Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase

A differential-type amperometric biosensor based on conventional thick-film technology has been developed for breath alcohol measurement. The amperometric breath alcohol biosensor utilizes the alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) cofactor which produce reduced NADH as a product of the oxidation of alcohol. The biosensor was designed in a differential format consisting of a common Ag/AgCl reference electrode, an active working electrode containing the ADH, and the inactive working electrode containing only bovine serum albumin instead of the ADH. The differential signal between the active working electrode and the inactive working electrode minimized the interference from a large number of oxidizable species present in a person's breath. Prior to the amperometric measurement the biosensor was hydrated simply by dipping it into a phosphate buffer solution at pH 7.4. The NADH produced from the enzymatic reaction was oxidized at the working electrode biased at a potential of 470 mV vs. an on-board Ag/AgCl reference electrode. The biosensor can measure a person's breath alcohol over the concentration range 20–800 ppm routinely required in a test of drunken driving.     

从酵母细胞中分离纯化醇脱氢酶

醇脱氢酶(ADH)用于食品工业，还是酶法测定乙醇或乙醛含量的工具酶。沉淀法和层析法至今仍然广泛用于分离蛋白质和酶。用离子交换层析、亲和层析等方法经过4～5个纯化步骤后，分离纯化ADH和其它蛋白质，纯化倍数可达100多倍。我们从酵母细胞中分离出醇脱氢酶，并通过硫酸铵分段盐析、凝胶层析以及离子交换层析对该酶进行纯化，用较少的步骤得到了相对高的纯化倍数。     

SnO2纳米材料的制备及其对丙烯醛还原反应的催化性能

 采用柠檬酸法制备了SnO2纳米材料,并采用差热-热重、 X射线衍射、透射电子显微镜和氮吸附等技术对其进行了表征,考察了其在丙烯醛还原制烯丙醇反应中的催化性能. 结果显示,制备的SnO2纳米材料粒径(尺寸为8～10 nm)均一, 并具有多孔性质(孔径4～6 nm); 在丙烯醛还原制烯丙醇反应中具有良好的催化性能,催化剂的初始活性很高,丙烯醛的初始转化率可达93.6%, 烯丙醇的最高选择性为60%, 烯丙醇的最高收率为47%. 表面结焦是造成催化剂失活的主要原因.     

CTAB-己醇-辛烷体系分离纯化醇脱氢酶的反萃研究

本文报道了用反胶束体系萃取醇脱氢酶(ADH)的研究结果。在此萃取体系中,以十六烷基三甲基溴化铵(CTAB)作为表面活性剂,辛烷和己醇作为溶剂和助溶剂。考察了表面活性剂及助溶剂浓度,水相pH值对ADH萃取的影响。详细讨论了离子强度、异丙醇浓度和振荡时间对ADH反萃的影响。确定了最佳反向萃取条件。     

Enantioselective oxidation of secondary alcohols at a quinohaemoprotein alcohol dehydrogenase electrode

Quinohaemoprotein alcohol dehydrogenase fromComamonas testosteroni was co-immobilized with a redox polymer (a poly(vinylpyridine) complex functionalized with osmium bis(bipyridine) chloride) on an electrode. The enzyme electrode readily oxidizes primary alcohols and secondary alcohols with maximum current densities varying between 0.43 and 0.98 A m^-2 depending on the substrate and the operation temperature. The affinity of the enzyme for aliphatic alcohols increases with the chain length of the substrate (i.e., 1-pentano1 [K_m = 0.006 mM] is a much better substrate than ethanol [K_m= 2.2 mM]). The same property is observed for secondary alcohols in the series 2-propanol (K_m = 22 mM) to 2-octano1 (K_m = 0.05 mM). The enzyme electrode is enantioselective in the oxidation of secondary alcohols. A strong preference is observed for the S-2-alcohols; the enantioselectivity increases with increasing chain length. The enantiomeric ratio (E) increases from 13 for (R,S)-2-butanol to approximately 80 for (R,S)-2-heptanol and (R,S)-2-octanol. This makes the enzyme electrode, potentially, a powerful tool for the preparation of a large range of alkanones and/or for the (kinetic) resolution of racemic alcohols.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

