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1.
Michaela Dina Stanescu Magdalena Fogorasi Boris L. Shaskolskiy Simona Gavrilas Vladimir I. Lozinsky 《Applied biochemistry and biotechnology》2010,160(7):1947-1954
Laccases are enzymes belonging to the Oxidoreductases class. These enzymes may be good biocatalysts for different processes, at laboratory and industrial levels. A successful
use at industrial scale demands a higher stability of the enzyme. As an easy way to obtain longer life biocatalysts, the immobilization
process is recommended. Thus, the paper presents different ways of obtaining new biocatalysts by a laccase covalent immobilization
on a macroporous carrier based on poly(vinyl alcohol) cryogel. Different procedures of covalent immobilization are described,
the newly obtained biocatalysts being characterized. According to the experimental data, the stability of the immobilized
enzyme increased and the pH profile changed, compared with those of the free enzyme. 相似文献
2.
3.
Nanofibrous and nanotubular materials, natural and synthetic, are important alternative matrices for the immobilization of metallocomplexes, especially metalloporphyrins, as oxidation catalysts. The process permits a regular and controllable distribution of the active phase at the outer and/or inner surfaces of the tubes, promoting a special environment for the approximation of a substrate to the catalytic active species. The immobilization also prevents the molecular aggregation and bimolecular self-destruction reactions, facilitates the recovery and reuse of the catalyst, reduce de cost of material preparation and environmental concerns. A variety of nanofibrous and nanotubular structures are presented and specific examples of immobilization of iron porphyrins in different supports and their oxidation catalytic activities are presented and discussed. 相似文献
4.
Enzymes exhibit high selectivity and reactivity under normal conditions but are sensitive to denaturation or inactivation
by pH and temperature extremes, organic solvents, and detergents. To extend the use of these biocatalysts for practical applications,
the technology of immobilization of enzymes on suitable supports was developed. Recently, these immobilized biomolecules have
been widely used and a variety of immobilization supports have been studied. The majority of these supports cover diverse
kinds of materials such as natural or synthetic polyhydroxylic matrives, porous in organic carriers, and all kinds of functional
polymers. Microporous molecular sieve, zeolite, has attracted extensive interest in research because of its distinctive physical
properties and geochemistry. Recently, with the discovery of a new family of mesoporous molecular sieves, MCM-41, this series
of materials shows great potential for various applications. Molecular sieves involve such a series of materials that can
discriminate between molecules, particularly on the basis of size. As support materials, they offer interesting properties,
such as high surface areas, hydrophobic or hydrophilic behavior, and electrostatic interaction, as well as mechanical and
chemical resistance, making them attractive for enzyme immobilization. In this article, different types of molecular sieves
used in different immobilization methods including physical adsorption on zeolite, entrapment in mesoporous and macroporous
MCM series, as well as chemically covalent binding to functionalized molecular sieves are reviewed. Key factors affecting
the application of this biotechnology are discussed systematically, and immobilization mechanisms combined with newly developed
techniques to elucidate the interactions between matrixes and enzyme molecules are also introduced. 相似文献
5.
