Optimal Immobilization Conditions Research Articles

Selection of CalB immobilization method to be used in continuous oil transesterification: Analysis of the economical impact

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size < 1000 μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2–10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments.

Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization

Sucrose phosphorylase from Bifidobacterium adolescentis was recombinantly expressed in Escherichia coli and purified by use of a His-tag. Kinetic characterization of the enzyme revealed an optimal temperature for phosphorolytic activity of 58 °C, which is surprisingly high for an enzyme from a mesophilic source. The temperature optimum could be further increased to 65 °C by multipoint covalent immobilization on Sepabeads EC-HFA. The optimal immobilization conditions were determined by surface response design. The highest immobilization yield (72%) was achieved in a phosphate buffer of 0.04 mM at pH 7.2, irrespective of the temperature. The immobilized enzyme was able to retain 65% of its activity after 16 h incubation at 60 °C. Furthermore, immobilization of the enzyme in the presence of its substrate sucrose, increased this value to 75%. The obtained biocatalyst should, therefore, be useful for application in carbohydrate conversions at high temperatures, as required by the industry.

Non-isothermal bioremediation of waters polluted by phenol and some of its derivatives by laccase covalently immobilized on polypropylene membranes

In view of the heath problems induced by the presence into the environment of endocrine disruptors, laccase from Trametes versicolor was covalently immobilized on a chemically modified polypropylene membrane in order to remove phenol and its derivatives from polluted waters. Using phenol as substrate model the optimal immobilization conditions were determined. The immobilized laccase exhibited maximal enzyme activity at pH 5.5 and optimal temperature at 55 °C. These operative parameters have been compared with those obtained with the soluble laccase in order to ascertain the immobilization effect. When employed in a bioreactor operating under isothermal conditions the immobilized laccase was able to oxidize a wide range of phenolic substrates. In particular it was found that some phenol derivatives (2-CP, 3-CP, 4-CP, NP and chlorophene) were oxidized at a similar rate than phenol, other derivatives (paracetamol, 3-MP and chloroxyphenol) at a smaller rate, while others (2,4-DCP and BPA) at higher rate. When the catalytic membrane was employed in a non-isothermal reactor the reaction rate increased with the increase of the applied temperature difference. Practically the increase of the laccase oxidative power under the non-isothermal conditions followed the same sequence observed under isothermal conditions. Interesting enough, the percentage increase of enzyme reaction rate under non-isothermal conditions resulted higher in the cases in which the isothermal reaction rate was smaller. When the reduction of the production times by the presence of a temperature gradient is considered, the measured values strongly candidate the technology of non-isothermal bioreactors as a useful tool in processes of detoxification of waste waters polluted by endocrine disruptors of phenolic origin.

Laccase Immobilization on D380 Macroporous Exchange Resin by Cross-Linking with Glutaraldehyde

Macroporous exchange resins have been applied as supports for enzyme immobilization. The supporters were activated by cross-linking with glutaraldehyde, then Laccase was immobilized. Parameters of the immobilization process were optimized. The results of repeated batch showed the optimal immobilization conditions (10% of glutaraldehyde, pH=3, temperature=45 °C). In addition, the biocatalyst also showed good operational stability, maintaining 45% of its initial activity after 10 cycles of oxidation of ABTS. The immobilized laccase had potential for application in the continuous decolorization of textile effluents.

Immobilization of Cellulase on a Reversibly Soluble−Insoluble Support: Properties and Application

Cellulase was coupled to N-succinyl-chitosan (NSC) showing soluble-insoluble characteristics with pH change. Cellulase immobilized on NSC (NSCC) is in a soluble state during the enzyme reaction, yet can be recovered in its insoluble form by lowering the pH of the reaction solution after the reaction. NSCC was obtained under the optimized immobilization conditions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) 10 mg, cellulase 15 mg, and pH 5.0. The retention activity of the immobilized cellulase was found to be 48.8%. The effects of pH and temperature on the activity and stability of NSCC were studied and compared with those of free cellulase. The optimum temperature and pH of NSCC was 45 degrees C and 4.0, respectively, which was found unchanged compared with the free one. The stability of cellulase against change in the pH and temperature was improved by the immobilization. The effectiveness of employing NSCC for extracting flavonoids from Ginkgo biloba leaf powder was investigated. Results showed that NSCC enhanced extraction yield up to 2.35-fold when compared with the conventional method. Moreover, NSCC retained 83.5% of its initial activity after five batches of hydrolysis reaction.

