An Amperometric Biosensor Based on Laccase Immobilized in Polymer Matrices for Determining Phenolic Compounds

An amperometric enzyme electrode based on laccase for determining phenolic compounds is proposed. The following three types of polymer materials were used for enzyme immobilization on the surface of a glassy-carbon electrode: positively charged cetyl ethyl poly(ethyleneimine) (CEPEI) and negatively charged commercial Nafion and Eastman AQ 29D polymers. The advantages and disadvantages of each of the above polymers for enzyme immobilization are discussed. The detection limits of the model phenolic compounds hydroquinone and pyrocatechol in a buffer solution on laccase immobilization in a Nafion membrane were 3.5 × 10⁻⁸ and 5.0 × 10⁻⁸ M, respectively, at a signal-to-noise ratio of 3. Electrodes with laccase immobilized in Nafion and Eastman AQ 29D membranes exhibited the shortest response time. The operating stability and the stability in storage can be significantly improved by the additional incorporation of gelatin in the polymer matrices. Gelatin prevents enzyme inactivation as a result of enzyme modification by the free-radical oxidation products of phenolic compounds.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 624–628.Original Russian Text Copyright © 2005 by Yaropolov, Shleev, Morozova, Zaitseva, Marko-Varga, Emneus, Gorton.Presented at the VI All-Russia Conference (with international participation) on Electrochemical Methods of Analysis (EMA-2004, Ufa, May 23–27, 2004).     

Kinetic study of catalase adsorption on disperse carbonaceous matrices

The effect of the main factors known to govern the kinetic regularities of enzyme adsorption, such as enzyme solution concentration, temperature, pH, specific surface of the adsorbent, etc., were studied. Two kinds of disperse carbonaceous materials-activated carbon NORIT and carbon black PM-100, were used as matrices for enzyme immobilization. For both immobilization matrices studied, the amount of the adsorbed enzyme was found to reach saturation at catalase (CAT) enzyme concentrations exceeding 20 mg·mL⁻¹ (∼100 μM). The pH of the solution affected the adsorption capacities of the selected immobilization matrices; larger amounts of CAT adsorbed were estimated in neutral and alkaline solutions than under acidic conditions for enzyme immobilization. UV-spectrophotometry was employed as a basic analytical approach in this study.     

Tobacco Peroxidase as a New Reagent for Amperometric Biosensors

The results of testing a new enzyme, anionic tobacco peroxidase (TOP), in various amperometric biosensors are summarized. The biochemical and electrochemical properties of the enzyme are briefly characterized. As compared to the commonly used cationic peroxidase from horseradish roots, TOP exhibits a wider optimum stability pH range, higher stability to inactivation with hydrogen peroxide, and higher efficiency in direct electron-transfer processes. The enzyme immobilized by adsorption on graphite is effective in determining aminophenols and aromatic diamines under flow conditions with a detection limit of 10 nM. Upon immobilization on graphite by incorporation into a gel of a redox-active polymer (crosslinked polyvinylimidazole with osmium 4,4′-dimethylbipyridinium chloride), TOP exhibited sensitivity and stability comparable to those of horseradish peroxidase and a wider linearity range. Upon immobilization on a self-assembled thiol monolayer at a gold electrode, TOP was much superior to horseradish peroxidase in the sensitivity of determining hydrogen peroxide, regardless of the charge of the monolayer. Prospects for the further use of the native enzyme and its genetically engineered unglycosylated form are considered.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 629–638.Original Russian Text Copyright © 2005 by Gazaryan, Gorton, Ruzgas, Csoregi, Schuhmann, Lagrimini, Khushpul’yan, Tishkov.Presented at the VI All-Russia Conference (with international participation) on Electrochemical Methods of Analysis (EMA-2004, Ufa, May 23–27, 2004).     

Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides: Comparative study

A comparison between several acetylcholinesterase (AChE) immobilization procedures on the 7,7,8,8-tetracyanoquinodimethane (TCNQ)-modified graphite working electrodes is presented. The immobilization methods employed crosslinking with glutaraldehyde in presence of BSA protein and photopolymerization with poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). The main variations were related to the enzyme charge in each electrode and the enzyme conditioning and storage conditions after immobilization. Initially, the enzyme-substrate reaction was carried out and the following parameters were chrono-amperometrically and -coulometrically monitored: current intensities, time to stabilize the current response, and the mass transfer represented by the Coulomb charge. The screen-printed biosensors that presented best characteristics were then used to perform the inhibition assays and to verify the sensitivity against the following NMC insecticides: aldicarb, carbaryl, carbofuran, and methomyl.In general, diffusion of electrons into the sensitive layer, mass transfer, and time to stabilize the current were adequate in all cases. The Cottrell law was followed before the 1 min of enzyme-substrate reaction. Adequate reproducibility within electrochemical measurements was also observed, with relative standard deviations varying from 6.5 to 18.6%.AChE immobilization with glutaraldehyde allow to obtain robust and reproducible biosensors, but they need a much higher enzyme content (80 mUA per electrode) to achieve current values comparable to that constructed by immobilizing the AChE through photopolymerization with PVA-SbQ (0.7 to 1 mUA per electrode). The limits of detection were determined with a minimum 10% inhibition, and varied from 10⁻⁹ to 8×10⁻⁹ M (0.2 to 1.5 ppb) by employing the enzyme immobilization through photopolymerization with PVA-SbQ. In practice, this kind of immobilization procedure is much simpler and produces good results: fast response, adequate reproducibility, large pesticides working ranges, and excellent sensitivities to N-methylcarbamates (NMCs) which in general do not present enzyme inhibition power as elevated as for the organophosphate pesticides.     

