Activity Of Immobilized Enzyme Research Articles

Characterization and manipulation of microscopic biochemically active regions by scanning electrochemical microscopy (SECM).

Preparation and characterization of microscopic biochemically active regions are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. Scanning electrochemical microscopy (SECM) has emerged as an ideal tool for prototyping such systems. The technique is based on electrochemical conversions of dissolved species at a micrometer-sized probe electrode. It offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activities. The working modes of the techniques are illustrated by recent studies from this laboratory for the design and characterization of patterned enzyme layers covalently linked to gold surfaces via thiol self-assembly chemistry.

Prediction of continuous reactors performance based on batch reactor deactivation kinetics data of immobilized lipase

Experiments on deactivation kinetics of immobilized lipase enzyme fromCandida cylindracea were performed in stirred batch reactor using rice bran oil as the substrate and temperature as the deactivation parameter. The data were fitted in first order deactivation model. The effect of temperature on deactivation rate was represented by Arrhenius equation. Theoretical equations were developed based on pseudo-steady state approximation and Michaelis-Menten rate expression to predict the time course of conversion due to enzyme deactivation and apparent half-life of the immobilized enzyme activity in PFR and CSTR under constant feed rate policy for no diffusion limitation and diffusion limitation of first order. Stability of enzyme in these continuous reactors was predicted and factors affecting the stability were analyzed.

Calorimetric determination of the enzyme activity immobilised on macroporous glass membranes

A direct method for the determination of the catalytic activity of immobilised enzymes by use of a miniaturised calorimeter is presented. The possibilities and advantages of the calorimetric method are demonstrated by investigations with catalase immobilised on macroporous glass membranes as model enzyme. The investigated enzyme carrier systems are characterised by a high catalytic activity, a marked long-term stability and by the possibility for the regeneration of the activity by repeated immobilisation.

Immobilization of Aspergillus oryzae β-galactosidase on tosylated cotton cloth

Immobilization of Aspergillus oryzae β-galactosidase on cotton cloth activated with p-toluenesulfonyl chloride (tosyl chloride) was studied. Enzyme immobilization on the tosylated cotton followed a nucleophilic substitution mechanism as evidenced by UV spectra. NaOH mercerization before tosylation and adding pyridine at a tosyl (g)/pyridine (ml) ratio of 0.2–0.4 increased the final activity of the immobilized enzyme by six-fold. The optimal pH for enzyme immobilization was found to be 4.5. Among different fibrous matrices tested, the knitted cotton cloth showed the highest immobilized enzyme activity. About 50 mg enzyme was immobilized onto each gram of cotton cloth with a protein coupling efficiency of ∼85% and enzyme activity yield of ∼55%. The immobilized enzyme had half-life of ∼50 days at 50 °C, and more than 1 year at 40 °C, an improvement of 25–28-fold as compared to free enzyme. The enzyme immobilized on cotton cloth can be used in a plug-flow reactor for continuous production of galacto-oligosaccharides (GOS) from lactose. The reactor performance was stable, and there was no loss in enzyme activity at 40 °C for the 15-day period studied. The immobilized enzyme also showed same pattern and level of GOS formation from lactose as those from free enzyme reaction, indicating that the reaction kinetics was not affected by immobilization and there was no significant diffusion limitation in the immobilized enzyme reactor. The method to prepare tosylated cotton cloth for enzyme immobilization is simple, inexpensive, and scaleable for industrial applications. Thus, tosylated cotton cloth can be used as a low cost support for highly active and stable biocatalyst.

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles

Magnetic bead cellulose particles and magnetic poly(HEMA–co–EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA–co–EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg 2+, Ca 2+, Mn 2+ and Co 2+) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co 2+ ions.

Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix

In this study, a hydrophobic group containing monomer, 2-methacrylamidophenyalanine (MAPA) was prepared by using methacrylochloride and phenylalanine. Then, poly(2-hydroxyethyl methacrylate-co-methacrylamido-phenlyalanine) (pHEMA-MAPA) membranes were prepared by UV-initiated photopolymerization of HEMA and MAPA in the presence of an initiator α-α ′-azobisisobutyronitrile (AIBN). The lipase was immobilized onto these membranes by covalent bonding through carbodiimide activation. The amount of enzyme loading on the membranes was increased as the MAPA ratio increased in the membrane structure. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 60 ° C the thermal stability constants were 1.1×10 −1min for free enzyme and 1.2×10 −2min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments.

