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,被广泛地应用于工业生产中,为了更好的提升游离酶的性能,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,且制备简单,绿色无污染受到广泛关注。本文综述了近年来纳米花型酶-无机杂化固定化酶的研究进展,根据纳米花型酶-无机杂化固定化酶的形成特点,将其分为单酶纳米花、双酶纳米花和负载型纳米花。阐述了纳米花型酶-无机杂化固定化酶的制备过程和形成机理并对纳米花型酶-无机杂化固定化酶在食品工业和检测领域的应用进展做出总结。最后,对纳米花型酶-无机杂化固定化酶的发展前景做出展望。     

淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质

分离得到产抗菌聚氨基酸--ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2,从中纯化了ε-聚赖氨酸降解酶,并对其性质进行了研究.结果表明,该酶为膜结合蛋白.为提取该降解酶,先收集菌体细胞并用超声波破碎,细胞膜部分用1.0 moL/L NaSCN溶液溶解.将粗酶液进行Sephadex G100凝胶柱层析分离.用100mmol/L磷酸缓冲液洗脱,收集活性部分.纯化后的样品用SDS-PAGE检测,酶亚基分子量约为54700.酶活力在pH=6.0～9.0间稳定,最适宜pH=7.0.酶的最适温度为30℃,在10～50℃水浴30 min酶活力未见明显下降.研究了不同金属离子对酶活力的影响,结果表明,Zn~(2+),Cu~(2+)和Fe_(3+)可分别提高酶活力29.72%,15.85%和15.08%;但Ag~(+),Hg~(2+),Co~(2+)和Mn~(2+)对酶活力有强烈的抑制作用.Ca2~(2+),K~+和Ba~(2+)对酶活力没有影响.添加4%Tween-80能提高酶活力10%,但EDTA能强烈抑制酶活力.研究结果表明,此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.     

Immobilizing redox enzymes at mesoporous and nanostructured electrodes

Three key challenges are stimulating intensive research in the development of productive direct electron transfer mode enzyme electrodes: proper enzyme orientation, high enzyme loading, and full retention of enzyme activity. In this review, we summarize some significant advances that have been reported in the last years on the design of mesoporous and nanostructured electrodes as enzyme scaffolds and of innovative methodologies for wiring enzymes to electrodes. Particular attention is given to investigations on physical factors that determine a favorable enzyme immobilization, to provide rational guidelines for the design of productive enzymatic electrodes. Finally, some emerging trends focused on the spatial organization of either single enzymes or enzyme cascades are also briefly addressed.     

Which Parameters Affect the Response of the Channel Biosensor?

The important parameters in defining the response of the portable channel biosensor described previously are explored by connecting the portable flow cell to a gravity feed flow system and using a highly defined enzyme immobilization protocol which ensures the enzyme reaction is a surface reaction. The enzyme glucose oxidase (GOD) was immobilized by covalent attachment to a self‐assembled monolayer modified gold surface. As a glucose solution flowed down the rectangular duct defined by the flow cell, it passed over the enzyme layer where the enzyme reaction produced hydrogen peroxide. The hydrogen peroxide was swept further downstream to the detector electrode. The response of such an enzyme electrode was shown to be limited by mass transport of the cosubstrate oxygen to the enzyme layer. Increasing the amount of oxygen in the sample meant the response of the biosensor became limited by the enzyme kinetics. The influence of parameters such as flow rate, height of the channel, enzyme layer length and the gap between the enzyme layer and the detector electrode were explored.     

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。     

Purification and Characterization of the Enzyme Cholesterol Oxidase from a New Isolate of Streptomyces sp.

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.     

二氧化硅纳米与微米颗粒作为固定化酶载体的生物效应

分别将二氧化硅纳米颗粒(SiNPs)与微米颗粒(SiMPs)作为固定化载体, 选择多聚酶牛肝过氧化氢酶(CAT)和单体酶辣根过氧化物酶(HRP)作为酶模型, 通过考察酶固定化后在酶活回收率、热稳定性、 酶促反应最适温度以及酶在水-有机溶剂混合体系中催化能力的变化, 对载体与酶所产生的生物效应差异进行了系统研究. 酶活回收率结果表明, SiNPs显示出比SiMPs优越的对酶无选择性的高生物亲和性, 而SiMPs则能使固定于其上的酶热稳定性大幅度提高, 且二者都能使固定化酶在有机相中的稳定性得到明显增强. 但酶促反应最适温度的变化结果表明, 对不同类型的酶所产生的生物效应则表现出无规律性.     

Parameters important in fabricating enzyme electrodes using self-assembled monolayers of alkanethiols.

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.     

