Nylon shavings enzyme reactor for batch determination of urea

The design and performance of an enzyme reactor (enzyme electrode) which features (i) incorporating nylon shavings onto which an enzyme is covalently bonded, (ii) a flat-surface combination pH electrode for proton monitoring, and (iii) a body providing an injection port for sample injection and washing and stirring capabilities is described. The reactor configuration described here offers good diffusional and partition characteristics which result in relatively fast response, good stability, simplicity of operation, low sample and reagent consumption, and adaptability to flow systems. Application to the determination of urea in standards and physiological salt solutions is demonstrated by use of immobilized urease (EC 3.5.1.5).     

肌苷酶电极生物传感器

为了构建肌苷酶电极生物传感器,以固定化核苷磷酸化酶(EC 2.4.2.1)、黄嘌呤氧化酶(EC 1.2.3.2)与过氧化氢电极组成电流型酶电极生物传感器,用于检测肌苷片中的肌苷,其输出电流可达500nA.结果发现,肌苷测定的线性范围为1-268 mg/L,精度:RSD小于0.14%,响应时间:60 s,使用寿命大于25 d,实际测定肌苷片中肌苷含量回收率:100.8%.由此表明:采用双酶电极法测定肌苷片中的肌苷含量,由于酶促反应专一性高、样品不需分离直接进样分析、处理条件温和、反应时间短暂因而结果较为可靠.     

Comparison of designed and randomly generated catalysts for simple chemical reactions

There has been recent success in designing enzymes for simple chemical reactions using a two-step protocol. In the first step, a geometric matching algorithm is used to identify naturally occurring protein scaffolds at which predefined idealized active sites can be realized. In the second step, the residues surrounding the transition state model are optimized to increase transition state binding affinity and to bolster the primary catalytic side chains. To improve the design methodology, we investigated how the set of solutions identified by the design calculations relate to the overall set of solutions for two different chemical reactions. Using a TIM barrel scaffold in which catalytically active Kemp eliminase and retroaldolase designs were obtained previously, we carried out activity screens of random libraries made to be compositionally similar to active designs. A small number of active catalysts were found in screens of 10(3) variants for each of the two reactions, which differ from the computational designs in that they reuse charged residues already present in the native scaffold. The results suggest that computational design considerably increases the frequency of catalyst generation for active sites involving newly introduced catalytic residues, highlighting the importance of interaction cooperativity in enzyme active sites.     

Model-based optimization of a conductive matrix enzyme electrode

A mathematical model has been developed to describe the mechanism for internal mass transfer and enzyme reaction kinetics of an amperometric conductive matrix enzyme electrode. The model is simplified and solved analytically to arrive at a representation for the response slope in the linear range as well as for the response time. This is the first time that the response time of an enzyme electrode is described by a mathematical model. Simulations give information on how the design parameters influence the performance of the electrode for a glucose oxidase catalyzed sensing reaction process. Based on this information, several designs were constructed and tested showing suitable agreement with theoretical predictions. Finally, an optimized electrode was designed and validated.     

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO₂, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.     

一种免受乙醇干扰的GOD的酶电极

将酶电极应用于发酵糖的测定时。与糖共存的乙醇常常会影响测糖的准确性,通过对葡萄糖氧化酶(GOD)电极的研究,探讨了乙醇对测糖酶电极测定影响,进而研制出抗干扰的GOD酶电极,若是GOD电极的酶膜通过夹心法制备．在乙醇含量为0．1％(V／V)时·即产生显著的影响,使测定结果偏大4．3%,且乙醇的影响随浓度的升高而增大,若用尼龙网固定GOD膜,GOD电极在测定20mmol／L和5mm01／L左右的葡萄糖溶液时,含量高达9％的乙醇仍未对测定产生显著的影响．表现出良好的不受乙醇干扰的特性,并且,该尼龙网GOD电极具有良好的重复性、稳定的响应活性及较长的保存期．     

提高1，1’-二甲基二茂铁石墨糊谷氨酸电极寿命的研究

自从Cass1984年首先报道了二茂铁介铁电极以来,已有不少文献报道了这方面的研究工作。还原态的二茂铁不溶于水,而氧化态的二茂铁却溶于水。这样介体电极在使用过程中会因介体的逐渐丢失而失去作用。为了提高介体的稳定性,已报道了多种方法。 提高酶膜的稳定性是生物传感器研制中的一个重要方面。以前一般采用改进固定化方法与选择固定化载体来提高酶膜的稳定性。本文将报道用DEAE-葡聚糖提高DcFe介体电极稳定性的新方法与用DEAE-葡聚糖和乳糖混合物稳定自产的谷氨酸氧化酶的研究结果。     

Anaerobic column chromatography in the presence of detergents and its application to a bacterial HCN-producing enzyme

An easy method to deoxygenize protein or detergent containing buffer solutions by stirring and sparge flushing with an inert gas is described. The deoxygenation of buffers and the establishment of anaerobic conditions in a column chromatography system was followed continuously with an oxygen electrode at the outlet. All polyethylene tubes were placed inside stainless steel tubes and the interspace flushed with nitrogen. A peristalic pump with silicone rubber tubing, mounted in a small plastic box, was likewise flushed with nitrogen. By this method a very unstable cyanide-producing enzyme preparation, solubilized with Triton X-100 from a Pseudomonas species, could be fractionated on a phenyl Sepharose CL-4B column without loss of activity.     

A rotating disk electrode for kinetic studies of superoxide dismutases: applicability in a wide pH range and for continuous monitoring of enzyme inactivation

A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.     

A simple, sensitive, and accurate alcohol electrode

The construction and performance of an enzyme electrode is described which specifically detects lower primary aliphatic alcohols in aqueous solutions. The electrode consists of a commercial Clark-type oxygen electrode on which alcohol oxidase (E.C. 1.1.3.13) and catalase were immobilized. The decrease in electrode current is linearly proportional to ethanol concentrations between 1 and 25 ppm. The response of the electrode remains constant during 400 assays over a period of two weeks. The response time is between 1 and 25 min. Assembly of the electrode takes less than 1 h.     

Enzyme-entrapping membranes for biosensors obtained by radiation-induced polymerization

A new method of physically immobilizing enzymes in poly(2-hydroxyethyl methacrylate) membranes was developed in order to obtain suitable biosensors. It was possible to prepare an enzyme sensor based on an oxygen Clark electrode and on glucose oxidase immobilized by low-temperature gamma radiation-induced polymerization. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the resulting biosensor are summarized. The determination of glucose in standard solutions was carried out and a linear calibration curve, with an R² value of 0·9993, from the detection limit 5 × 10⁻⁵ to 1·2 × 10⁻³ was obtained. The biosensor was employed to analysis of control sera and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

Kappa乘积法在寡聚体变构酶反应速度方程中的应用

寡聚体变构酶所催化的反应中,酶与底物的结合是逐步的,形成链式反应.因此,酶与底物结合的每一种形式可用一组相应的速度常数乘积来表示,这是根据平衡理论建立微分方程组所导出来的,本文应用Kappa乘积法简易导出MWC及KNF模式的速度方程,并对这种方法应用范围的扩大作了讨论.     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

A computational method for design of connected catalytic networks in proteins

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side‐chain functional groups which maximize transition‐state stabilization, and then searching for sites in protein scaffolds where the specified side‐chain–transition‐state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen‐bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw‐like two‐dimensional representation of the transition state with hydrogen‐bond donors, acceptors, and covalent interaction sites indicated, and all placements of side‐chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen‐bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully‐connected active sites in protein scaffolds. The method generates many fully‐connected active site solutions for a set of model reactions that are promising starting points for the design of fully‐preorganized enzyme catalysts.     

Partial characterization and post-feeding activity of midgut aminopeptidase in the human body louse, Pediculus humanus humanus

Abstract. A leucine aminopeptidase was found in the midgut of the human body louse, Pediculus humanus humanus L. (Anoplura: Pediculidae). The enzyme is activated by the bloodmeal with a pH optimum at 8. The enzyme is soluble in both aqueous and detergent-containing solutions. The two forms of the enzyme had the same Km but exhibited different catalytic activities with regard to Vmax values in these solutions. The enzyme is inhibited competitively by a substrate analogue 1,10-phenanthroline and by Mn²⁺ ions in the presence and absence of detergent.     

Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

Role of mediators in electron transport from glucose oxidase redox centre to electrode surface in a covalently coupled enzyme paste electrode

We have studied the glucose oxidase immobilized carbon paste electrodes in the presence and absence of small mediator molecules. We have used p-benzoquinone and riboflavin as mediators in our studies. The effect of mediator molecules on the electron transfer between the enzyme redox centre and the electrode surface was explained from the cyclic voltammograms and rotating disk electrode data. In the absence of oxygen, we have noted that the mediators play a central role in the electron transfer. We have also proposed a possible mechanism for the electron transfer from enzyme active site to the electrode surface via mediators, based on our observations. Dedicated to the memory of Professor J Das     

Design and response characteristics of an enzyme electrode for measurement of penicillin in fermentation broth

A principle for the construction of an autoclavable enzyme electrode is presented. Some characteristics of a penicillin electrode constructed according to this principle are given. The response time is ˜1 min. The response to increasing concentrations of penicillin is linear in buffered samples but logarithmic in unbuffered samples. The reason for the linearity is discussed. A local pH decrease in the enzyme, which is a is a consequence of the enzymatic reaction, reduces the buffering capacity within the electrode and thus increases the sensitivity. It is suggested that this increased sensitivity eliminates the logarithmic response predicted by the Nernst equation for a pH-based enzyme electrode.     

Electrochemical oxidation of water by a cellobiose dehydrogenase from <Emphasis Type="BoldItalic">Phanerochaete chrysosporium</Emphasis>

Cellobiose dehydrogenase (CDH) is a redox protein containing two electron transfer centers; a flavin coenzyme performing a two-electron transfer reaction and an iron-heme coenzyme facilitating single-electron transfer. Purified CDH from Phanerochaete chrysosporium was immobilized on a pyrolytic graphite electrode and electron transfer from cellobiose to the electrode was generated. With cellobiose present during cyclic voltammetry, this novel enzyme/electrode system exhibited sharp, stable oxidation peaks with slower, though equivalent, reduction peaks. During cyclic voltammetry without substrate, the enzyme was rapidly oxidized during the initial scan, with no corresponding enzyme reduction during the reducing half of the cycle. After resting for several hours in aqueous buffer, the full oxidation current appeared again. These results suggest that the CDH is reduced by water splitting, albeit at a slow rate.