Matrix For Enzyme Immobilization Research Articles

Controlling enzymatic activity by immobilization on graphene oxide

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells’ viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Construction of amperometric glucose biosensor based on in-situ fabricated hierarchical meso-macroporous SiO2 modified Au film electrodes

Gold nanoparticles (GNPs) modified hierarchical meso-macroporous (HMMP) SiO2 layer on the surface of Au film electrode was developed as a novel enzyme immobilization matrix for biosensors construction. HMMP SiO2-Au bilayer film electrodes were in-situ fabricated with magnetron sputtering process and templating method. The as-prepared HMMP SiO2 films were characterized by SEM, TEM, and cyclic voltammetry (CV). The modified layer of HMMP SiO2 has interconnected pore channels, and the sizes of macropores and mesopores are about 330 nm and 9 nm, respectively. The HMMP SiO2 modified gold film electrodes not only have no diffusion barrier for electrochemical probes, but also exhibit good electrochemical properties. In addition, the activity and stability of the immobilized enzyme can be commendably retained in HMMP SiO2. The biosensor exhibits an excellent bioelectrocatalytic response to glucose with a linear range of 1.0×10-4 M-1.0×10-2 M, high sensitivity of 18.0 μA·mM-1·cm-2, as well as good reproducibility and stability.

A Novel Xylan-Polyvinyl Alcohol Hydrogel Bead with Laccase Entrapment for Decolorization of Reactive Black 5

In an attempt to find a more efficient technique for biodegradation of the recalcitrant Reactive Black 5 (RB-5) dye, a composite xylan-polyvinyl alcohol (xylan-PVOH) hydrogel was used to immobilize laccase from the white-rot fungus Trametes versicolor. Xylan was prepared from the black liquor of pulp and paper effluent, and it was esterified with citric acid prior to cross-linking with polyvinyl alcohol (PVOH). The optimum composition for the immobilized laccase bead formation consisted of 4% (w/v) modified xylan, 10% (w/v) PVOH, and 15 U.mL-1 crude laccase. The maximum decolorization of RB-5 (98.45  1.96 %) was obtained within the first cycle (6 h) at 40 °C. In the eighth cycle, the reused beads were able to decolorize 55.35  2.46 % of the RB-5. Moreover, the xylan-PVOH beads extended the optimum pH range of laccase activity from 6 to 10 and tolerated a temperature up to 10 °C higher than that of the free enzyme. These results suggest that the xylan-PVOH bead has great potential as the polymer matrix for enzyme immobilization, which has applications in wastewater treatment.

A Co(II)-based metallo-supramolecular polymer as a novel enzyme immobilization matrix for electrochemical glucose biosensing

A Co(II)-based metallo-supramolecular polymer (polyCo) was used as a novel matrix to immobilize glucose oxidase (GOx) for enzymatic glucose sensing. A facile fabrication of a modified glassy carbon electrode (GCE) was achievable by a one-step co-immobilization method. UV–vis spectroscopy and cyclic voltammetry were used to characterize the electrochemical biosensor and the sensing mechanism of the reaction on the polyCo-modified GCE. After the optimization of composition and loading of the polyCo/GOx GCE, the fabricated sensor demonstrated excellent amperometric quantification of glucose, with high sensitivity (47.1μAmM−1cm−2) in the linear range of up to 1.4mM. The selectivity, based on the enzyme matrix without any pretreatment, was also sufficiently high. The outstanding stability of the sensor supported our assumption that metallo-supramolecular polymers can be used as electrode materials for glucose biosensors.

PVA-AWP/tyrosinase functionalized screen-printed electrodes for dopamine determination

Highly sensitive biosensors for dopamine determination were developed using commercially available SPEs in combination with the PVA-AWP photopolymer as the enzyme immobilization matrix.