Biochemical studies of soluble and immobilized aldehyde dehydrogenase from yeast

Aldehyde dehydrogenase from baker’s yeast was purified to homogeneity. The soluble and immobilized forms of this enzyme were characterized and compared biochemically. These included steady state kinetics, stability study, fluorescence, and NMR spectroscopy. Evidence of the coenzyme binding to this enzyme in the absence of aldehyde substrates was obtained by fluorescence and NMR studies. A significant quenching of protein fluorescence was observed upon the addition of coenzymes to the aldehyde-free enzyme solution. Significant shifts and broadening of coenzyme proton resonances in the presence of enzyme also indicate the enzyme-coenzyme interactions in the aldehyde-free solution. the enzyme was immobilized on glass beads by three different methods. The immobilized enzyme was found to exhibit physical and biochemical properties similar to those of the soluble enzyme. A system in which the immobilized alcohol, aldehyde, and steriod dehydrogenases are included in an enzyme reactor is described.     

Fiber-entrapped dipeptidyl aminopeptidase

The enzyme dipeptidyl aminopeptidase has been immobilized on cellulose triacetate fibers. The Michaelis-Menten parameters of the entrapped enzyme have been determined and compared to those of the literature for the native enzyme. The entrapped enzyme retains 13% of its activity toward synthetic substrate. The influence of mixing effects on the catalytic properties of the enzyme has been substantiated. The activity appears to be controlled by external mass-transfer.     

淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质

分离得到产抗菌聚氨基酸ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2, 从中纯化了ε-聚赖氨酸降解酶, 并对其性质进行了研究. 结果表明, 该酶为膜结合蛋白. 为提取该降解酶, 先收集菌体细胞并用超声波破碎, 细胞膜部分用1.0 mol/L NaSCN溶液溶解. 将粗酶液进行Sephadex G100凝胶柱层析分离. 用100 mmol/L磷酸缓冲液洗脱, 收集活性部分. 纯化后的样品用SDS-PAGE检测, 酶亚基分子量约为54700. 酶活力在pH=6.0~9.0间稳定, 最适宜pH=7.0. 酶的最适温度为30 ℃, 在10～50 ℃水浴30 min酶活力未见明显下降. 研究了不同金属离子对酶活力的影响, 结果表明, Zn²⁺, Cu²⁺和Fe³⁺可分别提高酶活力29.72%, 15.85%和15.08%; 但Ag⁺, Hg²⁺, Co²⁺和Mn²⁺对酶活力有强烈的抑制作用. Ca²⁺, K⁺和Ba²⁺对酶活力没有影响. 添加4%Tween-80能提高酶活力10%, 但EDTA能强烈抑制酶活力. 研究结果表明, 此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.     

Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory.Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer.Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.     

Comparative studies on physical properties concerned with the stability of unphosphorylated, phosphorylated and proteolytically modified L-type pyruvate kinase from pig liver

A study was made of the effects of some agents on the stability of unphosphorylated pyruvate kinase type L, the enzyme phosphorylated with cyclic 3',5'-AMP-stimulated protein kinase and the subtilisin-modified enzyme form from which the phosphorylatable site had been removed. The phosphorylated pyruvate kinase was found to be the most labile of the enzyme forms at high temperature and in the presence of urea. The circular dichroism spectrum of the phosphorylated enzyme also differed from that of the unphosphorylated and proteolytically modified forms. All three forms of the enzyme showed a high degree of stability over a wide pH range. The unphosphorylated enzyme seemed, however, to be the most sensitive to differences in pH. Only 10% of its maximal activity remained after incubation at pH 10 and 30 degrees C for 30 min, compared with 30% and 75% for the phosphorylated and proteolytically modified forms of the enzyme, respectively. Of the three enzyme forms tested the subtilisin-modified pyruvate kinase was most rapidly inactivated by trypsin. These results taken together suggest that the phosphorylated enzyme has a less ordered structure than the other two enzyme forms studied.     

Photolithographically patterned enzyme membranes for the detection of pesticides and copper(II) based on enzyme inhibition

Summary A non-aqueous and an aqueous photopolymer system with an enzyme are used to prepare photolithographically patterned enzyme membranes for amperometric (thinfilm platinum electrode) and potentiometric (ISFET) sensors based on enzyme inhibition. Flow methods for enzyme inhibition tests are described. The decrease in enzyme (AChE) activity after incubation in a solution of dichlorvos as inhibitor is detected amperometrically. The enzyme urease is immobilized onto the pH-sensitive gate area of an ISFET. Such a biosensor is able to detect copper-(II) in water in the ppm-range without preconcentration.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site‐directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.     

