毛细管电泳法在酶抑制动力学研究中的应用进展

毛细管电泳法因其分离高效、分析快速和进样微量且柱体不易受污染等特点,被广泛用于酶抑制剂的相关研究中,如酶抑制剂筛选、抑制活性评估及抑制类型判断等方面。本文介绍了近年来毛细管电泳法在酶抑制动力学研究中的发展,包括用于酶抑制动力学研究的测定模式和在酶抑制动力学研究的应用两个部分。     

毛细管电泳技术在线酶分析研究进展

生物酶是生物体内活细胞产生的生物催化剂,控制着生物体的新陈代谢、营养和能量转换等反应过程.建立生物酶活性及催化反应或抑制反应动力学的快速、准确和有效的测定方法,对于理解生物反应过程、药物研发以及疾病诊治等具有重要意义.毛细管电泳技术由于其具有分离效率高、分析速度快、操作简单和样品消耗少等优点,在酶分析研究中越来越受到关注,正逐渐发展为生物酶快速分析的技术平台.根据酶的存在形态,毛细管电泳在线酶分析可分为均相模式(包括电泳媒介微分析法和在线连续监测酶分析)和异相模式(固定化酶反应器),本文综述了近10年来这2种不同模式及其在酶抑制剂筛选、酶动力学研究和底物定量检测等方面的应用.     

电泳中介微分析及其应用进展

介绍了电泳中介微分析(EMMA)的特点及其原理。对EMMA的进样、混合模式、在线反应控制、分离动力学以及检测模式等进行了综合评述。对EMMA在酶活性、酶基体、酶抑制剂或激动剂测定和柱内衍生等方面的应用以及发展前景作了全面介绍。引用文献59篇。     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

3-氨丙基键合开管柱电色谱分离中性芳香族化合物

制备了3-氨丙基键合毛细管开管柱,在此柱上以氨水/甲醇(体积比30∶70)溶液为溶剂,采用毛细管电泳法有效地分离了八种中性芳香族化合物.实验中考察了电色谱条件如:氨水浓度、表面活性剂十二烷基硫酸钠(SDS)浓度及电压等对分离度的影响.结果表明八种中性芳香族化合物在该柱上的分离效果和重现性良好.     

毛细管电泳的原理及应用（第三讲）毛细管电泳的柱技术

毛细管电泳的原理及应用（第三讲）毛细管电泳的柱技术王义明,罗国安（清华大学化学系,清华大学生命科学与工程研究院北京１０００８４）关键词毛细管电泳,柱技术１概述毛细管电泳（ＣＥ）的分离和检测过程均在毛细管内完成,所以说毛细管是ＣＥ的核心部件之一。早期对毛细管的研究集中在毛细管直径、长度、形状和材料方面。采用细柱可减少电流及自热,而且能加快散热,以保持高效分离;但会造成进样、检测及清洗上的困难,也不利于对吸附的抑制,故现在一般采用２５～１００μｍ内径的毛细管。     

毛细管电泳涂层柱技术的进展

毛细管电泳涂层柱是解决蛋白质在毛细管壁吸附的最有效的方法。较为系统地综述了毛细管电泳涂层柱的几种制作方法,指出了毛细管电泳涂层柱（包括毛细管电色谱柱）的发展趋势,３９篇。     

多柱色谱技术进展

在色谱分析过程中，利用串联、并联或串并联结合的方式将多根色谱柱组合起来，可以实现高通量和高分辨的分离效果。相比于传统单柱色谱技术，多柱技术很好地满足了批量样品分析和复杂生物样品分离分析的需求，因此引起了广泛关注。该文对多柱技术在多维分离、芯片色谱、毛细管电泳、固定相筛选以及串联色谱等领域的应用进行了综述，并对其发展前景进行了展望。     

溶胶-凝胶法制备开管毛细管柱的研究进展

介绍了溶胶-凝胶法制备开管毛细管柱的方法,分析了溶胶-凝胶法制备开管毛细管柱的影响因素和特点,总结了溶胶-凝胶技术在毛细管气相色谱柱、毛细管液相色谱柱、毛细管电泳柱及电色谱柱方面的应用进展。     