Display of Thermotoga maritima MSB8 nitrilase on the spore surface of Bacillus subtilis using out coat protein CotG as the fusion partner

In the present study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. An Escherichia coli– Bacillus subtilis shuttle vector (pHS-CotG-nit) was constructed for the spore surface display of the nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The successful display of CotG-nit fusion protein on the spore surface of B. subtilis was verified by western blot analysis and activity measurement. The optimal temperature and pH of the spore surface-dispalyed nitrilase were observed to be 50°C and pH 8.0, respectively, which were higher than the free nitrilase (45°C and pH 7.5). The analysis of thermal and pH stability indicated that the spore surface-displayed nitrilase retained 79% and 97% at 75°C and pH 8.0 after 1h of incubation, whereas it were 32% and 52%, respectively, for free nitrilase. Furthermore, the reusability experiments indicated that the activity of the spore surface-displayed nitrilase was not significantly decreased throughout the reusability process, which still retained 83% of the initial activity at the fifth cycle. Above all, these results suggested that surface display of enzymes on the spore of B. subtilis might be an effective method for enzyme immobilization and help to meet the ever-increasing industrial demand for preparation and stabilization of biocatalysts.

Flexible 3D nitrogen-doped carbon nanotubes nanostructure: A good matrix for enzyme immobilization and biosensing

To increase the protein capability and simplify biosensing fabrication, a three-dimensional (3D) nitrogen-doped carbon nanotubes (NCNTs) on free-standing electrospun carbon nanofibers film (NCNT@CNFs) was prepared via pyrolysis of pyridine. The density, size and nitrogen content of the “quasi-aligned” NCNT arrays can be easily controlled by modulating the growth temperature. By directly dropping the free-standing and flexible composite onto the electrode surface with high glucose oxidase loading (3.2×10−9molcm−2), a glucose biosensor with remarkable sensitivity (24.8mAM−1cm−2), a low detection limit (6μM at a signal-to-noise ratio of 3), and a wide linear range can be simply constructed. In addition, the biosensor exhibited high reproducibility, good storage stability, satisfactory anti-interference ability, and strong applicability to real samples. The N-doping combining with abundant defective sites and the favorable 3D network structure could effectively avoid enzyme leakage, maintain the bioactivity of the enzyme, and facilitate electron transfer, which resulted in excellent electrocatalytic performance. The facile preparation method and attractive analytical performance make this robust electrode material promising for the development of effective electrochemical sensors.

Disposable urea biosensor based on nanoporous ZnO film fabricated from omissible polymeric substrate.

In the present study, a facile and simple fabrication method of a semiconductor based urea biosensor was reported via three steps: (i) producing a ZnO-PVA composite film by means of a polymer assisted electrodeposition of zinc oxide (ZnO) on the F-doped SnO2 conducting glass (FTO) using water soluble polyvinyl alcohol (PVA), (ii) obtaining a nanoporous ZnO film by PVA omission via a subsequent post-treatment by annealing of the ZnO-PVA film, and (iii) preparation of a FTO/ZnO/Urs biosensor by exploiting a nanoporous ZnO film as an efficient and excellent platform area for electrostatic immobilization of urease enzyme (Urs) which was forced by the difference in their isoelectric point (IEP). The characterization techniques focused on the analysis of the ZnO-PVA film surfaces before and after annealing, which had a prominent effect on the porosity of the prepared ZnO film. The surface characterization of the nanostructured ZnO film by a field emission-scanning electron microscopy (FE-SEM), exhibited a film surface area as an effective bio-sensing matrix for enzyme immobilization. The structural characterization and monitoring of the biosensor fabrication was performed using UV-Vis, Fourier Transform Infrared (FT-IR), Raman Spectroscopy, Thermogravimetric Analysis (TGA), Cyclic Voltammetry (CV), and Electrochemical Impedance Spectroscopy (EIS) techniques. The impedimetric results of the FTO/ZnO/Urs biosensor showed a high sensitivity for urea detection within 8.0-110.0mg dL(-1) with the limit of detection as 5.0mg dL(-1).

In Situ Formation of a Biocatalytic Alginate Membrane by Enhanced Concentration Polarization.

