Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.     

Multilayer enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors

Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.     

Flexible Assembly of an Enzyme Cascade on a DNA Triangle Prism Nanostructure for the Controlled Biomimetic Generation of Nitric Oxide

Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA‐binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H₂O₂ intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.     

Covalent immobilization of glucose oxidase to poly(O‐amino benzoic acid) for application to glucose biosensor

A biosensor for glucose utilizing glucose oxidase (GOX) covalently coupled to poly(o‐amino benzoic acid) (PAB; a carboxy‐group‐functionalized polyaniline) is described. Amperometric response measurements conducted via unmediated and mediated (with ferrocene carboxylic acid and tetrathiafulvalene) reoxidation of GOX show that glucose can be detected over a wide range of concentrations. An enzyme‐conducting polymer‐mediator model provides for better charge transport in a biosensor. The optimal response, obtained at pH 5.5 and 300 K, lies in the 1–40 mM range. A kinetic plot yields the value of the apparent Michaelis–Menten constant, K_m^app. The operational stability of the PAB‐based glucose biosensor was experimentally determined to be about 6 days. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 662–667, 2000     

Immobilization of glucose oxidase onto electrochemically prepared poly(aniline‐co‐fluoroaniline) films

Poly(aniline‐co‐fluoroaniline) [poly(An‐FAn)] films were electrochemically deposited on indium tin oxide glass plates. The characterization of these conducting copolymer films was carried out with ultraviolet–visible and Fourier transform infrared techniques. The enzyme glucose oxidase (GOX) was immobilized onto the conducting poly(An‐FAn) films by a physical adsorption method. Amperometric response experiments carried out with poly(An‐FAn)/GOX films revealed linearity from 0.5 to 22 mM glucose, and the Michaelis–Menten constant was found to be about 23.5 mM. The shelf life of these poly(An‐FAn)/GOX electrodes was measured to be about 15 days, and the electrodes were stable up to 45°C. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3999–4006, 2004     

Maximizing the Efficiency of Multienzyme Process by Stoichiometry Optimization

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three‐enzyme system catalyzing a five‐step chemical conversion. Kinetic models of pathways with wild‐type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one‐pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes.     

Separate or simultaneous immobilization of glucose oxidase and peroxidase on latex particles and their function

Glucose oxidase and peroxidase were immobilized individually or simultaneously on aminated latex particles by using sodium meta-periodide and borohydride. The amount of immobilized enzymes and their activity depended on the surface potential of particles and the surface density of their own, respectively. In the simultaneous immobilization of two enzymes, the predominant immobilization of peroxidase is attributed to higher carbohydrate content in peroxidase compared with that in glucose oxidase. Simultaneously immobilized enzymes worked better in the determination of glucose than the mixture of separately immobilized ones because of the close proximity of the two enzymes.     

Multienzyme‐immobilized modified polypropylene membrane for an amperometric creatinine biosensor

Creatinine has become an important clinical analyte that is used for the determination of renal and muscular dysfunction. It is essential to determine its concentration in the serum of patients suffering from renal insufficiency. Therefore, an amperometric creatinine biosensor fabricated from a covered platinum/silver electrode with a thin layer of an immobilized multienzyme membrane was studied. Poly(acrylic acid) was introduced onto an argon‐plasma‐treated porous polypropylene membrane surface by graft copolymerization. Subsequently, three different enzymes (sarcosine oxidase, creatinase, and creatininase) were immobilized onto this novel grafted membrane simultaneously via a carbodiimine agent to form a thin layer. The sensor performance was evaluated with a biochemistry analyzer. Moreover, attenuated total reflection/Fourier transform infrared, electron spectroscopy for chemical analysis, and scanning electron microscopy were used to confirm the progression of these reactions. The developed sensor showed a linear detection range of 3.2–320 μM for creatinine in a pH 7.4 buffered solution with 0.1M phosphate. The immobilized multienzyme membrane could be used for at least 3 weeks. The results obtained in our study will hopefully lead to the successful application of modified polypropylene for the development of a creatinine sensor. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3126–3134, 2004     

Survey of a Salivary Effector in Caterpillars: Glucose Oxidase Variation and Correlation with Host Range

Salivary glucose oxidase (GOX) has been reported in a few insect species where it plays a role in protection against infectious disease. Our recent research has focused on the role of this salivary enzyme in the noctuid Helicoverpa zea, where it functions as an effector to suppress the induced defenses of the host plant Nicotiana tabacum. In this study, we examined the labial gland GOX activities in 23 families of Lepidoptera (85 species) and two families of plant-feeding Hymenoptera (three species). We analyzed the relationship between host breadth and GOX activities, and we found a significant relationship, where highly polyphagous species were more likely to possess relatively high levels of GOX compared to species with more limited host range. We also examined the effect of diet on GOX activity and found that the host plant had a significant effect on enzyme activity. The significance of these findings is discussed in relation to caterpillar host breadth.     