Calmodulin-mediated reversible immobilization of enzymes 总被引:1,自引:0,他引:1
Daunert S Bachas LG Schauer-Vukasinovic V Gregory KJ Schrift G Deo S 《Colloids and surfaces. B, Biointerfaces》2007,58(1):20-27
This work demonstrates the use of the protein calmodulin, CaM, as an affinity tag for the reversible immobilization of enzymes on surfaces. Our strategy takes advantage of the of the reversible, calcium-mediated binding of CaM to its ligand phenothiazine and of the ability to produce fusion proteins between CaM and a variety of enzymes to reversibly immobilize enzymes in an oriented fashion to different surfaces. Specifically, we employed two different enzymes, organophosphorus hydrolase (OPH) and beta-lactamase and two different solid supports, a silica surface and cellulose membrane modified by covalently attaching a phenothiazine ligand, to demonstrate the versatility of our immobilization method. Fusion proteins between CaM-OPH and CaM-beta-lactamase were prepared by using genetic engineering strategies to introduce the calmodulin tail at the N-terminus of each of the two enzymes. In the presence of Ca(2+), CaM adopts a conformation that favors interaction between hydrophobic pockets in CaM and phenothiazine, while in the presence of a Ca(2+)-chelating agent such as EGTA, the interaction between CaM and phenothiazine is disrupted, thus allowing for removal of the CaM-fusion protein from the surface under mild conditions. CaM also acts as a spacer molecule, orienting the enzyme away from the surface and toward the solution, which minimizes enzyme interactions with the immobilization surface. Since the method is based on the highly selective binding of CaM to its phenothiazine ligand, and this is covalently immobilized on the surface, the method does not suffer from ligand leaching nor from interference from other proteins present in the cell extract. An additional advantage lies in that the support can be regenerated by passing through EGTA, and then reused for the immobilization of the same or, if desired, a different enzyme. Using a fusion protein approach for immobilization purposes avoids the use of harsh conditions in the immobilization and/or regeneration steps, which could cause inactivation of the immobilized enzyme. Moreover, we have demonstrated that the CaM affinity tag allows immobilization of enzymes on a variety of surfaces without compromising their enzymatic activity substantially; for example, the immobilized OPH retained more than 80% of the activity of the free enzyme. Our results with beta-lactamase showed the feasibility of using a phenothiazine surface in several consecutive loading and regeneration cycles. This can be advantageous when expensive and/or difficult to obtain immobilization surfaces have to be employed; the immobilization surface could be reused to immobilize the same or a different enzyme using the CaM affinity tail. We also determined that the phenothiazine-modified silica particles are stable for long periods of time, i.e., up to 2 years when stored at 4 degrees C. It is envisioned that this type of reversible immobilization may find applications in the development of reversible, reusable biosensors and bioreactors endowed with the additional advantage that the biological element at the surface of the sensor or bioreactor could be replaced under mild conditions when needed to sense or process a different target molecule. 相似文献
6.
共价有机框架(Covalent Organic Frameworks, COFs)是一种新型的多孔材料,具有结构规整、骨架稳定、孔径结构可调等特点,被视为固定化酶的理想载体。我们主要总结了近10年来COFs材料作为载体,通过物理吸附、共价连接、包埋的固定化策略制备固定化酶的研究进展与应用,并讨论了COFs材料在酶固定化领域所面临的机遇和挑战。 相似文献
7.
The gas permeability and diffusion time lag may exhibit varying degrees of pressure dependence for glassy polymers. The sorption isotherm appears to consist of contributions from both Langmuir and Henry's law terms in such systems. This “dual sorption” theory advanced in the literature pictures gas held by the Langmuir mode as being completely immobilized. In the present paper, this model is extended to accommodate different degrees of partial immobilization of gas sorbed by this mode using two different formulations. One uses a transport expression based on concentration gradients while the other is based on chemical potential gradients. The predictions are that: (1) total immobilization results in a constant permeability with a time lag which strongly decreases with pressure; (2) no immobilization results in a constant time lag with a permeability which decreases strongly with pressure; and (3) incomplete immobilization results in both the permeability and time lag decreasing with pressure but neither as strongly as in the other limiting cases. The differences which may arise by the two formulations of the model are discussed. 相似文献
8.
有机电极材料因其理论比容量高、低成本、环境友好以及分子结构可设计性强等特点,有望成为下一代可持续和多功能能量储存设备的有效电极材料。然而,根据“相似相溶”原理,该类材料极易溶解在有机电解液中,导致电池容量衰减快、循环稳定性和倍率性能也较差。目前已有许多研究致力于通过“固定化”过程解决有机电极材料的溶解问题。本综述针对有机电极材料的固定化策略展开评述,介绍了有机电极材料的固定化机理,以及各种固定化策略在不同种类有机电极材料中所起的作用,指出了有机电极材料面临的挑战,并对未来的研究和改进方向进行展望。 相似文献
9.