快速测定酶法动力学拆分中转化率和ee值的新方法

在动力学拆分反应中 ,转化率和对映体过量值( ee值 )是经常需要测量的两个重要参数 .传统的测定转化率的方法包括色谱法、分光光度法和滴定法等 .而测定 ee值主要应用手性色谱柱、手性毛细管电泳、核磁以及旋光法等 .在动力学拆分过程中 ,需要对反应进程进行监测 ,而应用传统的方法进行监测需要同时测定两个量 (转化率和 ee值 ) ,工作量较大 ,且使用手性色谱柱进行监测成本也较高 ,不适合大量实验数据的常规分析 .为此 ,需要发展一种快速、灵活、经济的测量方法 ,用以实验前期筛选条件时的常规分析 .基于此思路 ,我们借助于自己编制的计算机…     

单一粒度法研究固定化酶内扩散限制的酶应动力学

本文提出了用一种粒度的固定化酶,利用在高底物浓度与低底物浓度时分别表现为零级反应与一级反应的特点,研究在连续式反应器中内扩散限制下酶反应动力学的方法。通过在棒条状异构化酶上葡萄糖转化为果糖的反应,测求了酶反应的本征参数以及一级反应速度常数、表观米氏常数、底物扩散系数、效率因子等动力学参数。     

酶的催化多功能性及其在有机合成中的新进展

介绍了近几年来酶的催化多功能性及其应用于有机合成反应研究的最新进展. 包括酶催化多功能性的几种主要类型和相应催化多功能性在Michael加成、Markovnikov加成、羟醛缩合反应、氧化反应及串联反应中的应用.     

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。     

Engineering the Donor Selectivity of D‐Fructose‐6‐Phosphate Aldolase for Biocatalytic Asymmetric Cross‐Aldol Additions of Glycolaldehyde

D ‐Fructose‐6‐phosphate aldolase (FSA) is a unique catalyst for asymmetric cross‐aldol additions of glycolaldehyde. A combination of a structure‐guided approach of saturation mutagenesis, site‐directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800‐fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose‐like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross‐aldol additions of glycolaldehyde to N‐carbobenzyloxyaminoaldehydes to furnish between 80–98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.     

The first example of molecularly imprinted nanogels with aldolase type I activity

The molecular-imprinting approach was used to obtain a nanogel preparation capable of catalysing the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-based mechanism of aldolase type I enzymes. The diketone template used to create the cavity was designed to imitate the intermediate of the aldol reaction and was bound to the functional monomer using a reversible covalent interaction prior to polymerisation. By using a high-dilution polymerisation method, soluble imprinted nanogels were prepared with dimensions similar to those of an enzyme and with the advantage of solubility and flexibility previously unattainable with monolithic polymers. Following template removal and estimation of active-site concentrations, the kinetic characterisation of both imprinted and non-imprinted nanogels was carried out with catalyst concentrations between 0.7 and 3.5 mol %. Imprinted nanogel AS147 was found to have a k(cat) value of 0.25 x 10(-2) min(-1), the highest value ever achieved with imprinted polymers catalysing C--C bond formation. Comparison of the catalytic constants for both imprinted nanogel AS147 and non-imprinted nanogel AS133 gave a ratio of k(cat 147)/k(cat 133)=18.8, which is indicative of good imprinting efficiency and highlights the significance of the template during the imprinting process. This work represents a significant demonstration of the superiority of nanogels, when the molecular-imprinting approach is used, over "bulk" polymers for the generation of catalysts.     

Structure and Mechanism Leading to Formation of the Cysteine Sulfinate Product Complex of a Biomimetic Cysteine Dioxygenase Model

Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3‐His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme–substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work‐up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X‐ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys‐SO₂^? functional group was found to be bound in an η²‐O,O‐coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η²‐O,O‐binding mode for synthetic as well as the natural enzyme.     

An Unusual Terpene Cyclization Mechanism Involving a Carbon–Carbon Bond Rearrangement

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat‐9‐en‐7‐ol synthase (CotB2) synthesizes an unusual 5‐8‐5 fused‐ring structure with six chiral centers from the universal diterpene precursor, the achiral C₂₀ geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2‐catalyzed cyclization that involves a carbon–carbon backbone rearrangement and three long‐range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly ¹³C‐labeled glucose and in vitro reactions of regiospecifically deuterium‐substituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.