2.5μM, with a detection limit of 0.032μM. The proposed biosensor was tested in real water samples successfully.The experimental test results were applied to train ANNs by the back-propagation algorithm. The input and output parameters were current and catechol concentration, respectively. Results from ANN modeling complied well with the experiments, signifying its useful application in biosensor technology.

Self‐Assembly of Nucleobase, Nucleoside and Nucleotide Coordination Polymers: From Synthesis to Applications

AbstractWith multiple metal binding sites, nucleobases, nucleosides, and nucleotides form various coordination polymers (CPs) with metal ions. These self‐assembled materials are very interesting for their simplicity to prepare, highly tunable structure and properties, and excellent biocompatibility. Polymerization is achieved under ambient conditions without the need of free radical initiators or UV light. Different nucleobases have different metal‐binding preferences, but in general most of their coordination sites prefer borderline or soft metals, while the phosphate groups in the nucleotides prefer hard Lewis acids. In this Focus Review, recent developments in this field are summarized starting from the synthesis and characterization of these CPs. Various metal ions including gold, silver, lanthanides, zinc, copper, and iron are individually reviewed, and each metal brings its own property into the CP materials. While most of these materials are non‐crystalline nanoparticles, under certain conditions crystalline metal‐organic frameworks (MOFs) and hydrogels can also be prepared. The applications of such CPs are then described including enzyme entrapment, drug loading and delivery, biosensor development, catalysis, coating of nanoparticles, and luminescence. Finally, future research opportunities of this field are discussed.

New efficient laccase immobilization strategy using ionic liquids for biocatalysis and microbial fuel cells applications

AbstractBACKGROUNDLaccases belong to the family of blue multicopper oxidases, these enzymes are efficient in many applications, including in biofuel cells to produce electricity through chemical reactions. Several laccase immobilization methods have been elucidated, such as covalent link and entrapment.RESULTSThe present work describes how, for the first time, laccase was successfully immobilized by entrapment into polymer inclusion ionic liquid membranes (PILMs) based on ionic liquids and polyvinylchloride. The immobilization rate was very high in all cases (99.2 ± 0.6%), so that the enzyme entrapment strategy described can be considered as successful and one of the best methods for laccases immobilization that have been reported to date. The assayed membranes were evaluated as performing carrier for laccase by means of the ABTS oxidation. Four screened ionic liquids were used for the first time for this purpose: [OMIM][NTf2], [EMIM][NTf2], [Epy][NTf2] and [Chol][NTf2]. This work demonstrated the efficiency in terms of the activity rate and stability of the enzyme versus IL concentrations. The most suitable IL as regards activity [Chol][NTf2]. However, the reuse assays showed that the most stable PILM was the one constituted by 50% [Epy][NTf2]. For physical resistance and water insolubility, the [OMIM][NTf2] based PILM was selected for microbial fuel cell (MFC) application.CONCLUSIONThis is the first report describing this original entrapment strategy for laccase immobilization into PILM. The application of this laccase‐membrane in MFC showed high stability during almost one week of use and good efficiency in producing bio‐energy and removing COD from industrial wastewater. © 2017 Society of Chemical Industry

Novel Biocompatible Thermoresponsive Poly(N-vinyl Caprolactam)/Clay Nanocomposite Hydrogels with Macroporous Structure and Improved Mechanical Characteristics.

Poly(N-vinyl caprolactam) (PVCL) hydrogels usually suffer from the imporous structure and poor mechanical characteristics as well as the toxicity of cross-linkers, although PVCL itself is biocompatible. In this paper, novel biocompatible thermoresponsive poly(N-vinyl caprolactam)/clay nanocomposite (PVCL-Clay) hydrogels with macroporous structure and improved mechanical characteristics are developed for the first time. The macroporosity in the hydrogel is introduced by using Pickering emulsions as templates, which contain N-vinyl caprolactam (VCL) monomer as dispersed phase and clay sheets as stabilizers at the interface. After polymerization, macropores are formed inside the hydrogels with the residual unreacted VCL droplets as templates. The three-dimensional PVCL polymer networks are cross-linked by the clay nanosheets. Due to the nanocomposite structure, the hydrogel exhibits better mechanical characteristics in comparison to the conventional PVCL hydrogels cross-linked by N,N'-methylene diacrylamide (BIS). The prepared PVCL-Clay hydrogel possesses remarkable temperature-responsive characteristics with a volume phase transition temperature (VPTT) around 35 °C, and provides a feasible platform for cell culture. With macroporous structure and good mechanical characteristics as well as flexible assembly performance, the proposed biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogels are ideal material candidates for biomedical, analytical, and other applications such as entrapment of enzymes, cell culture, tissue engineering, and affinity and displacement chromatography.

