基于DNA定向固定化技术构建毛细管固定化酶微反应器的研究进展

生物酶影响着物质代谢和质能转换等生命活动,生物体内某些酶的活性变化会导致疾病的发生。发展新型的酶分析方法对深刻理解生物代谢过程、疾病诊断和药物研发等具有重要意义。毛细管电泳(CE)具有分离效率高、分析速度快、操作简单和样品消耗少以及可与多种检测手段联用等优点,在酶分析研究中越来越受到关注。CE酶分析主要包括离线和在线两种模式,其中,固定化酶微反应器与毛细管电泳联用(CE-IMER)的在线酶分析已经成为主要的酶分析方法之一。CE-IMER充分结合了固定化酶和CE的优势,将游离酶固定在毛细管内,不仅可以显著提高酶的稳定性和重复使用性,而且可以实现纳升规模溶液的自动化酶分析,进而显著降低酶分析成本。目前已有大量方法制备IMER用于CE酶分析,然而如何构建性能良好、可再生使用、酶固载量大、自动化程度高的CE-IMER一直是该领域重点研究的问题。DNA定向固定化技术(DDI)可以充分利用DNA分子的碱基互补配对(A-T, C-G),在温和的生理条件下特异性固定生物大分子。由于短链双螺旋DNA分子具有较强的机械刚性和物理化学稳定性,通过DDI将酶固定在载体表面,有利于降低传质阻力,提高酶与底物的接...     

New supports for enzyme immobilization based on copolymers of vinylene carbonate and acrylamide

In this study, a series of beadlike and hydrophilic supports containing reactive cyclic carbonate groups for enzyme immobilization were prepared via reverse-phase suspension copolymerization of the aqueous solutions of vinylene carbonate (VCA), acrylamide (AA), and N,N′-methylene bisacrylamide in paraffin oil. The supports were used as a matrix for immobilization of trypsin and showed a considerable capacity to couple with trypsin and reasonable retention of activity for the immobilized trypsin, depending on the immobilization conditions, such as the content of VCA structural units, reaction time, and pH of the medium.     

Isonitrile derivatives of polyacrylamide as supports for the immobilization of biomolecules

Isonitrile derivatives of crosslinked polyacrylamide beads (Biogell P-100) were prepared by a two-step procedure: a. N-hydroxymethylation (methylolation) of amide groups on the polymer by treatment with formaldehyde; and b. Attachment of side chains, containing isonitrile functional groups by a displacement reaction involving 1-tosyloxy-3-isocyanopropane (p-CH₃-C₆H₄·SO₂·O·(CH₂)₃ NC) and alkoxide ions generated on methylolated polyacrylamide by treatment with a strong base in a polar aprotic solvent. The modified polyacrylamide beads were tested as support for the immobilization of proteins, and low mol wt ligands by four component condensation (4CC) reactions. Trypsin-polyacrylamide acting on N-benzoyl-L-arginine ethylester exhibited nonlinear Michaelis Menten kinetics and distorted pH activity profiles. The kinetic anomalies could be reduced by increasing the concentration of buffer. The data were consistent with a model assuming “buffer facilitated proton transport” in a diffusionally constrained system.     

Chemically modified nylons as supports for enzyme immobilization

An isocyanide derivative of nylon, polyisonitrile-nylon (1,2), was used as a starting material whereby, through a series of modification reactions, different chemically reactive functional groups could be introduced on the polyamide backbone. The chemistry employed allowed for considerable flexibility in the choice of procedures for covalent fixation of proteins, all starting from the same chemically reactive parent polymer, polyisonitrile-nylon. Thus, polyisonitrilenylon could be used directly for the immobilization of enzymes via fourcomponent condensation reactions. The isocyanide functional groups of the parent polymer could be transformed, by treatment with bromine, into the strongly electrophilic dibromoisocyanide (—N=CBr^b2) groups. The selectivity of the —N=CBr^b2 group toward the various functional groups present in proteins could be regulated by appropriate control of the pH of the coupling reaction. Dibromoisocyanide-nylon was also further modified into other types of chemically reactive nylon derivatives.     

The use of an immobilized enzyme nylon tube reactor incorporating a four enzyme system for creatinine analysis

A single immobilized enzyme nylon tube reactor was produced incorporating a four enzyme system for the analysis of creatinine. The enzyme activity ratios in the coupling solution used to prepare the reactor were found to be of extreme importance in governing the activity of the latter. The reactor was incorporated into a continuous flow analysis system used to assay creatinine in urine samples and the results were correlated with a manual technique employing the same enzyme system in solution. The precision, correlation, high specificity, simplicity, and speed of the analysis were concluded to be factors in favor of the method's suitability for urine creatinine determinations.     

甲壳胺-硅胶复合载体的制备及其在脂肪酶固定化中的应用

固定化脂肪酶;甲壳胺-硅胶复合载体的制备及其在脂肪酶固定化中的应用     

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.     

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 μmol of NH₂/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-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 (55.8U/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 μmol of H₂O₂ reduced/(h·U_{immob HRP}).     

Combinatorial discovery of reusable noncovalent supports for enzyme immobilization and nonaqueous catalysis

A simple and effective method is described for the preparation of enzyme-containing materials that possess excellent catalytic activity, mechanical strength, and reusability. Uniform spherical beads were produced via the colyophilization of alpha-chymotrypsin with the support materials, leaving the active enzyme entrapped within the porous "ice-templated" support matrix. The composites were assayed for catalytic activity by monitoring a nonaqueous transesterification reaction. The mechanical strength for each composite was measured using a compression assay. Initial screens identified a set of six support materials that contributed favorably to either the enzyme activity or to the mechanical strength of the composite. A design of experiments (DoE) methodology was employed to screen 80 combinations of these six "base" materials. A model representing this formulation space was constructed which could be used to predict both the catalytic activity and mechanical strength with reasonable accuracy for any combination of the six base component materials. This model was used to predict optimized materials with an enzyme activity that was 50 times greater than that of the free enzyme. The model was also used to set a minimum acceptable mechanical stability for these composites, and the resulting materials were shown to be reusable for at least ten reaction cycles.     

