Topic
Immobilized enzyme
About: Immobilized enzyme is a research topic. Over the lifetime, 15282 publications have been published within this topic receiving 401860 citations.
Papers published on a yearly basis
Papers
More filters
••
TL;DR: This review presents some of the strategies that may give a synergism in the final results of chemical modification of enzymes, and discusses the use of different targets for chemical modifications with small molecules or multifunctional polymers.
Abstract: Chemical modification and immobilization of enzymes have been usually considered unrelated tools to improve biocatalyst features. However, there are many examples where a chemically modified enzyme is finally used in an immobilized form, and that exemplifies how both tools may be complementary resulting in a synergism in the final results. In this review we present some of the strategies that may give that result. For example, the chemical modification of soluble enzymes may be used to improve their immobilization (reinforcing adsorption or improving multipoint covalent attachment), or just to improve enzyme stability and facilitate the selection of the immobilization conditions. Chemical modification of previously immobilized enzymes benefits from solid-phase chemistry due to the nature of enzymes (e.g., prevention of inactivation, aggregation, etc.). The use of different targets for chemical modifications with small molecules or multifunctional polymers are also discussed: intramolecular or intersubunit cross-linking, one-point modification, generation of artificial microenvironments, etc.
306 citations
••
TL;DR: The concept of stabilization has been an important driving force for immobilizing enzymes and true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.
Abstract: The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.
302 citations
••
TL;DR: Sepabeads‐EP (a new epoxy support) has been utilized to immobilize‐stabilize the enzyme penicillin G acylase via multipoint covalent attachment and was hundreds‐fold more stable than Eupergit C derivatives when using a more sophisticated three‐step immobilization/stabilization/blockage procedure.
Abstract: Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).
297 citations
••
TL;DR: In this paper, the results of lipase-catalyzed, nonaqueous alcoholysis of sunflower oil under anhydrous conditions were examined and the overall content of tri-, di- and monoglycerides, as well as the corresponding alkyl esters, was measured.
Abstract: Lipase-catalyzed alcoholysis of sunflower oil under anhydrous conditions was examined. Lipases fromPseudomonas fluorescens and 2 immobilized enzymes fromMucor miehei and aCandida sp. gave sufficient conversion with petroleum ether as the solvent, even when methanol and ethanol were used. The overall content of tri-, di- and monoglycerides, as well as the corresponding alkyl esters, was measured. BecausePseudomonas lipase led to almost quantitative esterification, further studies were carried out with that enzyme varying the amounts of enzyme or the alcohols. Acceptable conversions were achieved even without solvent. Reaction rates of alcoholysis with 5 homologous alcohols, with or without the addition of water, were measured, and in all cases the reaction rates increased with higher chain length of the alcohol. In the case of methanol the highest rate was obtained without any addition of water, but a significantly higher rate was observed with 96% ethanol as opposed to absolute ethanol. The main advantages of lipasecatalyzed, nonaqueous alcoholysis as compared to classical procedures are the mild reaction conditions, the isolation of glycerin without further purification and without the formation of chemical waste, and the ability of lipases to catalyze the esterification of free fatty acids.
297 citations
••
TL;DR: Preparing and functionalization of magnetite (Fe3O4) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface are presented.
Abstract: Immobilization of bioactive molecules on the surface of magnetic nanoparticles is of great interest, because the magnetic properties of these bioconjugates promise to greatly improve the delivery and recovery of biomolecules in biomedical applications. Here we present the preparation and functionalization of magnetite (Fe3O4) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption. The enzymatic activity of the glucose oxidase-coated magnetic nanoparticles was investigated by monitoring oxygen consumption during the enzymatic oxidation of glucose using a ruthenium phenanthroline fluorescent complex for oxygen sensing. The glucose oxidase-coated magnetite nanoparticles could function as nanometric glucose sensors in glucose solutions of concentrations up to 20 mmol L−1. Immobilization of glucose oxidase on the nanoparticles also increased the stability of the enzyme. When stored at 4°C the nanoparticle suspensions maintained their bioactivity for up to 3 months.
295 citations