Kinetic behaviour of α-galactosidase produced by Absidia griseola: a comparison between free and immobilized forms of the enzyme

In this research the characteristics of free (partially purified) and immobilized (mould pellets of Absidia griseola) α-galactosidase have been investigated. Inhibition studies of the enzyme showed that p-nitrophenol and sucrose do not have any inhibitory effect on the enzyme, but that galactose is a competitive inhibitor. In the immobilized form, inhibition was lower than in the free enzyme and the level of inhibition decreased as the temperature increased. The activity and stability of free and immobilized enzyme were investigated with respect to temperature, and the results showed that the optimal temperature range of the free enzyme was 45–50 °C, while the immobilized enzyme had an optimum at 55–60 °C. The optimum pH for the free enzyme was 6.0 and the value was decreased to 5.0 by immobilizing. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including parameters such as pellet size, enzyme concentration in the pellets and substrate concentration. Both experimental and theoretical data concerning effectiveness factors are nearly the same.

Properties and Immobilization of Urease from Leaves of Chenopodium Album (C3)

In Chenopodium album, leaf excision and light both increase urease (EC. 3.5.1.5) activity. Dithiothreitol (DTT), reduced glutathione (GSH), cystcine and diazoinedicarboxylic acid bis(N, N-dimcthylamide) (diamide) activated the crude enzyme. In contrast, crude urease was inhibited by phenylmethylsuiphonyl fluoride (PMSF) and N- a-p -tosyl-L-lysine chloromethyl ketone HC1 (TLCK), suggesting the presence of serine and histidine residues in the active site. The enzyme is Ca dependent for its activity and exogenous calmodulin (CaM) did not stimulate it. However the enzyme is strongly inhibited by CaM antagonist fluphenazine, indicating the presence of a Ca-like domain. EGTA, LaC13 and tetraacetic acid, 3,4,5,-trimethoxybenzoic acid 8-(diethyl-amino)-octyl ester (TMB-8) inhibited urease activity in vivo, and the inhibition was restored by exogenous Ca. Urease was immobilized in gelatin by covalent cross-linking with formaldehyde as organic hardener. The results indicated enhanced resistance to thermal denaturation, increased temperature optima (from 30CC to 40CC), and a rapid rate of substrate saturation were achieved after immobilization. The free urease showed remarkable loss of activity in the presence of sodium dodecyl sulphate, sodium deoxycholate, sodium taurocholate, Triton X-100, and Tween 80. The free enzyme lost 68%, 75% and 81% of its activity in the presence of 5,Y-dithiobis-(2-nitrobenzoic acid) (NBS2), p-hydroxymercuribenzoate (PHMB) and phenylmercuric acetate (PMA) as thiol reagents. However, the immobilized enzyme was not affected significantly by these compounds. By increasing the incubation time, the activity of immobilized enzyme decreased faster than that of the free one.

Preparation and application of immobilized C60-glucose oxidase enzyme in fullerene C60-coated piezoelectric quartz crystal glucose sensor

Immobilized fullerene C60-glucose oxidase enzyme was prepared and applied in a C60-coated piezoelectric (PZ) quartz crystal glucose sensor. The activity of the immobilized C60-glucose oxidase enzyme was investigated with an oxygen electrode detector. An optimum pH at 7.0 and optimum temperature at 30°C for activity of the insoluble immobilized enzyme were found. The useful life of the immobilized C60-glucose oxidase enzyme was sufficient with an activity of 88% after 93 days. The fullerene C60-coated PZ quartz crystal membrane glucose sensor based on immobilized C60-glucose oxidase enzyme with a home-made computer interface for signal acquisition and data processing was prepared. The fullerene C60-coated crystal sensor was employed to detect gluconic acid, a product of glucose oxidation by immobilized C60-glucose oxidase coated on porous silica plates in glucose aqueous solutions. The oscillating frequency of the quartz crystal decreased due to the adsorption of gluconic acid on fullerene C60. The immobilized enzyme PZ glucose sensor exhibited a linear frequency response to the log concentration of glucose with a slope of 5.9×102Hz per decade (ΔHz/Δlog M) and a good lower limit of linearity (LLL) of approximately 3.9×10−5M for glucose in aqueous solutions. The interference of various common species in the human blood, e.g. urea, creatine, galactose, cysteine, tyrosine, ascorbic acid and metal ions, to the PZ glucose sensor was also investigated. These species showed nearly no interference with the PZ glucose sensor. Effects of coating load of C60 and amount of immobilized enzyme on the frequency response of the C60-coated PZ glucose immobilized enzyme sensor were also studied and discussed.

Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy.

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements.