A thin alginate layer induced on the surface of a commercial polysulfone membrane was used as a matrix for noncovalent immobilization of enzymes. Despite the expected decrease of flux across the membrane resulting from the coating, the initial hypothesis was that such a system should allow high immobilized enzyme loadings, which would benefit from the decreased flux in terms of increased enzyme/substrate contact time. The study was performed in a sequential fashion: first, the most suitable types of alginate able to induce a very thin, sustainable gel layer by pressure-driven membrane filtration were selected and evaluated. Then, an efficient method to make the gel layer adhere to the surface of the membrane was developed. Finally, and after confirming that the enzyme loading could remarkably be enhanced by using this method, several strategies to increase the permeate flux were evaluated. Alcohol dehydrogenase (EC 1.1.1.1), able to catalyze the conversion of formaldehyde into methanol, was selected as the model enzyme. An enzyme loading of 71.4% (44.8 μg/cm(2)) was attained under the optimal immobilization conditions, which resulted in a 40% conversion to methanol as compared to the control setup (without alginate) where only 10.8% (6.9 μg/cm(2)) enzyme was loaded, with less than 5% conversion. Such conversion increased to 60% when polyethylene glycol (PEG) was added during the construction of the gel layer, as a strategy to increase flux. No enzyme leakage was observed for both cases (with/without PEG addition). Modeling results showed that the dominant fouling mechanism during gel layer induction (involving enzyme entrapment) was cake layer formation in the initial and intermediate phases, while pore blocking was the dominant mechanism in the final phase. Such mechanisms had a direct consequence on the type of immobilization promoted in each phase. The results suggested that the strategy proposed could be efficiently used to enhance the enzyme loading on polymer membranes.

Development of tyrosinase biosensor based on quantum dots/chitosan nanocomposite for detection of phenolic compounds

A sensitive and simple amperometric biosensor for phenols was developed based on the immobilization of tyrosinase into CdS quantum dots/chitosan nanocomposite matrix. The nanocomposite film with porous nanostructure, excellent hydrophilicity and biocompatibility resulted in high enzyme loading, and the tyrosinase (Tyr) immobilized in this novel matrix retained its activity to a large extent. The CdS quantum dots/chitosan nanocomposite film was characterized by scanning electron microscopy and electrochemical impedance spectroscopy, and the parameters of the various experimental variables for the biosensor were optimized. Under the optimal conditions, the designed biosensor displayed a wide linear response to catechol over a concentration range of 1.0×10−9 to 2.0×10−5M with a high sensitivity of 561±9.7mAM−1 and a low detection limit down to 0.3 nM at a signal-to-noise ratio of 3. The CdS quantum dots/chitosan nanocomposites could provide a novel matrix for enzyme immobilization to promote the development of biosensing and biocatalysis.

Enhanced amperometric response of a glucose oxidase and horseradish peroxidase based bienzyme glucose biosensor modified with a film of polymerized toluidine blue containing reduced graphene oxide

Reduced graphene oxide (RGO) was used to construct a bienzyme biosensor containing horseradish peroxidase (HRP) and glucose oxidase (GOx). A poly(toluidine blue) (pTB) film containing RGO acted as both enzyme immobilization matrix and electron transfer mediator. The bienzyme biosensor was characterized by electrochemical techniques and displays a highly sensitive amperometric response to glucose and hydrogen peroxide (H2O2) at a potential as low as −0.1 V (vs. SCE). It is shown that use of RGO causes a strong enhancement on the amperometric responses. H2O2 formed by the action of GOx in the presence of oxygen can be further reduced by HRP in the pTB film contacting the RGO modified electrode. In the absence of oxygen, glucose oxidation proceeds by another mechanism in which electron transfer occurs from GOx to the electrode and with pTB acting as the mediator. Amperometric responses to glucose and H2O2 follow Michaelis-Menten kinetics. The experimental conditions were optimized, and under these conditions glucose can be determined in the 80 μM to 3.0 mM range with a detection limit of 50 μM. H2O2, in turn, can be quantified in up to 30.0 μM concentration with a detection limit of 0.2 μM. The bienzyme biosensor is reproducible, repeatable and stable. Finally, it has been successfully applied to the determination of glucose in plasma samples.

Mimetic biomembrane–AuNPs–graphene hybrid as matrix for enzyme immobilization and bioelectrocatalysis study