Phospholipase C-catalyzed sphingomyelin hydrolysis in a membrane reactor for ceramide production

A membrane reactor for the production of ceramide through sphingomyelin hydrolysis with phospholipase C from Clostridium perfringens was studied for the first time. Ceramide has raised a large interest as an active component in both pharmaceutical and cosmetic industry. The enzymatic hydrolysis of sphingomyelin has been proven to be a feasible method to produce ceramide. In the membrane reactor constructed, the aqueous phase and the organic phase were separated by a membrane containing the immobilized enzyme, while the organic phase was continuously circulated. Among the 10 selected membranes, the enzyme immobilized in membrane RC 70PP had low immobilization efficiency, but retained the highest catalytic activity. Three immobilization methods, i.e. filtration (adsorption/entrapment), covalent binding, and cross-linking, were compared. The enzyme immobilized by filtration had the highest activity even under the low fixation level (9.4%). The optimal flow rate of the organic phase was 5 ml/min. High initial enzyme amount in the immobilization led to the decrease in the fixation level. Both the initial reaction rate and the specific activity of the enzyme increased with increasing enzyme loading, and slightly decreased after the immobilized enzyme amount over 50 μg in 9.6 cm² membrane area. The immobilized enzyme retained 16% of the original activity after five cycles. Finally, the liquid enzyme, the enzyme immobilized on particle carriers, and the enzyme immobilized in the membrane were compared. The study demonstrated the improved enzyme reusability, the fast immobilization process, the straightforward up-scaling and the combination of the hydrolysis with the product separation in the membrane reactor developed.     

Preparation of a very stable immobilized Solanum tuberosum epoxide hydrolase

The covalent immobilization of the Solanum tuberosum epoxide hydrolase (StEH) was explored using highly activated Sepabeads-epoxy or Glyoxyl-agarose based supports. A Glyoxyl-agarose immobilizate, prepared under optimized experimental conditions, led to a material exhibiting excellent thermal and chemical stability. The key step of this immobilization process was the use of 164 kDa dextran as an additive during immobilization, which prevented the enzyme from inactivation at the high pH (pH 10) necessarily used for performing this immobilization. This afforded a Glyoxyl-agarose-StEH immobilizate with 80% initial enzymatic activity retention and a stabilization factor of at least 300 at 60 °C, as compared to the free enzyme. The high enantio- and regio-selectivity properties of this novel biocatalyst were shown to be nearly identical to those of the free enzyme.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone Activated Alginate Beads: I. Beads Preparation and Characterization

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.     

Immobilization of isolated and cellular hydrogenase ofD. desulfuricans in radiation polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.     

Surface functionalization of oxidized multi-walled carbon nanotubes: Candida rugosa lipase immobilization

This work examines two approaches for immobilization of lipase from Candida rugosa on oxidized multi-walled carbon nanotubes (o-MWCNTs). One method included the presence of activating agents to promote covalent bonding and the other the adsorption on o-MWCNTs to elucidate if non-specific bonding on the o-MWCNTs surface exists. The influence of the immobilization time and initial enzyme concentration on protein loading and the expressed lypolitic activity of the immobilized preparation were investigated. The results showed that the enzyme adsorbs on o-MWCNTs in a maximal amount of 37 μg mg⁻¹ CNTs, while the attached amount was more than 2-times higher under covalent promoting conditions (80 μg mg⁻¹ CNTs). Furthermore, similar trends were observed for the lypolitic activity, whereby preparations obtained under covalent promoting conditions had almost 3-times higher activity (560 IU g⁻¹ of immobilized enzyme). In addition, immobilization of the enzyme was confirmed by Fourier transformation infrared spectroscopy and thermogravimetric analysis.     

Influence of Silica-Derived Nano-Supporters on Cellobiase After Immobilization

Core shell magnetite nanoparticle (CSMN) was successfully synthesized with diameter around 125 nm according to the determination with scanning electronic microscopy. SBA-15 with diameter around 31 nm was synthesized in our previous work as another supporter for immobilized degradation enzymes. The aim of this study was to investigate the influence of silica-derived nano-supporters on cellobiase after immobilization. With covalent method, glutaraldehyde was introduced to immobilize cellobiase. The immobilized enzyme efficiency, specific activity, and its characterization, including optimum pH, pH stability, optimum temperature for enzyme reaction, and enzyme thermal stability were investigated. Results show that the method of enzyme immobilization on both nano-supporters could improve cellobiase stability under low pH and high temperature conditions compared with the free enzyme. In the aspect of immobilization efficiency, SBA had higher amount of bounded protein than that of CSMN, but had lower specific enzyme activity than CSMN, assumably due to the change in silica surface properties caused by process of supporter synthesis.     