Balamurugan S Obubuafo A Soper SA Spivak DA 《Analytical and bioanalytical chemistry》2008,390(4):1009-1021
In this review we examine various methods for the immobilization of aptamers onto different substrates that can be utilized
in a diverse array of analytical formats. In most cases, covalent linking to surfaces is preferred over physisorption, which
is reflected in the bulk of the reports covered within this review. Conjugation of aptamers with appropriate linkers directly
to gold films or particles is discussed first, followed by methods for conjugating aptamers to functionally modified surfaces.
In many aptamer-based applications, silicates and silicon oxide surfaces provide an advantage over metallic substrates, and
generally require surface modification prior to covalent attachment of the aptamers. Chemical protocols for covalent attachment
of aptamers to functionalized surfaces are summarized in the review, showing common pathways employed for aptamer immobilization
on different surfaces. Biocoatings, such as avidin or one of its derivatives, have been shown to be highly successful for
immobilizing biotin-tethered aptamers on various surfaces (e.g., gold, silicates, polymers). There are also a few examples
reported of aptamer immobilization on other novel substrates, such as quantum dots, carbon nanotubes, and carbohydrates. This
review covers the literature on aptamer immobilization up to March 2007, including comparison of different linkers of varying
size and chemical structure, 3′ versus 5′ attachment, and regeneration methods of aptamers on surfaces. 相似文献
10.
亲水金和憎水二氧化硅纳米颗粒对葡萄糖生物传感器响应灵敏度的增强作用 总被引:16,自引:0,他引:16
用亲水金、憎水二氧化硅纳米颗粒固定葡萄糖氧化酶(GOD),采用聚乙烯醇缩丁醛(PVB)为辅助固酶膜基质来制备葡萄糖生物传感器,并考察了亲水金、憎水二氧化硅纳米颗粒对酶电极电流响应的影响.实验表明,引入纳米粒子可显著增强电极响应灵敏度.并对两种不同性质纳米颗粒所起作用的可能机理进行讨论,从理论和实验上证明了纳米颗粒对固定酶的作用.为制备有实用价值的葡萄糖生物传感器提供了可供参考的实验和理论依据. 相似文献
11.
Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities
is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at
the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of
the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells,
proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely
categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization.
Each method has individual immobilization characteristics. Although every biomolecule–paper combination has to be optimized
before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and
biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based
analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries;
(3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial
paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends.
Current, emerging and future applications of bioactive paper are also discussed. 相似文献
12.
《Journal of membrane science》2001,181(1):29-37
Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis. 相似文献
13.
Yeo DS Panicker RC Tan LP Yao SQ 《Combinatorial chemistry & high throughput screening》2004,7(3):213-221
Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides. 相似文献
14.
Petrovykh DY Kimura-Suda H Whitman LJ Tarlov MJ 《Journal of the American Chemical Society》2003,125(17):5219-5226
We describe the complementary use of X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy to quantitatively characterize the immobilization of thiolated (dT)(25) single-stranded DNA (ssDNA) on gold. When electron attenuation effects are accurately accounted for in the XPS analysis, the relative coverage values obtained by the two methods are in excellent agreement, and the absolute coverage can be calculated on the basis of the XPS data. The evolution of chemically specific spectral signatures during immobilization indicates that at lower coverages much of the DNA lies flat on the surface, with a substantial fraction of the thymine bases chemisorbed. At higher immobilization densities, the (dT)(25) film consists of randomly coiled ssDNA molecules each anchored via the thiol group and at possibly one or two other bases. We use two examples to demonstrate how the quantitative analysis can be applied to practical problems: the effects of different buffer salts on the immobilization efficiency; the immobilization kinetics. Buffers with divalent salts dramatically increase the efficiency of immobilization and result in very high surface densities (>5 x 10(13)/ cm(2)), densities that may only be possible if the divalent counterions induce strong attractive intermolecular interactions. In contrast with previous reports of alkanethiol adsorption kinetics on gold, ssDNA immobilization in 1 M phosphate buffer does not occur with Langmuir kinetics, a result attributable to rearrangement within the film that follows the initial adsorption. 相似文献
15.
Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody-antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures. 相似文献
16.
Site-selective immobilization of dyes and different protein recognizing entities at the surface of zeolite L crystals using mild radical nitroxide exchange reactions is reported. Exposure of these crystals to aqueous protein solutions leads to site-selective immobilization of proteins onto the crystals. 相似文献
17.
Marianna Portaccio Rosario Esposito Ines Delfino Maria Lepore 《Journal of Sol-Gel Science and Technology》2014,71(3):580-588
Immobilization procedures are a fundamental step in the technological use of enzymes. Among the different immobilization procedures sol–gel technique is widely recognised as a valuable approach to obtain very high quality catalytic supports. In this paper different optical techniques have been used and compared to investigate structural and dynamic properties of glucose oxidase (GOD) prior and after sol–gel immobilization process. In particular, using Fourier Transform infrared micro-spectroscopy and time-resolved fluorescence the secondary structure of GOD and the flavin adenine dinucleotide (FAD) conformational changes have been respectively investigated. Infrared spectroscopy measurements have confirmed that enzymatic activity is preserved and a predominant β-sheet subcomponent is retained by immobilized GOD. By time-resolved FAD fluorescence a three-exponential decaying behaviour has been observed for both free and immobilized enzymes with three different lifetimes, each being characteristic of a peculiar conformational state of the FAD structure. The comparison between lifetime values for free and immobilized GOD has not shown significant differences, while the fractional steady-state intensities of the single exponential components have been changed by immobilization procedure. All results reported and discussed in this paper have confirmed once again the efficacy of the adopted sol–gel immobilization procedure for enzymes and proteins. In addition, the joint use of different optical spectroscopic techniques has shown to be a very valuable tool for getting a better insight into structural and dynamic properties of immobilized enzymes. 相似文献
18.
Lingbing Yang Yilin Zhang Zeyun Xiao Wenbo Zhang Linhao Li Yubo Fan 《Macromolecular bioscience》2023,23(11):2300190
Hollow polymer microcapsules as drug carriers have the advantages of drug protection, storage, and controlled release. Microcapsules combined with tissue engineering scaffolds such as electrospun microfibers can enhance long-term local drug retention. However, the combination methods of microcapsules and fibers still need to be further explored. Here, different technical approaches to functionalize electrospun polycaprolactone (PCL) microfibers with silk fibroin (SF) microcapsules through encapsulation and surface immobilization are developed, including direct blending and emulsion electrospinning for encapsulation, as well as covalent and cleavable disulfide-linkage for surface immobilization. The results of “blending” approach show that silk microcapsules with different sizes could be uniformly encapsulated inside electrospun fibers without aggregation. To further reduce the use of organic solvents, the microcapsules in the aqueous phase can be uniformly distributed in the PCL organic phase and successfully electrospun into fibers using surfactant span-80. For surface immobilization, silk microcapsules are efficiently covalent binding to the surface of electrospun PCL fibers via click chemistry and exhibited noncytotoxic. Based on this method, with the incorporation of a disulfide bond, the linkages between microcapsule and fiber could be cleaved under reducing conditions. These microcapsule-electrospun fiber combination methods provide sufficient options for different drug delivery requirements. 相似文献
19.
Savina A. A. Garnashevich L. S. Zaitsev I. S. Tsarkova M. S. Zaitsev S. Yu. 《Moscow University Chemistry Bulletin》2019,74(6):306-309
Moscow University Chemistry Bulletin - Abstract—Both synthetic and natural polymers are promising carriers for the immobilization of enzymes, including lipases from different sources. This... 相似文献
20.
Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application. 相似文献