Metal–Organic Framework Disintegrants: Enzyme Preparation Platforms with Boosted Activity

An enzyme formulation using customized enzyme activators (metal ions) to directly construct metal–organic frameworks (MOFs) as enzyme protective carriers is presented. These MOF carriers can also serve as the disintegrating agents to simultaneously release enzymes and their activators during biocatalysis with boosted activities. This highly efficient enzyme preparation combines enzyme immobilization (enhanced stability, easy operation) and homogeneous biocatalysis (fast diffusion, high activity). The MOF serves as an ion pump that continuously provides metal ion activators that greatly promote the enzymatic activities (up to 251 %). This MOF–enzyme composite demonstrated an excellent protective effect against various perturbation environments. A mechanistic investigation revealed that the spontaneous activator/enzyme release and ion pumping enable enzymes to sufficiently interact with their activators owing to the proximity effects, leading to a boost in biocatalytic performance.     

Immobilization of Candida antarctica lipase B on the surface of modified sol–gel matrix

The use of modified sol–gel matrix to immobilize the enzyme Candida antartica lipase B (CALB) was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol–gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88 % attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol–gel supports has higher thermal stability with 2.7 times higher half-life compared to soluble enzymes at 60 °C. This enzyme immobilization system retains the enzyme residual activity even for repetitive use. Hence, the immobilization approach developed recommends its further application.     

Properties of chicken liver phosphofructokinase-2.

Phosphofructokinase-2 was purified to homogeneity from chicken livers by homogenization, polyethylene glycol fractionation and column chromatography on DEAE-Sephadex A-50 and Blue-Sepharose 4B. Some properties of the enzyme were as follows: (i) The saturation curve of the enzyme for fructose 6-phosphate showed hyperbolic and the Km of fructose 6-phosphate was affected by inorganic phosphate while Vmax was not; (ii) the binding of ATP to the enzyme was of negative cooperativity with a Hill coefficient of 0.56; (iii) the activity of the enzyme was completely lost in the presence of EDTA. The enzyme was activated by Mg2+ at low concentrations, but inhibited by Mg2+ at high concentrations; (iv) the enzyme was stable below 30 degrees C and easily lost its activity when the temperature was above 40 degrees C; (v) the activity of the enzyme was stable at the range of pH 7-9, increased at pH 9.0-9.5 and decreased when pH was over 9.5; (vi) the enzyme was sensitive to trypsin and ATP protected the enzyme against the proteolysis of trypsin.     

Purification and characterization of thermostableD-hydantoinase from thermophilicbacillus stearothermophilus SD-1

A thermostable D-hydantoinase of thermophilicBacillus stearothermophilus SD-1 was purified to homogeneity using an immuno-affinity chromatography. The affinity chromatography that employed polyclonal antibody immobilized on Sepharose 4B was simple to operate and gave a purification yield of 60% of enzyme activity. Molecular mass of the enzyme was determined to be about 133.9 kDa by gel filtration chromatography and the molecular mass of the subunit was 54 kDa on SDS-PAGE. Mass spectrometric analyses were also performed for the determination of the molecular mass of the native enzyme and its subunit. The apparent molecular masses were 51.1 and 102.1 kDa for the subunit and native enzyme, respectively. Based on the molecular masses determined by these two methods, it is suggested that the D-hydantoinase exists as a dimeric conformation in the cell. Isoelectric pH of the enzyme was observed to be 4.47. It was found that the enzyme requires one manganese ion per molecule of enzyme for the activity. The optimal pH and temperature for the catalytic activity were about 8.0 and 65‡C., respectively. The half-life of the enzyme was estimated to be 30 min at 80‡C., confirming that the enzyme purified is one of the most thermostable D-hydantoinase reported so far. Kinetic constants of the enzyme for different substrates were also determined.     

Metal–Organic Framework Disintegrants: Enzyme Preparation Platforms with Boosted Activity

An enzyme formulation using customized enzyme activators (metal ions) to directly construct metal–organic frameworks (MOFs) as enzyme protective carriers is presented. These MOF carriers can also serve as the disintegrating agents to simultaneously release enzymes and their activators during biocatalysis with boosted activities. This highly efficient enzyme preparation combines enzyme immobilization (enhanced stability, easy operation) and homogeneous biocatalysis (fast diffusion, high activity). The MOF serves as an ion pump that continuously provides metal ion activators that greatly promote the enzymatic activities (up to 251 %). This MOF–enzyme composite demonstrated an excellent protective effect against various perturbation environments. A mechanistic investigation revealed that the spontaneous activator/enzyme release and ion pumping enable enzymes to sufficiently interact with their activators owing to the proximity effects, leading to a boost in biocatalytic performance.