Cell Disruption Optimization and Covalent Immobilization of β-D-Galactosidase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YW-1 for Lactose Hydrolysis in Milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Improvement in the Thermostability of a Recombinant β-Glucosidase Immobilized in Zeolite under Different Conditions

β-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant β-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant β-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower K_M value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.     

Immobilization of Fungal β-Glucosidase on Silica Gel and Kaolin Carriers

β-Glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal β-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 °C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20–25% loss.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe₃O₄@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

     

Development of a new biosensor for superoxide radicals

A superoxide dismutase (SOD) biosensor for determination of superoxide radicals has been developed by immobilization of superoxide dismutase within gelatin (G) on a Pt electrode surface. The properties of the biosensor have been investigated and optimum conditions–enzyme concentration, glutaraldehyde concentration, and pH–were determined. The response of the G-SOD biosensor was proportional to concentration and the detection limit was 0.01 mmol L⁻¹ at a signal-to-noise ratio of 3. The biosensor retained 89% and 60% of its sensitivity after use for three and four weeks, respectively. Immobilization of SOD on gelatin provides a biocompatible microenvironment around the enzyme and stabilizes the activity of the enzyme very efficiently. The superoxide dismutase biosensor was used to determine the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Studies of Penicillin G Acylase Immobilization Using Highly Porous Cellulose-Based Polymeric Membrane

The different ionic molecules/compounds were used as a ligand for the immobilization of penicillin G acylase on the highly porous cellulose-based polymeric membrane having buffer flux 1,746 LMH (L m⁻² h⁻¹) at 0.5 bar pressure. The immobilized enzyme activity around 250 U_App was obtained with the ligand such as proline, tryptophan, casein acid hydrolysate, and brilliant green. Comparatively, proline showed less IMY% (percentage immobilization yield—58) but higher RTA% (percentage of activity retention—71) and specific activity (145 U_App g⁻¹). However, the crosslinked preparation of brilliant green obtained using glutaraldehyde showed 82 ± 2.7% immobilized enzyme activity after the completion of successive five cycles. In comparison with the free enzyme, the enzyme immobilized on the brilliant green coupled membrane showed around 2.4-fold increase in K _m value (47.4 mM) as well as similar optimum pH (7.2) and temperature (40 °C). The immobilized enzyme retained almost 50% activity after 107 days and 50 cycles of operation. Almost 50% decrease in buffer flux after enzyme immobilization was observed. At the end of the 30 cycles, flux pattern shows around 38% decrease in buffer flux however, after 16 cycles of operation flux moves closer towards the steady state.     

Cellulose filter paper immobilized α-glucosidase and its application to screening inhibitors from traditional Chinese medicine

In this study, α-glucosidase was successfully immobilized on cellulose filter paper and further applied to screening inhibitors from traditional Chinese medicines combined with capillary electrophoresis analysis. For α-glucosidase immobilization, a cellulose filter paper was used as the carrier and grafted with amino groups by coating chitosan, then α-glucosidase was covalently bonded on the amino-modified carrier via epoxy ring-opening reaction using polyethylene glycol diglycidyl ether as the crosslinker. Several parameters influencing the enzyme immobilization were optimized and the optimal values were enzyme concentration of 4 U/mL, polyethylene glycol diglycidyl ether concentration of 1.25%, chitosan concentration of 7.5 mg/mL, immobilization pH 7.0, crosslinking time of 4 h and immobilization time of 2 h. The immobilized α-glucosidase exhibited good batch-to-batch reproducibility (RSD = 2.1%, n = 5), excellent storage stability (73.5% of its initial activity after being stored at 4°C for 15 days), and reusability (75% of its initial activity after 10 repeated cycles). The Michaelis constant of immobilized α-glucosidase and half-maximal inhibitory concentration of acarbose were calculated to be 1.12 mM and 0.38 μM, respectively. Finally, the immobilized α-glucosidase was used for screening inhibitors from 14 kinds of Traditional Chinese Medicine extracts, and Sanguisorbae Radix showed the strongest inhibitory effect on α-glucosidase.     

Immobilization of Cyclodextringlycosyltransferase (CGTase) from Bacillus firmus in Commercial Chitosan

The enzyme cyclodextringlycosyltransferase (CGTase) was immobilized in commercial chitosan with different methods of immobilization at different temperatures, with the aim of obtaining a product with improved activity and higher recovery of the free enzyme activity. Three immobilization methods were tested: adsorption, covalent bonding with -aminopropyltriethoxysilane (CB-APTS), and covalent bonding with hexamethylenediamine (BC-HEMDA). Two test conditions were used, 7 °C without agitation and 26 °C with stirring. The best results were obtained with the method that uses HEMDA as the bifunctional covalent binding agent, giving the highest immobilized enzyme specific activity, 0.263 mol -CD/min mg of protein, and highest enzyme activity recovery, 5.2%, when immobilization was carried out at 7 °C.