Effects of copper on the activity and kinetics of free and immobilized acid phosphatase

Heavy metal pollution presents a major hazard to the soil environment. Studies have shown that the activities of a variety of soil enzymes are inhibited by heavy metals. However, little information is available concerning the effect of heavy metals on the activity of enzymes immobilized by different soil constituents. The main objective of this work was to investigate the effects of copper on the activity and kinetic properties of acid phosphatase both free and immobilized on two variable-charge soil clays and the minerals kaolin, goethite and manganese oxide. The effect of different forms of copper on enzyme activity was also examined. In the presence of copper chloride, the activity of free and immobilized enzymes was inhibited at copper concentrations of 0.005–0.8 mM at pH 5.0 and inhibition increased at pH 6.0. The inhibitory effect of copper chloride was greater on the enzymes bound by the two soil clays and kaolin than those by goethite and MnO 2. Addition of copper chloride decreased both the K m values and the V max/ K m ratios of free and all forms of immobilized enzymes, and showed mixed type inhibition kinetics. Comparing the effect of different forms of Cu, the residual activities of free enzyme and soil clay–enzyme and kaolin–enzyme complexes were higher when copper citrate was used than with copper chloride. The reverse was true for the enzymes immobilized on goethite and MnO 2. These results indicate that the inhibition by Cu of enzymes immobilized on soil components are influenced by the properties of the adsorbent and the form of Cu, as well as pH.

In situ entrapment of α‐chymotrypsin in the network of acrylamide and 2‐hydroxyethyl methacrylate copolymers

A mild and reproducible method has been developed for the entrapment of α-chymotrypsin into a crosslinked copolymer. A porous copolymer was synthesized at 293 K by solution copolymerization of acrylamide and 2-hydroxyethyl methacrylate. α-Chymotrypsin was entrapped during copolymerization at different polymerization stages. The effect of crosslinking on enzyme loading and retention of its activity was examined. Copolymer with 2% crosslinking could entrap >90% of the enzyme. The activity of free and immobilized α-chymotrypsin was determined by using N-benzoyl-L-tyrosine ethyl ester and casein as low and high molecular weight substrates respectively. Storage as well as thermal stability of the immobilized enzyme was superior to that of the free one. Effect of calcium and heavy metal ions was studied on immobilized enzyme activity. The immobilized enzyme showed little variation in activity with pH and retained 50% activity after nine cycles. The Michaelis constant Km of the free and immobilized enzyme was estimated to be 2.7 and 4.2 × 10−3 mM, respectively, indicating no conformational changes during entrapment. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2996–3002, 2000

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 1. Isothermal conditions

The catalytic behaviour under isothermal conditions of two different membranes loaded with β-galactosidase was investigated. One membrane (M 1) was constituted by a nylon sheet grafted with methylmethacrylate by means of chemical grafting. The other, (M 2), was prepared by a double chemical grafting: the first one with styrene (Sty) and the second one with methylmethacrylate. Membrane activity was characterized as a function of temperature, pH and substrate concentration. The role of Sty in increasing membrane hydrophobicity has been discussed. Membrane M 2 was found to be better suited for employment in non-isothermal bioreactors.

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 2: Non-isothermal conditions

Lactose hydrolysis by β-galactosidase immobilized on two nylon membranes, differently grafted, has been studied in a bioreactor operating under isothermal and non-isothermal conditions. One membrane (M 1) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M 2) by a double chemical grafting: styrene (Sty) and MAA. Hexamethylenediamine was used as a spacer between the grafted membranes and the enzyme. Both membranes have been physically characterized studying their permeabilities in presence of pressure or temperature gradients. Under non-isothermal conditions, the increase in activity of membrane M 2 was higher than that of membrane M 1. The α and β coefficients, giving the percentage of activity increase when a temperature difference of 1°C is applied across the catalytic membranes, have been calculated. Results have been discussed with reference to the greater hydrophobicity of membrane M 2 with respect to membrane M 1, the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.