京尼平交联磁性壳聚糖微球的制备及其脂肪酶的固定化

采用反相悬浮法与溶胶凝胶法结合制备磁性壳聚糖微球,并以此为载体,京尼平为交联剂,脂肪酶为模型酶进行固定化,研究了酶固定化的最优条件和固定化酶的性质。结果表明,在京尼平浓度为0.6 g/L、交联温度为55 ℃、交联时间8 h,固定化酶的比活力最大,为4.31 U/g。固定化酶在25~35 ℃,pH值在8.0有最大活性,其米氏常数Km为0.26 mol/L。同时,固定化酶具有良好的热稳定性及pH稳定性,可重复利用,且能进行磁分离。     

Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.     

固定化酶扩散-反应问题的近似解

固定化酶在生物技术领域具有重要的理论和实际意义。由于固定化酶内部存在扩散传质阻力,因此固定化酶系统的化学反应速率不同于游离酶的反应速率。固定化酶反应动力学一般是基质浓度的非线性函数。通过求解固定化酶扩散 反应微分方程,可以得到固定化酶有效因子,而有效因子是固定化酶反应系统设计和模拟的重要参数,也是评定固定化酶系统性能优劣的重要因素之一。有效因子的计算通常采用将扩散 反应微分方程离散化的方法,例如正交配置法求解[1,2]。本文基于大参数的假设,在寻求固定化酶扩散 反应问题近似解的基础上,导出计算固定化酶有效…     

Recent advances in immobilization methods of antibodies on solid supports

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.     

Automation of a heterogeneous enzyme immunoassay for atrazine. Comparison of three immobilization supports

Intensive agriculture associated with the use of large amounts of different pesticides, together with the growing concern about the potential contamination of ground water, have brought about the need for developing fast screening methods. This work presents the automation of an enzyme-linked immunosorbent assay for atrazine by means of a flow-through system. Three different solid supports for antibody immobilization were compared in a direct competitive assay format. Sensitivity reached in all cases was below the maximum level allowed in the EU (100 ng L^–1). Cross-reactivity of atrazine-related compounds was also studied. The performance of the different supports is discussed regarding sensitivity and immunosurface regeneration.     

Automation of a heterogeneous enzyme immunoassay for atrazine. Comparison of three immobilization supports

Intensive agriculture associated with the use of large amounts of different pesticides, together with the growing concern about the potential contamination of ground water, have brought about the need for developing fast screening methods. This work presents the automation of an enzyme-linked immunosorbent assay for atrazine by means of a flow-through system. Three different solid supports for antibody immobilization were compared in a direct competitive assay format. Sensitivity reached in all cases was below the maximum level allowed in the EU (100 ng L^–1). Cross-reactivity of atrazine-related compounds was also studied. The performance of the different supports is discussed regarding sensitivity and immunosurface regeneration. Received: 21 July 1997 / Revised: 7 November 1997 / Accepted: 11 November 1997     

Diffusion and reaction in an immobilized enzyme starch saccharification catalyst

Effectiveness factors were predicted from measurements of basic parameters made on single oligosaccharides, and the prediction was compared to experimental effec tiveness factors for the reaction of each oligosaccharide in the immobilized enzyme catalyst. Kinetic parameters were obtained for the hydrolysis of each oligosaccharide catalyzed by soluble glucoamylase, and were fit with a subsite model equation capable of generalization to all sizes of oligosaccharide. Diffusion coefficients in free solution were determined from movement out of a capillary tube. Spatial characteristics of the immobilized enzyme bed were obtained from pulse response experiments, allowing the calculation of effective diffusivities. Experimental effectiveness factors plotted against modulus were in reasonable agreement with the predictions.     

Effects of carbodiimide structure on the immobilization of enzymes

A series of water-soluble disubstituted carbodiimides of different structure was tested for enzyme immobilization. In the experiments, a polyacrylamide-type bead polymer possessing carboxylic functional groups was used as support. The enzymes immobilized were aminoacylase (N-acylamino acid amidohydrolase; EC 3.5.1.14), arginase (L-arginine amidinohydrolase; EC 3.5.3.1), cyclodextrin glycosyltransferase (alpha-1,4-glucan 4-glycosyltransferase, cyclizing; EC 3.2.1.19), glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3), and carboxypeptidase B (peptidyl-L-lysine [L-arginine] hydrolase; EC 3.4.17.2). It was found that the degree of immobilization strongly depended on the structure of carbodiimide used.     

蛋白质组学中的固定化酶反应器制备的研究进展

固定化酶反应器作为蛋白质组学研究中"bottom-up"策略重要的组件,具有酶解快速、酶解效率高、酶稳定性和活性高、简单易操作、能够与多种检测方式联用等优点,对于发展高效快速的蛋白质组学分析方法具有重要意义。本文就固定化酶反应器的制备方法及其在蛋白质组学中的应用做简单的概述,着重介绍酶的固定方法、固定化酶的载体、用于固定的酶的种类。近几年固定化酶反应器的研究集中于提高固酶量、保持酶活性、增加酶解效率、减小非特异性吸附等方面。研究结果表明,采用纳米材料、整体材料等新型载体,提高载体亲水性,采用多酶同时酶解等方法能够有效改善固定化酶反应器的性能,提高蛋白质的鉴定效率。     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.