Immobilization conditions of ketoreductase on enantioselective reduction in a gas‐solid bioreactor

The immobilization conditions of commercial ketoreductase for continuous enantioselective reduction in the gas-phase reaction were investigated with respect to the immobilization efficiency (residual activity and protein loading) and the gas-phase reaction efficiency (initial reaction rate, half-life, and enantioselectivity). For the analyses, ketoreductase was first immobilized by physical deposition on glass supports and the reduction of 2-butanone to (S)-2-butanol with the concomitant regeneration of NADH by 2-propanol was used as a model reaction. The optimal conditions of enzyme immobilization were obtained using an absolute pressure of 100 hPa for drying, a pH between 6.5 and 7.0, and a buffer concentration of 50 mM. The buffer concentration in particular had a strong effect on both the enzyme activity and enantioselectivity. Under optimal immobilization conditions, the thermostability of ketoreductase in the gas-phase system was enhanced compared to the aqueous-phase system, while the enantioselectivity was successfully maintained at a level identical to that of the native enzyme. These results indicate that the gas-phase reaction has a great potential for industrial production of chiral compounds, but requires careful optimization of immobilization conditions for the reaction to progress effectively.

Preparation of bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide) hollow fiber cation-exchange membranes and immobilization of cellulase thereon

For cellulase immobilization, a hollow fiber cation-exchange membrane (HFCEM) as a support were prepared from bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide) (BPPO) by amination and carboxylation, and the two-step modification was confirmed by Fourier transform infrared spectroscopy (FTIR) spectra and cation-exchange capacity measurement. The prepared membrane exhibited similar cross-section morphologies as the BPPO base membranes: water uptake, 22.3–40.1%; cation-exchange capacity, 0.53–2.31 mmol/g; mechanical stress, 0.18–5.89 MPa. Then, cellulase was covalently immobilized onto the membrane, and the optimum immobilization conditions were as follows: EDC ( N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide) concentration, 40 mg/mL; activation time, 20 h; immobilization time, 25 h; enzyme concentration, 5.0 mg/mL; pH3.8. As compared to free cellulase, the immobilized cellulase exhibited greater stability against heat and pH, suggesting that the membrane was suitable as a support for enzyme immobilization.

Nuclease p1 immobilized on deae cellulose

Effects of various factors, such as pH, ionic strength, glutaraldehyde concentration, enzyme amount and immobilization time, on enzyme activity were investigated. The immobilization conditions were optimized by orthogonal experiments. Characterizations of immobilized nuclease p1 were also evaluated. Through orthogonal optimization, the optimal immobilization conditions were as follows: pH 5.6, ionic strength 0.125, glutaraldehyde concentration 0.20% and immobilization time 2.0 h. Optimal pH of immobilized enzyme was 5.8. Optimal temperature of immobilized enzyme was 70oC. Thermal, operational and storage stabilities of the enzyme were improved after it was immobilized on DEAE cellulose. Michaelis constant Km of immobilized enzyme at 69oC was found to be 27.21 g/l by the Lineweaver-Burk plot.

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh). A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C E: 70 U mL −1; C Con A: 1.5 mg mL −1 and C MWCN: 11 mg mL −1, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM −1 cm −2. The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10 −12 g L −1 of Paraoxon, as well as the interval (10 −11 to 10 −8 g L −1) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.

Optimization of Polyphenol Oxidase Immobilization in Copper Alginate Beads

Polyphenol oxidase (PPO, EC 1.14.18.1) was isolated from artichoke head (Cynara scolymus L.) by using 0.1 M Tris-HCl buffer (pH 7.0), concentrated by (NH4)2SO4 precipitation, and immobilized in copper-alginate beads. Immobilization yield was determined to be 70%. The cresolase and catecholase activities of enzyme immobilized at optimum immobilization conditions were found to be 13.3 and 670 U g beads min(-1), respectively. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration, amount of loading enzyme, bead size, and amount of beads on enzymatic activity were investigated. Optimum alginate and CuCl2 concentration were found to be 2 % and 3 % (w/v), respectively. Using bead (diameter 3 mm) amount of 0.25 g maximum enzyme activities were observed for both polyphenol activities. The initial concentrations of loading free enzyme were 6.5 U mL(-1) and 5815 U mL(-1) for cresolase activity and catecholase activities, respectively. Beads prepared at optimum immobilization conditions were suitable for up to 8 repeated uses.

Study of Immobilization of Nuclease P1 on Paper Cellulose

ABSTRACTIn this study, immobilization of nuclease p1 on paper cellulose was studied. Effect of various factors, such as etherification pH, couple pH and time, (NH4)2SO4 concentration, enzyme amount et al, on the activity of immobilized enzyme was investigated. The immobilization conditions were optimized by orthogonal experiment. Characterizations of immobilized nuclease p1 were also evaluated. Through orthogonal experiment, the optimal immobilization conditions were as follows: couple pH 12, etherification pH 7.0, (NHJ4)2SO4 concentration 10% and enzyme amount 6.0 ml. Optimal pH of immobilized enzyme was 5.8. Optimal temperature of immobilized enzyme was 80°C. Thermal, operational and storage stabilities of the enzyme were improved after it was immobilized on paper cellulose. Michaelis constant Km of immobilized enzyme was disclosed as 5.72 g/l by the Lineweaver-Burk plot at 70°C.