Fluorescence and NMR spectroscopy together with molecular simulations reveal amphiphilic characteristics of a Burkholderia biofilm exopolysaccharide

Biofilms are a collective mode of bacterial life in which a self-produced matrix confines cells in close proximity to each other. Biofilms confer many advantages, including protection from chemicals (including antibiotics), entrapment of useful extracellular enzymes and nutrients, as well as opportunities for efficient recycling of molecules from dead cells. Biofilm matrices are aqueous gel-like structures composed of polysaccharides, proteins, and DNA stabilized by intermolecular interactions that may include non-polar connections. Recently, polysaccharides extracted from biofilms produced by species of the Burkholderia cepacia complex were shown to possess clusters of rhamnose, a 6-deoxy sugar with non-polar characteristics. Molecular dynamics simulations are well suited to characterizing the structure and dynamics of polysaccharides, but only relatively few such studies exist of their interaction with non-polar molecules. Here we report an investigation into the hydrophobic properties of the exopolysaccharide produced by Burkholderia multivorans strain C1576. Fluorescence experiments with two hydrophobic fluorescent probes established that this polysaccharide complexes hydrophobic species, and NMR experiments confirmed these interactions. Molecular simulations to model the hydrodynamics of the polysaccharide and the interaction with guest species revealed a very flexible, amphiphilic carbohydrate chain that has frequent dynamic interactions with apolar molecules; both hexane and a long-chain fatty acid belonging to the quorum-sensing system of B. multivorans were tested. A possible role of the non-polar domains of the exopolysaccharide in facilitating the diffusion of aliphatic species toward specific targets within the biofilm aqueous matrix is proposed.

Immobilization and Characterization of Bacillus Thuringiensis HCB6 Amylase in Calcium Alginate Matrix

Free enzyme in solution react with substrates to result in products which cannot be recovered for reuse. These problems can be overcome to a certain extent by the use of enzyme immobilization method. Immobilized enzymes are more robust and more resistant to condition changes. More importantly, the heterogeneous immobilized enzyme systems allow an easy recovery of both enzymes and products, multiple re-uses of enzymes, and continuous operation of enzymatic processes. Entrapment of enzymes in Ca-alginate is one of the simplest methods of immobilization. The aim of this research was to obtain the optimum condition of the making of immobilized amylase beads using a Ca-alginate bead and to determine its characteristics. The optimization of immobilized amylase beads includes variation of sodium alginates and variations of enzyme contact time with CaCl2. The characterization of immobilized amylase includes determination of optimum substrate concentration, optimum pH, and optimum incubation time as well as amylase stability test. Amylase activity was determined by using dinitro salicylic (DNS) method. The results showed that the optimum immobilized amylase obtained at alginate concentrations of 5% (w/v), contact time of 60 minutes and immobilization efficiency of 67.5%. Furthermore, immobilized amylase showed optimum substrate concentration of 1.5-2.5% (w/v), optimum pH of 6, an optimum incubation time of 20 minutes with the activity of 179.8 U/mL. The KM value for free amylase and immobilized amylases were 0.3 mM and 0.12 mM respectively. Vmax value for free amylase and immobilized amylases were 105.3 U/mL and 10.1 U/mL respectively. Immobilized Amylase can be used up to six times with the residual activity of 52.7%.

Electrodepositable alginate membranes for enzymatic sensors: An amperometric glucose biosensor for whole blood analysis

Simple and disposable point of care systems are usually the best solution for chronic patients to get a rapid diagnosis in home care context. However, their main drawback relies on the poor reliability derived from the low stability of the bio-recognition elements and low quality of the transducers. In the current work, we study the use of electrodeposited calcium alginate hydrogels as a biocompatible matrix in the development of enzymatic amperometric biosensors for whole blood analysis, to enhance the enzymes stability and to protect the transducer from biofouling. The alginate electrodeposition involves the controlled Ca2+ release, so the gel thickness can be modulated. In the biosensor, horseradish peroxidase (HRP) and glucose oxidase (GOD) were electrodeposited within the hydrogel and the activity of the bi-enzymatic system was analyzed chronoamperometrically using 3,3′,5,5′-Tetramethylbenzidine (TMB) as the mediator. Besides enzyme entrapment, the obtained gels protected the transducer from biofouling, enabling the reuse of the transducer after hydrogel removal and re-electrodeposition. The biosensors showed good analytical characteristics to glucose determination in whole blood samples, discriminating among healthy and hyperglycemic samples, with good sensitivity (− 0.27µAcm−2mM−1), low limit of detection (126µM) and long lineal range (2–12mM).

