Activity Of Immobilized Enzyme Research Articles

Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films

Lactoperoxidase (LPS), purified directly from bovine rennet whey by Toyopearl-SP cation-exchange chromatography and lyophilized by using dextran as supporting material, maintained almost 70 and 60% of its activity after almost 2 and 5 months storage at −18 °C, respectively. Incorporation of the prepared LPS into alginate films between 0.08 and 0.69 mg/cm 2 (516–4325 U/cm 2) caused the immobilization of most of the enzyme and gave films with LPS activity between 0.05 and 2.8 U/cm 2, determined in the presence of 8 μM H 2O 2. Between 2 and 24 μM H 2O 2 concentrations, a two-fold increase in H 2O 2 concentration caused 1.5–2.5-fold increase in LPS activity of films incorporated with 0.24–0.28 mg/cm 2 (1200 U/cm 2) LPS. The Q 10 and E a of immobilized enzyme activity between 4 and 16 °C were 1.69 and 34.6 kJ/mol, respectively. However, in the 16–30 °C range, the temperature change had almost no effect on LPS activity of films. The optimal activity of immobilized LPS was observed at pH 6.0, but the enzyme maintained 30–85% of its activity between pH 3.0 and 7.0. The immobilized LPS also had a high stability between pH 4.0 and 6.0. The results of this study showed the good potential of LPS-incorporated alginate films in forming a natural antimicrobial mechanism in different foods.

SECM visualization of spatial variability of enzyme–polymer spots: 1. Discretisation and interference elimination using artificial neural networks

Enzyme–polymer layers immobilized on an electrode surface often serve as basis for amperometric biosensors. Caused by the formation process they show spatial variability in the polymer thickness which corresponds to a variability of immobilized enzyme activity. The relationship between topography and localized enzymatic activity of enzyme–polymer spots was studied using scanning electrochemical microscopy (SECM) in the feedback mode and generator-collector mode. Discretisation with a grid size corresponding to the scanning parameters defined substructures which can be treated as individual microsensors with specific response characteristics. The local responses are mainly governed by the polymer thickness but also influenced by neighbouring sites. Thus, discretisation allowed us to treat an enzyme–polymer spot with dimensions of about 300 μm diameter like an array of more than 400 individual microsensors. Using suitable selection criteria and multivariate calibration it was possible to identify sensing sites which are optimal for the determination of glucose. It was demonstrated that an artificial neural network which was trained with the data provided by SECM images well predicted glucose concentration in the presence of ascorbic acid.

Proteomic methods applied to the analysis of immobilized biocatalysts

Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.

Immobilization of Pancreatic Lipase on Polyvinyl Alcohol by Cyanuric Chloride

Porcine pancreatic lipase (EC 3.1.1.3) was covalently immobilized onto 2,4,6‐trichloro‐s‐triazine (cyanuric chloride) activated polyvinyl alcohol (PVA). The influence of activating agent and enzyme concentration on the immobilization process were evaluated. Hydrolytic activities of free and immobilized enzyme were determined and the immobilization yield was estimated by measuring the quantity of protein, both in free enzyme solution and in washing solutions after immobilization. After the optimization of immobilization process, the physical and chemical characterization of immobilized enzyme was performed. Additionally, the thermal, pH, storage, and operational stability of the immobilized and free enzymes were tested. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme.

Development of a high analytical performance-tyrosinase biosensor based on a composite graphite–Teflon electrode modified with gold nanoparticles

The design of a new tyrosinase biosensor with improved stability and sensitivity is reported. The biosensor design is based on the construction of a graphite-Teflon composite electrode matrix in which the enzyme and colloidal gold nanoparticles are incorporated by simple physical inclusion. Experimental variables such as the colloidal gold loading into the composite matrix, the enzyme loading and the potential applied to the bioelectrode were optimized. The Tyr-Au(coll)-graphite-Teflon biosensor exhibited suitable amperometric responses at -0.10 V for the different phenolic compounds tested (catechol; phenol; 3,4-dimethylphenol; 4-chloro-3-methylphenol; 4-chlorophenol; 4-chloro-2-methylphenol; 3-methylphenol and 4-methylphenol). The limits of detection obtained were 3 nM for catechol, 3.3 microM for 4-chloro-2-methylphenol, and approximately 20 nM for the rest of phenolic compounds. The presence of colloidal gold into the composite matrix gives rise to enhanced kinetics of both the enzyme reaction and the electrochemical reduction of the corresponding o-quinones at the electrode surface, thus allowing the achievement of a high sensitivity. The biosensor exhibited an excellent renewability by simple polishing, with a lifetime of at least 39 days without apparent loss of the immobilized enzyme activity. The usefulness of the biosensor for the analysis of real samples was evaluated by performing the estimation of the content of phenolic compounds in water samples of different characteristics.