Pyrene-adamantane-β-cyclodextrin: An efficient host–guest system for the biofunctionalization of SWCNT electrodes

The design of nanostructured biological architectures based on host–guest interactions between β-cyclodextrin and adamantane was investigated on SWCNT coatings using glucose oxidase (GOX) as biomolecule model. β-Cyclodextrin tagged GOX was immobilized on adamantane functionalized carbon nanotubes, deposited on platinum electrodes. Different functionalization techniques to attach “pyrene adamantane” on nanotubes were studied and compared in terms of the performances of the subsequently constructed glucose biosensors. The best results were obtained by dipping the nanotube deposit into a pyrene-adamantane solution followed by electropolymerization of the adsorbed pyrene monolayer. The constructed biosensor exhibited a good linear response toward glucose concentrations between 2 × 10⁻⁷ M and 1.6 × 10⁻³ M. The maximum current density and glucose sensitivity were 154.9 μA cm⁻² and 14.4 mA M⁻¹ cm⁻², respectively.     

Preparation of protein microarrays on non‐fouling and hydrated poly(ethylene glycol) hydrogel substrates using photochemical surface modification

BACKGROUND: Conventional protein microarrays prepared on hard, dry substrates, such as glass and silicone, have several limitations, as proteins may easily denature and lose their structure. To overcome such problems, the fabrication of wet protein microarrays on non‐fouling and hydrated PEG‐based hydrogels was investigated. RESULT: Bovine serum albumin (BSA) and glucose oxidase (GOX), chosen as model proteins, were covalently immobilized on PEG hydrogel surfaces via 5‐azidonitrobenzoyloxy N‐hydroxysuccinimide, a photoreactive bifunctional linker. Successful fixation of the bifunctional linker and subsequent immobilization of the proteins on the PEG hydrogel surfaces were confirmed with X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) studies. GOX immobilized on the hydrogel surface maintained approximately 50% of its initial activity after 24 h when left in dry conditions, but maintained only 20% when immobilized on a dry substrate. Photochemical fixation combined with photolithography produced well‐defined protein micropatterns with sizes ranging from 50–500 µm, and molecular recognition‐mediated specific binding between biotin and streptavidin was successfully assayed using microarrays on PEG hydrogels. CONCLUSION: A protein‐repellent PEG hydrogel surface was photochemically modified to covalently immobilize proteins and create protein microarrays. The use of hydrated hydrogels as substrates for protein microarrays could minimize the deactivation of proteins in dry conditions, and the non‐fouling property of PEG hydrogels allows the passivation step of protein microarray preparation to be skipped. Copyright © 2008 Society of Chemical Industry     

Chromatic detection of glucose using polymerization of diacetylene vesicle

A polydiacetylene vesicle was used to fabricate glucose sensor, allowing feasible colorimetric detection. The vesicle was formed by sonication of 10,12‐pentacosadiynoic acid (PCDA). H₂O₂ formed by the reaction between glucose and glucose oxidase functioned as the initiator for the polymerization of PCDA in the presence of horseradish peroxidase. The solution turned blue after the polymerization of PCDA vesicle. Thus, the glucose concentration could be detected to the concentration level that turns the solution to blue. The ultraviolet absorbance of glucose solution was proportional to glucose concentration. The results of this study indicate that glucose concentration upto 1 mM can be detected by change in blue color by eyes. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46394.     

Immobilization of glucose oxidase in alginate-chitosan microcapsules

In order to improve its stability and catalytic rate in flour, the immobilization of glucose oxidase (GOX) was investigated in this work. The enzyme was encapsulated in calcium alginate-chitosan microspheres (CACM) using an emulsification-internal gelation-GOX adsorption-chitosan coating method. The interaction between alginate and chitosan was confirmed by infrared spectroscopy (IR). The resultant CACM in wet state, whose morphology was investigated by scanning electron microscopy (SEM), was spherical with a mean diameter of about 26 μm. The GOX load, encapsulation efficiency and activity of the CACM-GOX were influenced by concentration of chitosan, encapsulation time and encapsulation pH. The highest total enzymatic activity and encapsulation efficiency was achieved when the pH of the adsorption medium was near the isoelectric point (pI) of GOX, approximately pH 4.0. In addition, the molecular weight of chitosan also evidently influenced the encapsulation efficiency. Storage stabilities of GOX samples were investigated continuously over two months and the retained activity of CACM-GOX was 70.4%, markedly higher than the 7.5% of free enzyme. The results reveal the great potential of CACM-GOX as a flour improver.     

Multienzyme co-immobilization-based bioelectrode: Design of principles and bioelectrochemical applications

Enzyme cascade reactions play significant roles in bioelectrochemical processes because they permit more complex reactions. Co-immobilization of multienzyme on the electrode could help to facilitate substrate/intermediate transfer among different enzymes and electron transfer from enzyme active sites to the electrode with high stability and retrievability. Different co-immobilization strategies to construct multienzyme bioelectrodes have been widely reported, however, up to now, they have barely been reviewed. In this review, we focus on recent state-of-the-art techniques for constructing co-immobilized multienzyme electrodes including random and positional co-immobilization. Particular attention is given to strategies such as multienzyme complex and surface display. Cofactor co-immobilization on the electrode is also crucial for the enhancement of catalytic reaction and electron transfer, yet, few studies have been reported. The up-to-date advances in bioelectrochemical applications of multienzyme bioelectrodes are also presented. Finally, key challenges and future perspectives are discussed.     