Invertase immobilized on spacer-arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

Microporous poly(2-hydroxyethyl methacrylate) (pHEMA) membrane was prepared by UV-initiated photopolymerization. The spacer arm (i.e., hexamethylene diamine) was attached covalently and then invertase was immobilized by the condensation reaction of the amino groups of the spacer arm with carboxyl groups of the enzyme in the presence of carbodiimides. The values of the Michael's constant Km of invertase were significantly larger (ca. 2.5 times) upon immobilization, indicating decreased affinity by the enzyme for its substrate, whereas Vmax was smaller for the immobilized invertase. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 70°C the half times for the activity decay were 12 min for the free enzyme and 41 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1685–1692, 2000

In situ entrapment of ?-chymotrypsin in the network of acrylamide and 2-hydroxyethyl methacrylate copolymers

A mild and reproducible method has been developed for the entrapment of α-chymotrypsin into a crosslinked copolymer. A porous copolymer was synthesized at 293 K by solution copolymerization of acrylamide and 2-hydroxyethyl methacrylate. α-Chymotrypsin was entrapped during copolymerization at different polymerization stages. The effect of crosslinking on enzyme loading and retention of its activity was examined. Copolymer with 2% crosslinking could entrap >90% of the enzyme. The activity of free and immobilized α-chymotrypsin was determined by using N-benzoyl-L-tyrosine ethyl ester and casein as low and high molecular weight substrates respectively. Storage as well as thermal stability of the immobilized enzyme was superior to that of the free one. Effect of calcium and heavy metal ions was studied on immobilized enzyme activity. The immobilized enzyme showed little variation in activity with pH and retained 50% activity after nine cycles. The Michaelis constant Km of the free and immobilized enzyme was estimated to be 2.7 and 4.2 × 10−3 mM, respectively, indicating no conformational changes during entrapment. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2996–3002, 2000

Development of sensors for direct detection of organophosphates. Part I: immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (<1 mm 2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol–gel process to create highly porous and stable silica matrices. Surface porosity of sol–gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol–gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-γ-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol–gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.

Practical enzymology course based on bioluminescence.

We describe our experience with laboratory courses in enzymology based on the phenomenon of bioluminescence. The soluble and immobilized enzymes of luminous bacteria are used and the practical enzymological course consists of four main courses: (1) training in measuring the activities of soluble and immobilized enzymes; (2) the investigation of kinetic characteristics (kinetic constants) and enzyme-substrate and enzyme-inhibitor interactions in the bacterial bioluminescent reaction; (3) The testing of physico-chemical characteristics of enzymes (pH, temperature, ion strength, etc.); (4) the effect of inhibitors on enzymes. Training is possible in groups of about ten persons. Our practice work has been introduced in the biological, pedagogical and physical departments of Krasnoyarsk State University. Students of the pedagogical department have created a popular and interesting series of laboratory works for high school children aged 14-17 years.

Influence of activation on the multipoint immobilization of penicillin G acylase on macroporous silica

Penicillin G acylase is the second most important enzyme used by industry in an immobilized form. Penicillin hydrolysis is its main application. This reaction is used to produce 6-aminopenicillanic acid (6-APA), an intermediate in the synthesis of semisynthetic antibiotics. This work aims to compare catalytic properties of different penicillin G acylase (PGA) derivatives obtained by multipoint immobilization of the enzyme on macroporous silica. Enzyme amino groups react with different aldehyde groups produced in the support using either glutaraldehyde or glyoxyl activation. In the former method, silica reacts with <FONT FACE="Symbol">g</FONT>-aminopropyltriethoxysilane (<FONT FACE="Symbol">g</FONT>-APTS) and glutaraldehyde; in the latter, a reaction with glycidoxypropyltrimethoxysilane (GPTMS) is followed by acid hydrolysis and oxidation using sodium periodate. This work determines the influence of degree of activation, using glutaraldehyde, on immobilization parameters. PGA was immobilized on these two different supports. Maximum enzyme load, immobilized enzyme activity (derivative activity), rate of immobilization and thermal stability were checked for both cases. For glutaraldehyde activation, the results showed that 0.5% of the <FONT FACE="Symbol">g</FONT>-APTS is sufficient for all the hydroxyl groups in the silica to react. They also showed that degree of activation only affects immobilization yield and reaction velocity and that reduction of the glutaraldehyde derivatives with sodium borohydride does not affect their thermal stability. In comparing the derivatives obtained using glyoxyl and glutaraldehyde activation, it was observed that the glyoxyl derivatives presented better immobilization parameters, with a maximum enzyme load of 264 IU/g silica and a half-life of 20 minutes at 60 °C.

Preparation and Properties of Immobilized Pig Kidney Aminoacylase and Optical Resolution of N-Acyl-DL-Alanine

Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-DL-alanine. The effects of pH, temperature, and Co2+ concentration on the activity of free and immobilized enzymes were investigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-L-alanine in the DL-isomer mixture were 8.0 and 65 degrees C, respectively. Co2+ was an activator for the immobilized enzyme in a similar role as for the free enzyme. No significant loss of activity was observed for at least 300 h of continuous operation. The yield of L-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.