A hybrid composite constructed of phospholipids bilayer membrane, gold nanoparticles and graphene was prepared and used as matrices for microperoxidase-11 (MP11) immobilization. The direct electrochemistry and corresponding bioelectrocatalysis of the enzyme electrode was further investigated. Phospholipid bilayer membrane protected gold nanoparticles (AuNPs) were assembled on polyelectrolyte functionalized graphene sheets through electrostatic attraction to form a hybrid bionanocomposite. Owing to the biocompatible microenvironment provided by the mimetic biomembrane, microperoxidase-11 entrapped in this matrix well retained its native structure and exhibited high bioactivity. Moreover, the AuNPs–graphene assemblies could efficiently promote the direct electron transfer between the immobilized MP11 and the substrate electrode. The as-prepared enzyme electrode presented good direct electrochemistry and electrocatalytic responses to the reduction of hydrogen peroxide (H2O2). The resulting H2O2 biosensor showed a wide linear range (2.0×10−5–2.8×10−4M), a low detection limit (2.6×10−6M), good reproducibility and stability. Furthermore, this sensor was used for real-time detection of H2O2 dynamically released from the tumor cells MCF-7 in response to a pro-inflammatory stimulant.

Preparation of α-amylase-immobilized freeze-dried poly(vinyl alcohol) foam and its application to microfluidic enzymatic reactor

Freeze-drying aqueous poly(vinyl alcohol) (PVA) solutions affords porous foam materials that show potential as an interesting entrapping matrix for enzyme immobilization. When an enzyme–PVA solution is freeze-dried, the enzymes are automatically entrapped in the architecture of the PVA matrix. The obtained PVA foams barely disperse in aqueous solution because of physically cross-linked PVA cryogel formation. This method is an in situ immobilization technique such that supporting materials can be produced in any space where the precursor solutions can be loaded. In this study, an attempt was made to prepare freeze-dried PVA foams in a microchannel to prepare an α-amylase–immobilized microreactor. Freeze-dried foams for amylase immobilization were demonstrated to be successfully prepared in microreactors with a ca. 18-μL microchannel volume, and were evaluated by conducting continuous starch hydrolysis reactions over 8d. The macroscopic pore structures seemed to be only minimally affected by the apparent activities of the immobilized enzymes; however, the cryogel structures, affected by the freezing protocol, and the microchannel materials employed were suggested as the key to determining reaction performances.

Construction of novel xanthine biosensor by using polymeric mediator/MWCNT nanocomposite layer for fish freshness detection

A novel nanocomposite host matrix for enzyme immobilization of xanthine oxidase was developed by incorporating MWCNT in poly(GMA-co-VFc) copolymer film. In the food industry fish is a product with a very low commercial life, and a high variability as well elevated level of xanthine is an important biomarker as a sign of spoilage. The fabricated process was characterized by scanning electron microscopy (SEM), and the electrochemical behaviors of the biosensor were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The prepared enzyme electrodes exhibited maximum response at pH 7.0 and 45°C +0.35V and reached 95% of steady-state current in about ∼4s and its sensitivity was 16mAM−1. Linear ranges (2–28μM, 28–46 and 46–86μM), analytical performance and a low detection limit 0.12μM obtained from the xanthine biosensor gives reliable results in measuring xanthine concentration in the fish meat. All the results indicating that the resulting biosensor exhibited a good response to xanthine that was related to the addition of MWCNT in the polymeric mediator film which played an important role in the biosensor performance. In addition, the biosensor exhibited high good storage stability and satisfactory anti-interference ability.

Immobilization of glucose oxidase on polydopamine-functionalized graphene oxide.

In this study, graphene oxide (GO) was modified with dopamine to create a matrix for enzyme immobilization. Dopamine can self-polymerize to get polydopamine (PDA) and coated on GO surface. At the same time, GO was reduced to get PDA/rGO biocomposite. The PDA/rGO may offer adherent surface for enzyme immobilization. Glucose oxidase (GOD), an oxidoreductase, was chosen as model enzyme and can be easily immobilized on PDA/rGO. Experimental results indicated that the thermal and pH stability as well as the storage stability and resistance toward the denaturing agents of GOD were significantly improved after immobilization. The Michaelis constant (K m) of the immobilized GOD was very close to that of the free GOD. This study offers a versatile approach for deposition of biopolymer on GO and provides a way for enzyme immobilization. Hopefully, the immobilized GOD may be further applied for biosensor and biofuel cell applications.

A novel and effective surface design: conducting polymer/β-cyclodextrin host-guest system for cholesterol biosensor.