Multienzyme co-immobilization-based bioelectrode:Design of principles and bioelectrochemical applications

Enzyme cascade reactions play significant roles in bioelectrochemical processes because they permit more complex reactions. Co-immobilization of multienzyme on the electrode could help to facilitate substrate/intermediate transfer among different enzymes and electron transfer from enzyme active sites to the electrode with high stability and retrievability. Different co-immobilization strategies to construct multienzyme bioelectrodes have been widely reported, however, up to now, they have barely been reviewed. In this review, we focus on recent state-of-the-art techniques for constructing co-immobilized multienzyme electrodes including random and positional co-immobilization. Particular attention is given to strategies such as multienzyme complex and surface display. Cofactor co-immobilization on the electrode is also crucial for the enhancement of catalytic reaction and electron transfer, yet, few studies have been reported. The up-to-date advances in bioelectrochemical applications of multienzyme bioelectrodes are also presented. Finally, key challenges and future perspectives are discussed.     

Immobilised multienzyme systems and organelles

Enzyme technology has demonstrated its economic and industrial potentials by the successful development of the ‘first generation’ of immobilised enzymes which concern simple degradative enzymes which by hydrolysis, oxidation and isomerisation yield products with rather limited added values. A new objective is to prepare industries to develop a ‘second generation’ of enzyme reactors in which sophisticated multienzyme systems will catalyse the synthesis of fine chemicals of high added value. There are two kinds of solutions to develop such new systems: immobilisation of subcellular organelles such as mitochondria, whole bacteria or fragments (chromatophores), chloroplasts (thylakoids). etc.; immobilisation of multienzyme systems including cofactor regeneration and realisation of multienzyme reactors like continuous stirred-tank reactor. Experimental examples dealing with both topics are described.     

Synthetic Multienzyme Assemblies for Natural Product Biosynthesis

In nature, enzymes that catalyze sequential reactions are often assembled as clusters or complexes. The formation of multienzyme complexes, or metabolons, brings the enzyme active sites into proximity to promote intermediate transfer, decrease intermediate leakage, and streamline the metabolic flux towards the desired products. We and others have developed synthetic versions of metabolons through various strategies to enhance the catalytic rates for synthesizing valuable chemicals inside microbes. Synthetic multienzyme complexes range from static enzyme nanostructures to dynamic enzyme coacervates. Enzyme complexation optimizes the metabolic fluxes inside microbes, increases the product titer, and supplies the field with high-yield microbe strains that are amenable to large-scale fermentation. Enzyme complexes constructed inside microbial cells can be separated as independent entities and catalyze biosynthetic reactions ex vivo; such a feature gains these complexes another name, “synthetic organelles” – new subcellular entities with independent structures and functions. Still, the field is seeking new strategies to better balance dynamicity and confinement and to achieve finer control of local compartmentalization in the cells, as the natural multienzyme complexes do. Industrial applications of synthetic multienzyme complexes for the large-scale production of valuable chemicals are yet to be realized. This review focuses on synthetic multienzyme complexes that are constructed and function inside microbial cells.     

Dielectric spectroscopic studies on polypyrrole glucose oxidase films

An investigation was made into the dielectric spectroscopic characteristics of p-toluene sulfonate (PTS) doped polypyrrole (PPY) films in the presence and absence of immobilized glucose oxidase (GOX) in three different configurations: Al-PTS-PPY-Al-PTS-PPY/GOX-Al, and Al-PTS-PPY/GOX/β-D -glucose-Al, respectively. Measurement of dielectric loss and capacitance yielded valuable information about the dielectric properties of GOX immobilized in PTS doped PPY films. The effect of both the temperature and varying βD -glucose concentrations on the mobility of the charge carriers in these films was also systematically studied. © 1996 John Wiley & Sons, Inc.     

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that α‐amylase (1,4‐α–D‐glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4‐β‐N‐acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg^?1, whereas cellulase complex (1,4‐(1,3:14)‐β‐D‐glucan 4‐glucano‐hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for α‐amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. Copyright © 2010 Society of Chemical Industry     

Toxicity of soluble products from the peroxidase‐catalysed polymerization of substituted phenolic compounds

The residual toxicities of aqueous solutions of phenol and substituted phenols were investigated following polymerization under the catalytic action of soybean peroxidase (SBP) and horseradish peroxidase (HRP) enzymes. The treated mixtures obtained from the enzymatic polymerization of these phenols were usually significantly more toxic than expected, and in several cases, the residual toxicity exceeded the initial toxicity of the solution of untreated parent compound. However, this residual toxicity tended to be lower when combinations of these phenols were co‐polymerized. The decrease in toxicity was attributed to the different polymeric products which form as a result of a cross‐coupling between products of the enzyme‐catalysed oxidation of parent phenols. The residual toxicities obtained using either SBP or HRP were not significantly different in most cases. © 2000 Society of Chemical Industry