Synthesis with immobilized enzyme of the most important sialic acid.

Starting from a crude mixture of N-acetylmannosamine and N-acetylglucosamine, N-acetylneuraminic acid has been easily synthesized on 2.8 millimoles scale, with one immobilized enzyme.     

Functionality of dielectrophoretically immobilized enzyme molecules

The enzyme horseradish peroxidase has been immobilized on nanoelectrode arrays by alternating current dielectrophoresis (DEP). Preservation of its enzymatic function after field application was demonstrated by oxidizing dihydrorhodamine 123 with hydrogen peroxide as co‐oxidant to create its fluorescent form, rhodamine 123 (Rh123). Localization of the fluorescently labeled enzyme and its product was conducted by fluorescence microscopy. Nanoelectrodes were prepared as tungsten pins arranged in square arrays. Experimental parameters for dielectrophoretic immobilization were optimized for even enzyme distribution and for enzymatic efficiency. Enzyme activity was quantified by determination of fluorescence intensities of immobilized enzyme molecules and of Rh123 produced. These results demonstrate that DEP can be applied to immobilize enzyme molecules while retaining their activity and rendering any chemical modifications unnecessary. This introduces a novel way for the preparation of bioactive surfaces for processes such as biosensing.     

On the nature of immobilized tris(carboxymethyl)ethylenediamine

Immobilized tris(carboxymethyl)ethylenediamine (TED) (also known as ethylenediamine-N,N,N'-triacetic acid) serves as a stationary phase for fractionation of proteins and the preparation of metal-free proteins by immobilized metal-ion affinity chromatography. The Cu(II) complex of commercially available immobilized TED has been characterized by elemental, electrochemical and spectroscopic techniques. There is a large discrepancy between the theoretical capacity determined from the nitrogen content and the experimental capacities determined by atomic-absorption spectroscopy and anodic stripping voltammetry (ASV), indicating that a substantial portion of the immobilized ligand is not binding Cu(II). In addition, the titration of immobilized TED with Cu(II), monitored by ASV, suggests that more than one ligand is involved in binding Cu(II). A comparison of the EPR spectrum for immobilized Cu(II)-TED with spectra for various model complexes shows that the major immobilized ligand is ethylenediamine-N,N'-diacetic acid (EDDA) with immobilized TED present only as a minor component. The complexation constant for Cu(II) is close to the value for Cu(II)-EDDA in solution. The formation of EDDA is consistent with the method for synthesizing immobilized TED.     

Flow-injection method for the determination of serine using immobilized enzyme

A flow-injection method for the determination of serine using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver is described. Ammonia produced from the enzymatic reaction is coupled with hypochlorite and phenol in an alkaline medium yields a blue product due to the indophenol anion formation, which is the basis of a spectrophotometric detection at 640 nm. The limit of detection (2×blank noise) is 0.01 mM with a sample throughput of 25 h⁻¹. Calibration graph is linear in the range 0.2–1.0 mM, with relative standard deviation 0.6–1.0%.     

Isotope effects and the nature of enantioselectivity in the shi epoxidation. The importance of asynchronicity

The epoxidation of beta-methylstyrene catalyzed by the Shi fructose-derived ketone is studied using experimental kinetic isotope effects and DFT calculations. The observation of a large beta olefinic (13)C isotope effect and small alpha carbon isotope effect is indicative of an asynchronous transition state with more advanced formation of the C-O bond to the beta olefinic carbon. By varying the catalyst conformation and alkene orientation, diverse transition structure geometries were located calculationally, and the lowest-energy structure leads to an accurate prediction of the isotope effects. Given this support for the accuracy of the calculations employed, the nature of enantioselectivity in this and related epoxidations is considered. The lowest-energy transition structures are generally those in which the differential formation of the incipient C-O bonds, the "asynchronicity," resembles that of an unhindered model, and the imposition of greater or less asynchronicity leads to higher barriers. In reactions of cis-disubstituted and terminal alkenes using Shi's oxazolidinone catalyst, the asynchronicity of the epoxidation transition state leads to increased steric interaction with the oxazolidinone when a pi-conjugating substituent is distal to the oxazolidinone but decreased steric interaction when the pi-conjugating substituent is proximal to the oxazolidinone. Overall, the asynchronicity of the transition state must be considered carefully to understand the enantioselectivity.     