Enzyme Inhibitor Screening by Capillary Electrophoresis with an on-Column Immobilized Enzyme Microreactor Created by an Ionic Binding Technique

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.

Bioorganically doped sol-gel materials containing amyloglucosidase activity

Amyloglucosidase (AMG) from Aspergillus niger was encapsulated in various matrices derived from tetraethoxysilane, methyltriethoxysilane, phenyltriethoxysilane and vinyltriacetoxysilane by different methods of immobilization. The immobilized enzyme was prepared by entrapment in two steps, in one-step and entrapment/deposition, respectively. The activities of the immobilized AMG were assayed and compared with that of the native enzyme. The effects of the organosilaneprecursors and their molar ratios, the immobilization method, the inorganic support (white ceramic, red ceramic, purolite, alumina, TiO2, celite, zeolite) and enzyme loading upon the immobilized enzyme activity were tested. The efficiency of the sol-gel biocomposites can be improved through combination of the fundamental immobilization techniques and selection of the precursors.

Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins

A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 microm x 125 microm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml −1, 1.2% (w/v or v/v) enzyme concentration and acetic–HCl/glycine–HCl buffer at pH 3.6 or tris–HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 °C. Thermal stability was not significantly altered by immobilization, especially at 40 °C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm 2 s −1, respectively, after 30 min at 40 °C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.

Polyacrylamide–gelatine carrier system used for invertase immobilization

Invertase was immobilized into polyacrylamide–gelatin carrier system by chemical cross-linking with chromium (III) acetate, chromium (III) sulphate, and potassium chromium (III) sulphate. The optimum conditions, namely substrate concentration, temperature, and pH were determined. The effect of polyacrylamide–gelatin ratio and cross-linker concentration on immobilized enzyme activity were analysed. Maximum immobilized enzyme activities were obtained with chromium (III) acetate (0.01 mol dm −3), chromium (III) sulphate (0.004 mol dm −3) and potassium chromium (III) sulphate (0.001 mol dm −3) for 0.177 (w/w) polyacrylamide–gelatin carrier ratio as 79%, 72% and 79%, respectively. The K m values were 86 and 166 mM for free and immobilized enzyme, respectively. All immobilized samples were used 20 times over a period of 2 months without a considerable loss of activity.

Cross-Correlation of Optical Microcavity Biosensor Response with Immobilized Enzyme Activity. Insights into Biosensor Sensitivity

Porous silicon multilayer structures have remarkable optical and morphological properties that can be exploited for biosensing. In particular, a high internal surface area (>100 m(2)/cm(3)) and a linear response profile to changes in the dielectric environment enable fabrication of sensitive devices and a straightforward quantitation of the optical response. These essential operating characteristics are illustrated for p+ mesoporous silicon (pore diameter 15-20 nm) optical microcavities. A series of devices were prepared to permit the immobilization of glutathione-S-transferase ( approximately 50 kDa) within the porous matrix. Enzyme activity was exploited as an indirect means to quantitate the amount of protein immobilized. Activity was positively correlated with the optical sensor response. However, at high enzyme load the activity becomes nonlinear while the microcavity response remains linear. These data were used to determine the transduction limit (minimum amount of protein required to transduce an optical response), which is reported as areal mass sensitivity ranging between 50 and 250 pg/mm(2). This value is considered in context with the dynamic range of the bulk sensitivity, defined as the magnitude of the wavelength shift per refractive index unit, which was measured as a function of microcavity design parameters. This work has uncovered key parameters that can be tuned to improve the detection limit of this sensor modality. Because of the ever increasing number of emerging new biosensor technologies, defining sensor detection limits has become an ambiguous topic and a need exists to standardize measurements and sensitivity units. For chip-based devices, it seems appropriate to report sensitivity in terms of the minimum number of grams of bound target per surface area.

Enzyme Immobilization in Porous Silicon: Quantitative Analysis of the Kinetic Parameters for Glutathione-S-transferases

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.

Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst

In this study, functionalization of large-pore nanoporous silica materials was carried out by condensation of tetraethylorthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), phenyltrimethoxysilane (PTMS), vinyltriethoxysilane (VTES), and 4-(triethoxysilyl)butyronitrile (TSBN), respectively, in the presence of non-ionic surfactant under acidic conditions. The TSBN functionality was subsequently converted to carboxyl group while APTES was further functionalized with glutardialdehyde, a cross linker. The various functionalized materials were used as supports for immobilization of enzyme penicillin G acylase (PGA). Experimental data showed that the functionalized materials except for the material functionalized with MPTMS possess a faster loading kinetics and a higher loading amount of enzyme PGA than the pure-silica counterpart. The enzymatic catalytic activities of the immobilized biocatalysts varied from 52.2 to 167.5 U/g of solid. The glutardialdehyde-activated material displayed the highest initial immobilized enzyme activity and the most stable activity among all the support materials. PGA immobilized on VTES-functionalized nanoporous silica showed the highest initial enzymatic activity (67.7 U/mg of PGA, much higher than that of free PGA (300 U/mg of PGA). Experimental data along with theoretical analysis results indicate that glutardialdehyde is a good cross linker, offering covalent binding of PGA with the support materials while VTES-functionalized nanoporous silica is a very good potential support for physical entrapment of PGA enzyme.

Preparation and characterization of magnetic duolite–polystyrene composite particles for enzyme immobilization

Duolite A568, which is a good support for enzyme immobilization was processed with magnetite and polystyrene by a solvent evaporation method. The most appropriate polyvinyl alcohol and sodium dodecyl sulphate contents of dispersion medium and process time were determined as 1 g/300 mL, 0.5 g/300 mL and 1–2 h respectively, to obtain maximum immobilized enzyme activity. These magnetic duolite–polystyrene particles were characterized as high spin complex with the g factor of 3.5 and as ferromagnetic. Pectinex Ultra SP-L was used as a model enzyme and it is found that 0.41 μL of enzyme was immobilized on 1 g of particles.

Enzyme immobilization in novel alginate–chitosan core-shell microcapsules

Alginate–chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, β-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl- β- d-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000 Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate–chitosan microcapsules as compared to barium alginate–chitosan microcapsules. Barium alginate–chitosan microcapsules, however, did improve the stability of the enzyme at 37°C relative to calcium alginate–chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

Covalent immobilization of invertase on microporous pHEMA–GMA membrane

Poly (2-hydroxyethyl methacrylate–glycidyl methacrylate) (pHEMA–GMA) membrane was prepared by UV-initiated photopolymerization. Invertase was immobilized by the condensation reaction of the epoxy groups of glycidyl methacrylate in the membrane structure with amino groups of the enzyme. The K m values were 22 mM and 58 mM for free and immobilized enzyme, respectively. Immobilization improved the pH stability and temperature stability of the enzyme. Thermal stability was found to increase with immobilization. The half times for the activity decay at 70 °C were found to be 11 and 38 min for the free and immobilized enzyme, respectively. The immobilized enzyme activity was found to be quite stable in later experiments.

Lactose hydrolysis by immobilized β-galactosidase: the effect of the supports and the kinetics

The kinetic behaviour of β-galactosidase from Kluyveromices marxianus ( Saccharomyces) lactis, immobilized on different oxides supports, such as alumina, silica, and silicated alumina has been studied. We observed a strong dependence of the immobilized enzyme activities on the chemical nature and physical structure of the supports. In particular, when the particle sizes of the supports are increased, the enzymatic activity strongly decreases. The hydrolysis of lactose, promoted by the mentioned enzyme immobilized on small commercial silica spheres, has been studied in different operative conditions, by changing: feed rate, reagent and products concentration and temperature, while pH has been kept constant (7.0). A depressing effect originated by both the reaction products, has been observed. Therefore, a kinetic model based on the Michaelis–Menten mechanism, in which the depressing effects of both the hydrolysis products (galactose and glucose) are also considered, have been developed and the related parameters determined.

Determination of Kinetic Constants and Enzyme Activity from a Biosensor Transient Signal

The kinetic constants and activity of soluble and immobilized enzymes can be determined electrochemically with a biosensor by applying the dynamic integrated model, which includes the inertia of the registering device, for data management and calculations. This model of electrochemical diffusion-limited sensors allows the prediction of steady-state parameters for enzyme-catalyzed reactions from a biosensor transient response and reproducible calculation of real physico-chemical constants without any additional experimental data. This approach has been used for the estimation of activity parameters of soluble and immobilized on mica glucose oxidase and for the evaluation of immobilization efficiency.

Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports.

A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 micromol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8 U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 micromol of H2O2 reduced/(h x Uimmob HRP).

Poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) reactive membrane utilised for cholesterol oxidase immobilisation

AbstractPoly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm2of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated aski (free)1.06 × 10−1min−1andki (imm)2.68 × 10−2min−1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments.© 2002 Society of Chemical Industry