Flow Injection Analysis for Amperometric Detection of Glucose with Immobilized Enzyme Reactor

ABSTRACTGOD was covalently immobilized on polymer/silica gel hybrid support prepared by coating high surface area of silica gel with modified acrylonitrile co-polymer. The relationship between immobilization factors and enzyme activity were examined by the series of contour plots. The selections of the immobilization variable range were extremely precise in the 3-level-3-factor fractional design. The results indicated that the optimal conditions for GOD immobilization were: 0.1% enzyme solution, immobilization temperature- 4°C and immobilization time- 24 h. Immobilized GOD was applied to amperometric determination of glucose using flow-injection analysis. The optimal flow rate was determined as 4.0 ml/min when injecting 100 μl sample volumes. The linear response range for the on-line detection of glucose using immobilized GOD in column minibioreactor was estimated to be from 0.01 mM to 20 mM (a correlation coefficient of 0.985). Moreover, its experimental detection limit is 10 μM (S/N=3) and the apparent Michaelis-Menten constant was calculated to be 20.15 mM. The proposed method for glucose measurement was validated in real samples of fruit juices.

A novel method for the continuous production of γ-aminobutyric acid using immobilized rice bran

A novel method for the continuous production of γ-aminobutyric acid (GABA) using immobilized rice bran was developed. Calcium alginate gel was used to entrap rice bran directly and orthogonal array design was used to obtain the optimal conditions for immobilization. Under these conditions, the preservation of glutamate decarboxylase (GAD) activity was 48.94%. The optimum temperature for immobilized rice bran GAD activity was found to be 45°C with the optimum pH at 5.5. The thermostability, pH stability, operation and storage stability of GAD were improved by immobilization on rice bran. Based on these characteristics, a column reactor of immobilized rice bran was applied to produce GABA continuously. The GABA produced in 60 h by this method was 88.11% of the GABA produced by the free rice bran method, while the protein and sugar contents of the product were greatly reduced. The high output and purity of the product permit its biotechnological potential to be exploited in food applications.

Immobilization of catalase onto Eupergit C and its characterization

Bovine liver catalase was covalently immobilized onto Eupergit C. Optimum conditions of immobilization: pH, buffer concentration, temperature, coupling time and initial catalase amount per gram of carrier were determined as 7.5, 1.0 M, 25 °C, 24 h and 4.0 mg/g, respectively. V max and K m were determined as 1.4(±0.2) × 10 5 U/mg protein and 28.6 ± 3.6 mM, respectively, for free catalase, and as 3.7(±0.4) × 10 3 U/mg protein and 95.9 ± 0.6 mM, respectively, for immobilized catalase. The thermal stability of the immobilized catalase in terms of half-life time (29.1 h) was comparably higher than that of the free catalase (9.0 h) at 40 °C. Comparison of storage stabilities showed that the free catalase completely lost its activity at the end of 11 days both at room temperature and 5 °C. However, immobilized catalase retained 68% of its initial activity when stored at room temperature and 79% of its initial activity when stored at 5 °C at the end of 28 days. The highest reuse number of immobilized catalase was 22 cycles of batch operation when 40 mg of immobilized catalase loaded into the reactor retaining about 50% of its original activity. In the plug flow type reactor, the longest operation time was found as 82 min at a substrate flow rate of 2.3 mL/min when the remaining activity of 40 mg immobilized catalase was about 50% of its original activity. The resulting immobilized catalase onto Eupergit C has good reusability, thermal stability and long-term storage stability.

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10 3, 98 × 10 3, 147 × 10 3 and 196 × 10 3 U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters ( K m and V max ) were found using Michaelis–Menten enzyme kinetics. The K m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.

9044 Curative surgery in oligometastatic non-small cell lung cancer patients

A novel, sensitive, disposable indium tin oxide (ITO)-based electrochemical immunosensor was developed firstly for simple, rapid determination of Sex-determining region Y-box 2 (SOX2). SOX2 is a cancer biomarker and used for detecting small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. In this study, a disposable ITO thin film based electrode was used as working electrode for biosensing the interaction between SOX2 antigen and anti-SOX2 antibody. In this study, carboxyethylsilanetriol (CTES) was also utilized for electrode modifying so as to obtain self-assembled monolayers. The formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemistry and they were used as a heterobifunctional crosslinker and activator, respectively. Anti-SOX2 antibody was used as a biorecognition molecule and was covalently immobilized onto the ITO thin film modified with CTES. Immobilization steps were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The optimum immobilization conditions such as antibody concentration, antibody and antigen incubation times were examined for the best sensitivity of the immunosensor. Under optimal conditions, this immunosensor had a wide linear detection range (25 fg/mL–2 pg/mL) with a detection limit as low as 7 fg/mL SOX2. Furthermore, the developed SOX2 immunosensor had good storage stability (79.36% of initial activity after 9 weeks), repeatability (3.88% of RSD) and reproducibility (4.25% of RSD). Our developed immunosensor has an acceptable performance for detection of SOX2 antigen, exhibits low detection limit, and has selective and reproducible results in immunoreaction analysis.

