固定化酶微反应器的制备及应用

固定化酶微反应器是将生物分子固定技术与生化微反应相结合制备的一种固定化催化装置。这种微型化的反应系统由于兼具固定化酶的特异性催化、可重复利用及微分析的低消耗、易分离等优点,在生命科学如蛋白质组学、酶抑制剂的筛选、生物催化等领域具有非常重要的作用。固定化酶微反应器的性能与其制作方法关系密切。本文着重从酶与固定化载体结合方式的角度,对近年来固定化酶微反应器的各种制备方法和应用进行了较为详细的评述。重点讨论了各种方法的优缺点和最新的发展情况,并对其发展前景进行了展望。     

采用磁性载体制备的固定化酶反应器的载体粒径对蛋白质酶解性能的影响

研究了以不同粒径的磁性颗粒为载体的固定化酶反应器在蛋白质酶解过程中,其粒径大小对团聚、酶解效率和漏切位点等的影响。实验结果表明,纳米级颗粒的酶负载量为亚微米级的3.5倍左右。但当酶固定量相同时,酶解效率基本相当。而在一定程度上加大磁性颗粒的粒径后,团聚现象得到明显改善。选择磁性载体粒径为20 nm的固定化酶反应器,对其性能进一步考察。结果显示胰蛋白酶与牛血清白蛋白(BSA)的质量比为1:1时,即能于1 min内实现快速酶解;当酶解10 min时,其零漏切位点肽段数和蛋白质序列覆盖率基本达到稳定,并明显优于溶液酶解水平。通过对漏切位点的统计分析比较,发现固定化酶解与溶液酶解时的漏切位点规律基本类似。因此,采用不同粒径磁性载体制备的固定化酶反应器均可在蛋白质组学研究中提供快速、高效的酶解。     

酶固定化技术用载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一.作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响着所得固定化酶的催化活性及操作稳定性.综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容.通过对传统材料的改性和新型载体材料的研究开发,必将促进固定化酶在各个领域的广泛应用.本文综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势.     

整体固定化酶反应器的研制

制作了微型整体柱型的固定化酶反应器。在500μm内径毛细管内,以乙烯基三甲氧基硅烷处理形成端基烯键,采用原位合成法,以甲基丙烯酸2-羟乙酯为功能单体,以乙二醇二甲基丙烯酸酯为交联剂制备了整体柱。整体柱表面的羟基经NaIO4氧化形成醛基后与胰蛋白酶的氨基进一步反应,实现胰蛋白酶的固定。在24s内,该酶反应器实现了肌红蛋白和细胞色素c的酶解,经MALDI-TOF MS鉴定,序列覆盖率分别达到65%和79%。     

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...     

酶和细胞的固定化

本文对酶和细胞固定化技术的近期发展作了较为全面的扼要综述。     

固定化酶反应器在高效液相色谱分析中的应用

高效液相色谱法(HPLC)是分离含多组分的复杂体系的最有效手段之一,但检测体系的灵敏度总不能令人满意。折光率检测器通用,但灵敏度不高;紫外、荧光、电化学检测器要求化合物须有生色团、荧光团和电活性基团。近年来,柱前和柱后标记方法发展较快,扩大了这些检测器的应用范围。在标记方     

固定化多酶级联反应器

多酶级联反应在生命活动过程中发挥着重要作用。固定化多酶级联反应器是将不同功能的酶通过物理化学或生物手段固定于特定载体上,以之模拟生物体内多种酶协同作用方式促使底物发生降解和转化等反应的新型仿生催化技术。该技术具有固定化酶的稳定性、可重复利用以及酶级联的高效协同催化等优点,近年来在生物传感、模拟生物学以及生物转化等领域得到越来越多的关注。本文从多酶级联反应原理、反应器制备、级联反应的影响因素及应用等方面对近年来固定化多酶级联反应器的进展进行详细评述,并展望其发展前景。     

原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用

针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其为载体制备了一种新型的固定化酶反应器。用质谱考察了银丝固定化酶反应器(SW-Trypsin)的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的SW-Trypsin的酶解效率较高,酶解标准蛋白牛血清白蛋白(BSA)20 min后,肽段的氨基酸序列覆盖率可达93%,高于传统溶液酶解方法酶解16 h所得79%的覆盖率。使用该固定化酶反应器于一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出良好的稳定性。另外,该固定化酶反应器酶解马心肌红蛋白(MYO)的回收率为87.67%。最后,用SW-Trypsin酶解腾冲嗜热菌全蛋白20 min,所鉴定到的氨基酸序列覆盖率和蛋白数量与同样条件下溶液酶解16 h的结果接近,且零漏切位点肽段的比例更高。加之容易分离的优点,SW-Trypsin在蛋白质组学的应用中具有良好的前景。     

溶解性可调节的酶载体制备和固定化酶的研究

本文利用自由基沉淀聚合反应,合成了甲基丙烯酸-丙烯酰胺-顺丁烯二酸酐三元共聚物,测定了这些共聚物形成水不溶性的大分子氢键复合物的临界pH值.利用共聚物上的酸酐基团,直接进行了木瓜蛋白酶的固定化,得到了具有液相酶与固相酶两者优点的新型修饰酶。     

Analytical applications of reactors containing immobilized enzymes

The theory for analytical packed-enzyme reactors is discussed and it is shown that a 100% conversion efficiency gives many advantages. This concept has been applied to methods for substrate determinations of urea, amino acids, and glucose. Enzyme reactors have also been used in the effluent from a Chromatographic column to enhance selectivity and sensitivity for cholesterol. Enzyme reactors for the determination of inhibitors, e.g. mercury ions, should be designed differently. A low conversion efficiency gives high sensitivity and a linear response.     

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.     

Immobilized‐enzyme reactors integrated with capillary electrophoresis for pharmaceutical research

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized‐enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis‐integrated immobilized‐enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug–enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.     

Immobilized enzyme reactors in proteomics

Fast, efficient characterization of proteins is becoming one of the hottest topics in the bioanalytical community, especially for large-scale proteomic studies. As an attractive approach, protein digestion by enzymes supported on various matrices (referred to as immobilized enzyme reactors, IMERs) has recently attracted much attention.In this article, we present a critical overview of some highly efficient IMERs and related analytical systems. We give major coverage to applications of IMERs in proteomic analysis, including protein-expression profiling, characterization of proteins with post-translational modifications, and protein quantification. We also comment on promising trends for IMERs in proteomics.     

纳米花型酶-无机杂化固定化酶研究进展

酶是一种绿色高效的生物催化剂,被广泛应用于工业生产中,为了更好地提升游离酶的性能,酶固定化技术应运而生.然而,与游离酶相比,固定化酶活性下降以及传质受限一直是酶固定化技术亟待解决的关键问题.作为一种新型酶固定化技术,纳米花型酶-无机杂化固定化酶因具有高比表面积、高酶活性和高催化效率且制备过程简单、绿色无污染而受到广泛关...     

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.     

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.     

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…