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…     

Activity of an enzyme immobilized on polyelectrolyte multilayers

The immobilization of α-chymotrypsin on the surface of boron silicate glass microspheres is conducted via the technique of multilayer adsorption of polyelectrolytes. It is shown that the enzyme is adsorbed on both positively and negatively charged surfaces and its activity is partially preserved relative to that in solution. The activity of the enzyme depends on the number of polyectrolyte layers preliminarily adsorbed on glass microspheres and on the charge of the surface. The activity of α-chymotrypsin adsorbed on the negatively charged surface is four times higher than the activity of this enzyme adsorbed on a positively charged surface.     

Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process.     

Comparison of immobilized enzyme reactors for flow-injection systems

Straight, coiled, beaded, and packed-bed reactors containing immobilized glucose oxidase or l-(+)-lactate dehydrogenase were compared in terms of reaction rate, sensitivity, sample throughput, and sample dispersion. The enzymes were covalently attached to the inside walls of 5.0-cm long, 1.12-mm i.d. nylon tubes, and the resulting reactors were tested in a flow-injection system. The beaded enzymatically-active reactors (BEARs) were filled with solid glass beads of 0.5-mm or 1.0-mm diameter. Reactors with the larger beads had 2–4 times the activity, twice the sensitivity, and better throughput than the open reactors; they also minimized the physical and chemical contributions to dispersion. Packed-bed reactors were superior in the lactate determination, but the beaded reactors were better for the determination of glucose. With BEARs containing 1.0-mm beads, glucose was determined in the range 10–800 μM with a conversion efficiency of 0.056 mol of product per mole of substrate; for lactate, the range was 8–64 μM with a conversion efficiency of 0.13 mol mol^?1.     

Multiquantum processes in the enzyme molecules immobilized on biological membranes

A physical model of an oxidation–reduction reaction for immobilized enzyme is considered. The influence of the electric-field intensity of a biological membrane on the enzyme reaction rate is analyzed. It is shown that the low-frequency dipole-active vibration of the oppostite charge groups of the substrate and enzyme relative to each other leads to multiquantum excitation of the system over the conformational degree of freedom. This excitation provides an abrupt increasing of the tunnel decay rate of the substrate–enzyme complex on a free product and a free enzyme. This way of the reaction is more probable in comparison with the usual overbarrier process. Some consequences for the immobilized cytochrome P-450 are discussed. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 66 : 255–260, 1998     

Development and characterization of an immobilized enzyme reactor based on glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.     

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。     

A physico-chemical analysis of soluble and immobilized enzyme stabilization

The literature and our experimental data on the effect of chemical modification and immobilization on the thermostability of enzymes are analyzed. The effect of various factors causing changes in the stability of enzymes after their modification or immobilization is demonstrated. It is shown that changes in the temperature dependence of the inactivation rate constant are associated with the change in the effective values of thermodynamic activation parameters for the inactivation processes. An increase in the activation energy of thermoinactivation, E_a, leads to the stabilization of a modified or immobilized enzyme at temperatures below the iso-kinetic temperature (“low-temperature” stabilization) and a decrease inE _a entails a “high-temperature” stabilization of enzymes. It is shown that with immobilized enzymes the high-temperature stabilization is invariably observed.     

Preparation of chitosan nanoparticles as carrier for immobilized enzyme

This work investigated the preparation of chitosan nanoparticles used as carriers for immobilized enzyme. The morphologic characterization of chitosan nanoparticles was evaluated by scanning electron microscope. The various preparation methods of chitosan nanoparticles were discussed and chosen. The effect of factors such as molecular weight of chitosan, chitosan concentration, TPP concentration, and solution pH on the size of chitosan nanoparticles was studied. Based on these results, response surface methodology was emploved. The results showed that solution pH, TPP concentration, and chitosan concentration significantly affected the size of chitosan nanoparticles. The adequacy of the predictive model equation for predicting the magnitude orders of the size of chitosan nanoparticles was verified effectively by the validation data. Immobilization conditions were investigated as well. The minimum particles size was about 42±5 nm under the optimized conditions. The optimal conditions of immobilization were as follow: one milligram of neutral proteinase was immobilized on chitosan nanoparticles for about 15 min at 40°C. Under the optimized conditions, the enzyme activity yield was 84.3%.     

Development of novel microscale system as immobilized enzyme bioreactor

This study involves a novel method for immobilized enzyme catalysis. The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry. Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material. Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/SU-8 molds. The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system. Transverse features also containing enzymes were molded directly into the channels.     

Integration of on-column immobilized enzyme reactor in microchip electrophoresis

A simple method integrating an immobilized enzyme reactor into a microchip electrophoresis device was developed. The enzyme immobilization into a microchip was performed by spotting and drying a drop of dissolved nitrocellulose (NC) on a glass substrate, and adsorbing enzyme on the reconstituted NC membrane. This enzyme-immobilized glass plate was assembled with a polydimethylsiloxane substrate on which the separation channel was fabricated. The advantage of this method is the ability to easily change the position and size of the reactor within the microchip electrophoresis device. A beta-galactosidase reaction was demonstrated with fluorescein di-beta-D-galactopyranoside using this integrated on-column enzyme reactor. A successful electrophoretic separation of its hydrolysis products, i.e., fluorescein mono-beta-D-galactopyranoside (FMG) and fluorescein, was achieved. Enzyme kinetics and inhibition of the beta-galactosidase using FMG and 2-phenylethyl beta-D-thiogalactoside, respectively, were also studied with microchip electrophoresis.     

Dispersive analysis of immobilized enzyme reactors by flow-through microfluorometry

The performance of immobilized enzyme reactors depends not only on the intrinsic activity of the enzymes used, but also upon the attributes of the particles used for the immobilization of enzymes. Investigations of the reaction kinetics of these systems involve the analysis of their heterogeneity.     

共轭效应和芳香性本质的争论和它们的历史发展

“共轭效应是稳定的”是有机化学的最最基本原理之一。但是,自30年代起,键长平均化,4N＋2芳香性理论,苯环D~6~h构架的起因,分子的构象和共轭效应的因果关系,π-电子离域的结构效应等已经受到了广泛的质疑。其中,最引人注目的是Vollhardt等合成了中心苯环具有环己三烯几何特征的亚苯类化合物,Stanger等合成了键长平均化,但长度在0.143～0.148nm的苯并类衍生物。最近(1999年),Stanger又获得了在苯环中具有单键键长的苯并类化合物。在理论计算领域,争论主要表现在计算方法上,集中在如何将作用能分解成π和σ两部分。随着论战的发展,作用能分解法在有机化学中的应用不断地发展和完善,Huckel理论在有机化学中的绝对权威也受到了挑战。为此,简要地介绍了能量分解法的发展史,对kollma法的合理性提出了质疑。此外特别介绍了我们新的能量分解法,及在共轭效应和芳香性的研究中的新观点和新的思维模式。     

The importance of model studies in computational organic synthesis.

We explore a model, the "synthesis engine", for synthesis of arbitrarily complex organic structures in the context of library construction of Grignard cycle compounds. We use the simplest possible governing logic and show that random synthesis produces an extremely uneven distribution of products over several target structure types. We show that the question of "synthetic power" may be addressed computationally in this model system.     

Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (termed FLAG), was genetically fused to the C-terminus of subtilisin BPN′ (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, k_cat/K_M, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.