Optimum immobilization of urease on modified acrylonitrile copolymer membranes: Inactivation by heavy metal ions

Poly(acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to chemical modification with hydroxyl ammonium sulfate (NH 2OH·H 2SO 4) and the amount of functional amidoxime groups was determined in the modified membranes. Urease was covalently immobilized on the modified membranes. The relationship between immobilization factors and enzyme activity was examined by a series of contour plots. The selections of the immobilization variable range were extremely precise in the 3-level-3-factor fractional design. The results indicated that the optimal conditions for urease immobilization were: 0.1% enzyme solution, immobilization temperature – 4 °C and immobilization time – 20 h. The inhibitory effect (IC 50) of Cu(II), Cd(II), Zn(II), Ni(II) and Pb(II) was studied on free and immobilized urease. The behavior of the immobilized urease in model solutions, containing different mixtures of heavy metals was represented in a 3D model. By studying the inhibition effect of two different mixtures Cu(II) and Cd(II); Cu(II) and Zn(II) it was found that the linear range of urease inhibition by Cu(II) ions for the first model mixture was from 0 to 1 mg l −1 and by Cd(II) ions from 0 to 1.5 mg l −1, as for the second model mixture the linear range of urease inhibition by Cu(II) ions was from 0 to 1 mg l −1 and by Zn(II) ions from 0 to 3.0 mg l −1. This linear dependence is very important from a practical point of view, regarding the application of immobilized urease in the construction of a biosensor for the detection of low concentrations of heavy metal ions.

Immobilization of acetylcholinesterase on nanostructure polyacrylonitrile membranes

PAN membranes with integrated gold nanoparticles (GNPs) into membrane pores were prepared for immobilization of acetylcholinesterase. Two types of polymer membranes were used—non-modified and chemically modified with NaOH. PAN + GNP membranes were investigated with respect to their water-permeation and electron-conducting properties and the results were compared to those, obtained for the initial membranes without GNPs. The SEM analyses showed morphology change in the different membranes. The chemical modification was an essential step in the preparation procedure in order to obtain more homogeneous distribution of the GNPs. AChE was covalently immobilized onto nanostructured membranes using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride. The relationship between immobilization factors and enzyme activity were examined by the series of contour plots. The selections of the immobilization variable range were extremely precise in the 3-level-3-factor fractional design. The results indicated that the optimal conditions for AChE immobilization were: 0.025% enzyme solution; immobilization temperature, 4 °C and immobilization time, 15 h. The biochemical characteristics and kinetic parameters of immobilized enzyme were determined.

Biological sulfide oxidation using autotrophicThiobacillussp.: evaluation of different immobilization methods and bioreactors

Evaluation of various immobilization methods and bioreactors for sulfide oxidation using Thiobacillus sp. was studied. Ca-alginate, K-carrageenan and agar gel matrices (entrapment) and polyurethane foam and granular activated carbon (adsorption) efficacy was tested for the sulfide oxidation and biomass leakage using immobilized Thiobacillus sp. Maximum sulfide oxidation of 96% was achieved with alginate matrix followed by K-carrageenan (88%). Different parameters viz. alginate concentration (1%, 2%, 3%, 4% and 5%), CaCl(2) concentration (1%, 2%, 3%, 4% and 5%), bead diameter (1, 2, 3, 4 and 5 mm), and curing time (1, 3, 6, 12 and 18 h) were studied for optimal immobilization conditions. Repeated batch experiments were carried out to test reusability of Ca-alginate immobilized beads for sulfide oxidation in stirred tank reactor and fluidized bed reactor (FBR) at different sulfide concentrations. The results proved to be promising for sulfide oxidation using Ca-alginate gel matrix immobilized Thiobacillus sp. for better sulfide oxidation with less biomass leakage. Biological sulfide oxidation is gaining more importance because of its simple operation. Present investigations will help in successful design and operation of pilot and industrial level FBR for sulfide oxidation.

Preparation of magnetic chitosan nanoparticles and immobilization of laccase

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and characterizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows: 10 mL of phosphate buffer (0.1 M, pH 7.0) containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg · mL−1 of laccase and 1% (v/v) glutaraldehyde, immobilization temperature of 4°C and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability (480 units · g−1 support) and had good storage stability and operation stability. The Km of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer (0.1 M, pH 3.0) at 37 °C, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.