The combination of supramolecules and conducting polymers (CPs) has gained much attention for the development of new immobilization matrices for biomolecules. Herein, an amperometric biosensor based on a novel conducting polymer, poly(2-(2-octyldodecyl)-4,7-di(selenoph-2-yl)-2H-benzo[d][1,2,3]triazole)) (PSBTz) and β-cyclodextrin (β-CD) for the detection of cholesterol, was constructed. The PSBTz film with β-CD was deposited on a graphite electrode by electropolymerization technique to achieve a suitable matrix for enzyme immobilization. Moreover, to justify the immobilization, alkyl chain containing conducting polymer (PSBTz) was designed, synthesized and electrochemically polymerized on the transducer surface. Alkyl chains in the structure of SBTz and hydroxyl groups of β-CD contributed to effective immobilization while protecting the suitable orientation of the biomolecule. Cholesterol oxidase (ChOx) was covalently immobilized onto the modified surface using N,N′-carbonyldiimidazole (CDI) as the cross-linking agent. After successful immobilization, amperometric biosensor responses were recorded at −0.7 V vs Ag/AgCl in phosphate buffer (pH 7.0). The apparent Michaelis-Menten constant (KM(app)), maximum current (Imax), limit of detection (LOD), and sensitivity values were determined: 28.9 μM, 12.1 μA, 0.005 μM, and 5.77 μA/μM cm(2), respectively. The fabricated biosensor was characterized using scanning electron microscopy (SEM) and cyclic voltammetry (CV) techniques. Finally, the prepared biosensor was successfully applied for the determination of cholesterol in blood samples.

Retrofitting of Fixed-Bed Heterogeneous Reactors for Glucose Isomerization

A retrofitting strategy is proposed here that can enhance the conversion level while significantly reducing the pressure drop in fixed-bed glucose isomerization reactors. The proposed strategy utilizes hollow support matrix for enzyme immobilization instead of commonly used solid spherical support. The absence of core in hollow catalytic support helps to lower diffusional resistances, thus ensuring a better utilization of the catalytic material. This leads to higher conversion in the reactor. Moreover, the use of hollow packing yields high bed void fraction, which helps to substantially lower the pressure drop. Specifying the proposed hollow support matrix as being either cylindrical or ring shaped, a rigorous heterogeneous model for the glucose isomerization reactor is developed here. The model validity is first tested by using reported experimental data for spherical support. Using the present model, the reactor performance can be optimized in terms of shape parameters of the proposed hollow support for enzyme immobilization.

Development of highly sensitive amperometric biosensor for glucose using carbon nanosphere/sodium alginate composite matrix for enzyme immobilization.

In this study, we developed a highly sensitive amperometric biosensor for glucose detection based on glucose oxidase immobilized in a novel carbon nanosphere (CNS)/sodium alginate (SA) composite matrix. This hybrid material combined the advantages of CNS and natural biopolymer SA. This composite film was characterized by scanning electron microscope, electrochemical impedance spectroscopy and UV-vis, which indicated that the hybrid material was suitable for immobilization of glucose oxidase. Various experimental conditions were investigated that influenced the performance of the biosensor, such as pH, applied potential and temperature. Under the optimum conditions, the biosensor showed excellent performance for glucose over a wide linear concentration range from 1.0 × 10(-6) to 4.6 × 10(-3) M with a detection limit of 0.5 μM based on a signal-to-noise ratio of 3. Furthermore, the biosensor exhibited excellent long-term stability and satisfactory reproducibility.

New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization

Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360μg/1.2×1011 spore. However, maximum activity was achieved at an enzyme concentration of approximately 60μg/.4×1010, corresponding to an estimated activity of 8×103IUmg−1/1.2×1011 spore for covalent immobilization and 8.53×103 for the adsorption method. After washing the enzyme with 1M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60°C and 80°C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The Km and Vmax values were determined by the GraphPad Prism software, which showed that the Vmax value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface.

CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production.

Backgroundγ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened.ResultsWe prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The Vmax and kcat value of Avicel immobilized Gla-linker-Cbd were 381 U mg-1 and 4.7 × 105 s-1 respectively. And the values of the free Gla-linker-Cbd were 151 U mg-1 and 1.8 × 105 s-1 respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value.ConclusionsCellulose is the most abundant and renewable material on the Earth. The absorption between Cbd domain and cellulose is a bio-green process. The cellulose immobilized fusion Gla exhibited good catalytic characters, therefore we think the cellulose immobilized Gla is a promising catalyst for the industrial preparation of (-) - γ-lactam.