Back Cover: Poly(benzoxazine) as an Immobilization Matrix for Miniaturized ATP and Glucose Biosensors (ChemElectroChem 4/2017)

The Back Cover picture shows a scheme of the improved ATP microbiosensor based on poly(benzoxazine) entrapment of enzymes for the detection of ATP at mechanically stimulated osteoblastic cells. More information can be found in the Article by C. Kranz and co-workers on page 864 in Issue 4, 2017 (DOI: 10.1002/celc.201600765).

Oyster mushroom’s lipase enzyme entrapment on calcium alginate as biocatalyst in the synthesis of lauryl diethanolamide

Lipase is an enzyme with large biotechnology applications, such as hydrolysis in the food industry, applications in chemical industry, synthesis of polymers and surfactants. Lipase was isolated from oyster mushroom with activity 0,93 U/mg and protein content 1,1234 mg/mL. Lipase was immobilized by entrapment method in a matrix of Ca-alginate. This report describes that we have developed for the synthesis of lauryl diethanolamide The result showed that the optimum condition of lipase immobilization was achieved on 3% Na-alginate solution with protein content 0,84 mg/mL and the activity 3,33 U/mg. An amide (22.911%) formed from the amidation of lauric acid and diethanolamine.

Liposomal Encapsulation Enzymes: From Medical Applications to Kinetic Characteristics.

Liposomes and nanoliposomes as small vesicles composed of phospholipid bilayer (entrapping one or more hydrophilic or lipophilic components) have recently found several potential applications in medicine and food industry. These vesicles may protect the core materials from moisture, heat and other extreme conditions. They may also provide controlled release of various bioactive agents, including food ingredients at the right place and time. Potential applications of enzyme-loaded liposomes are in the medical or biomedical field, particularly for the enzymereplacement therapy, as well as cheese industry for production of functional foods with improved health beneficial impacts on the consumer. Encapsulation process has a recondite impact on enzymes. In fact, liposome preparation techniques may alter the pH and temperature optima, affinity of the enzyme to substrate (Km), and maximum rate of reaction (Vmax). In addition, in this paper, the impact of process variables on the kinetic characteristics of enzymes encapsulated in liposomes was investigated. Also, the effects of enzyme entrapment in liposomes, prepared by different methods, on the catalytic efficiency of enzyme, as well as its kinetic properties and stability compared to native (free) enzymes has been reviewed.

Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

Immobilization engineering – How to design advanced sol–gel systems for biocatalysis?

An immobilization engineering approach using bioinformatics and experimental design tools was applied to improve the sol–gel enzyme entrapment methodology.

A high-performance enzyme entrapment platform facilitated by a cationic polymer for the efficient electrochemical sensing of ethanol.

Entrapment is one of the major approaches for enzyme immobilization; however, it suffers a few critical drawbacks including leakage and high mass transfer resistance to substrates. To address these challenges, herein we report on a new facile and effective enzyme entrapment platform using a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) (MADQUAT) on a single-wall carbon nanotube and reduced graphene oxide (SWCNT-rGO) nanohybrid thin film. To demonstrate this new approach, alcohol dehydrogenase (ADH) is employed as a model enzyme for the entrapment toward the design of an efficient electrochemical biosensor for the detection of ethanol. MADQUAT possesses strong electrostatic affinity with various negatively charged biomolecules; and our FTIR study has shown that there are no structural changes in the enzyme following the entrapment, with an excellent secondary structure association (r = 0.92). Our electrochemical measurements have shown that the entrapped ADH exhibits high ability to exchange electrons in the presence of the NAD+/NADH cofactor and that the SWCNT-rGO nanohybrid significantly enhances the biocatalytic activity of the immobilized ADH and the electrochemical oxidation of NADH in comparison with either SWCNTs or rGO. The ethanol biosensor developed in this study exhibits a fast response, wide linearity range, high sensitivity (26.27 μA mM-1 cm-2), remarkable low limit of detection (0.16 μM), high selectivity and high stability. The optimized biosensor has been further tested with real samples including wine, beer and blood alcohol, showing promising analytical and biomedical applications.

Enzyme Stabilization via Bio-Templated Silicification Reactions.

Effective entrapment of enzymes in solid phase materials is critical to their practical application. The entrapment generally stabilizes biological activity compared to soluble molecules and the material simplifies catalyst integration compared to other methods. A silica sol-gel process based upon biological mechanisms of inorganic material formation (biomineralization) supports protein immobilization reactions within minutes. The material has high protein binding capacity and the catalytic activity of the enzyme is retained. We have demonstrated that both oligopeptides and selected proteins will mediate the biomineralization of silica and allow effective co-encapsulation of other proteins present in the reaction mixture. The detailed methods described here provide a simple and effective approach for molecular biologists, biochemists and bioengineers to create stable, solid phase biocatalysts that may be integrated within sensors, synthetic processes, reactive barriers, energy conversion, and other biotechnology concepts.

Adding Biomolecular Recognition Capability to 3D Printed Objects.

Three-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems.