Enzymatic methods of analysis: Novel approaches and applications

The results of a series of investigations dealing with the development of enzymatic methods for determination of biologically active compounds, viz., inhibitors, activators, and substrates of native and immobilized enzymes of the oxidoreductase (peroxidases, alcohol dehydrogenases) and hydrolase (alkaline and acid phosphatases) classes isolated from diverse sources are summarized. Novel original approaches, proposed by the authors, for improving the sensitivity, selectivity, and rapidity of the methods are discussed. Numerous examples of application of the developed enzymatic procedures for the analysis of a wide range of samples are given. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 583–597, April, 2007.     

Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis

For most separations-based analyses of glycoprotein oligosaccharides, the first step is release of the oligosaccharides from the polypeptide. Historically, O-linked and N-linked oligosaccharides have been released from glycoproteins using chemical means, such as alkaline degradation (β-elimination) or hydrazinolysis. In the last two decades, a growing repertoire of enzymes, including endoglycosidases and glycoamidases, able to release glycoprotein oligosaccharides under mild conditions, have become available. This review traces the discovery characterization and use of these glycoprotein oligosaccharide releasing enzymes. Emphasis is placed on providing information of practical value for the researcher wishing to incorporate enzymatic oligosaccharide release into their study of glycoprotein oligosaccharide structure and function.     

Enzymatic digestion and chromatographic analysis of arsenic species released from proteins

A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAs^III or MMA^III during the protease digestion process. Up to 61% DMA^III (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMA^V). The arsenic species in the red blood cells of control rats was present as DMA^III complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity.     

Enzymatic self-wiring

Redox conducting polymers “Wires” have been widely used as a electron mediators between enzyme active sites and electrode surfaces in electrochemical biosensors. We report that a peroxidase is able to generate a molecular wire through its own enzymatic catalyzed reaction. The catalytic reaction is the polymerization of aniline to form conducting polyaniline. The polyaniline molecular wire is then capable of transducing the enzyme’s catalytic turnover into an electrochemical signal. In effect we demonstrate the selective bridging of the gap between nano and macroscales in a functional fashion (electrical conductivity) using the catalytic capabilities of the nanostructure.     

Enzymatic activity enhancement of non-covalent modified superoxide dismutase and molecular docking analysis

The enzyme activity of superoxide dismutase was improved in the pyrogallol autoxidation system by about 27%, after interaction between hydroxypropyl-β-cyclo-dextrin and superoxide dismutase. Fluorescence spectrometry was used to study the interaction between hydroxypropyl-β-cyclodextrin and superoxide dismutase at different temperatures. By doing this, it can be found that these interactions increase fluorescence sensitivity. In the meantime, the synchronous fluorescence intensity revealed the interaction sites to be close to the tryptophan (Trp) and tyrosine (Tyr) residues of superoxide dismutase. Furthermore, molecular docking was applied to explore the binding mode between the ligands and the receptor. This suggested that HP-β-CD interacted with the B ring, G ring and the O ring and revealed that the lysine (Lys) residues enter the nanocavity. It was concluded that the HP-β-CD caused specific conformational changes in SOD by non-covalent modification.     

Enzymatic analysis by means of the air-gap electrode determination of urea in blood

The newly developed air-gap electrode has been used for enzymatic assay of urea in serum and whole blood; analyses can be done accurately, reliably, simply and quickly. The determination is highly selective because the electrode senses only the ammonium ion, which is selectively released from urea by urease in a preliminary rapid incubation step. The reproducibility of the determination (standard deviation less than 2.4%) is sufficient for clinical purposes; the linear range of the method is 10^-2–lO^-4M urea. Since the electrode actually never touches the sample solution, the problems caused by the presence of proteins, blood cells etc. do not arise.     

酶生物燃料电池

综述了酶生物燃料电池的研究进展,特别是近年来在电池结构、性能以及电池的正极和负极研究等方面的成果。详细介绍了酶燃料电池中酶电极的制备方法和用于酶电极修饰材料的研究现状,对酶燃料电池研究中需要解决的问题和发展方向进行了探讨。     

Enzymatic microreactors for the determination of ethanol by an automatic sequential injection analysis system

A sequential injection analysis system with two enzymatic microreactors for the determination of ethanol has been designed. Alcohol oxidase and horseradish peroxidase were separately immobilized on glass aminopropyl beads, and packed in 0.91-mL volume microreactors, working in line with the sequential injection analysis system. A stop flow of 120 s was selected for a linear ethanol range of 0.005-0.04 g/L +/- 0.6% relative standard deviation with a throughput of seven analyses per hour. The system was applied to measure ethanol concentrations in samples of distilled and nondistilled alcoholic beverages, and of alcoholic fermentation with good performance and no significant difference compared with other analytical procedures (gas chromatography and high-performance liquid chromatography).     

酶催化反应研究进展

简要评述了近年来酶催化反应的进展,以及近年来5种不同反应体系(有机溶剂体系、反胶束体系、低共熔体系、超临界流体体系和气相体系)中酶催化反应的进展,并对不同体系中酶促反应的主要影响因素、酶催化底物的拓宽和模拟酶研究及酶催化的手性合成等作了简要概述。参考文献52篇。     

Enzymatic synthesis of monolaurin

The aim of this study was to produce monolaurin utilizing a commercial immobilized lipase (Lipozyme IM-20; Novo Nordisk, Bagsvaerd, Denmark) through the direct esterification of lauric acid and glycerol in a solvent-free system. The influence of fatty acid/glycerol molar ratio, temperature, and Lipozyme (IM-20) concentration on the molar fraction of monolaurin were determined using an experimental design. The best conditions employed were 55°C, lauric acid/glycerol molar ratio of 1.0, and 3.0% (w/w) enzyme concentration. The final product, obtained after 6 h of reaction, was 45.5% monolaurin, 26.8% dilaurin, 3.1% trilaurin, and 24.6% lauric acid. The reusability of the enzyme was also studied.     

Enzymatic determination of anions

Publications devoted to the determination of a number of anions by enzymatic methods are reviewed. The opportunities of utilizing native and immobilized enzymes for the determination of anions in medical, environmental, and food samples are considered.     

Enzymatic polymerization of phenols

The horseradish peroxidase-catalyzed oxidative polymerization of substituted phenols with the use of hydrogen peroxide as an oxidizer in an aqueous-organic medium has been studied. The presence of an aqueous buffer controlling activity of the catalyst is the necessary condition for this reaction. The rate of the process decreases with a reduction in the fraction of water in the reaction mixture. It has been shown that the oxidizer should be gradually added in portions to the reaction mixture. The resulting oligomers convert into high-molecular-mass crosslinked products under heating.