The O to S substitution in urea brings inhibition activity against thiocyanate dehydrogenase

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微波固相法制备ZnCl2/NaY催化剂ZnCl2/NaY在月桂烯Diels-Alder反应中的催化性能

比较了微波固相法、普通加热法和简单混合法制备的ZnCl2/NaY及溶液离子交换法制备的ZnNaY在月桂烯与丙烯醛Diels-Alder反应中的催化性能,发现微波固相法制备的催化剂表现出最高的环加成选择性和区域选择性.通过对微波固相法制备的催化剂中ZnCl2负载量的考察,揭示了微波诱导ZnCl2在NaY分子筛表面形成了不同催化活性中心的特点,分散在分子筛表面的ZnCl2具有更高的催化活性和区域选择性.微波辐射可成功地将ZnCl2固载于NaY分子筛中,当ZnCl2负载量为6.3%时,ZnCl2与NaY分子筛主要发生固态离子交换反应;当ZnCl2负载量达25%时,催化活性提高了3倍,区域选择性提高了1倍以上.而且,以微波固相法制备的ZnCl2/NaY催化剂,其环加成选择性和区域选择性随反应温度的升高而下降.     

Preparation and partial characterization of a soluble site-to-site directed enzyme complex composed of alcohol dehydrogenase and lactate dehydrogenase

A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase. By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase, the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site oriented.     

Effect of methyltin trichloride on the activity of lactate dehydrogenase

The effect of MeSnCl₃, which is a highly toxic compound, on the activity of L-lactate:NAD oxidoreductase (lactate dehydrogenase) in the extract from the liver of Russian sturgeon (Asipenser gueldenstaedtiB.). Noncompetitive inhibition of the enzymatic reaction was discovered. This can be due to a change in the enzyme conformation caused by the action on the thiol groups, important for enzyme activity.     

Alcohol sensor based on membrane-bound alcohol dehydrogenase

Ethanol is determined by a sensor system using purified, immobilized mernbrane-bound alcohol dehydrogenase frorn Gluconobacter suboxydans, attached to a platinum disk electrode (3 mm diameter), and covered with a dialysis membrane. Hexacyanoferrate (III) is used as the redox acceptor. To correct for the influence of interfering substances, this alcohol sensor is compensated by a control electrode which has no immobilized enzyme. The potential of these platinum electrodes was set at + 350 mV vs. Ag/AgCl. Linearity was observed in the range 0.1–5 mM ethanol, the response time was less than 5 min, the maximum sensitivity was obtained at 45°C and the optimum pH was in the range 4.5–5.5. The sensitivity decreased to 80% of the initial value after 1 month at 30°C. When the alcohol sensor system was applied to the determination of ethanol in alcoholic beverages, a good correlation was obtained between the results and those obtained by gas chromatography.     

Recent advances in quantum mechanical/molecular mechanical calculations of enzyme catalysis: hydrogen tunnelling in liver alcohol dehydrogenase and inhibition of elastase by α-ketoheterocycles

 Hybrid quantum mechanical (QM)/molecular mechanical (MM) calculations are used to study two aspects of enzyme catalysis, Kinetic isotope effects associated with the hydride ion transfer step in the reduction of benzyl alcohol by liver alcohol dehydrogenase are studied by employing variational transition-state theory and optimised multidimensional tunnelling. With the smaller QM region, described at the Hartree–Fock ab initio level, together with a parameterised zinc atom charge, good agreement with experiment is obtained. A comparison is made with the proton transfer in methylamine dehydrogenase. The origin of the large range in pharmacological activity shown by a series of α-ketoheterocycle inhibitors of the serine protease, elastase, is investigated by both force field and QM/MM calculations. Both models point to two different inhibition mechanisms being operative. Initial QM/MM calculations suggest that these are binding, and reaction to form a tetrahedral intermediate, the latter process occurring for only the more potent set of inhibitors. Recieved 3 October 2001 / Accepted: 6 September 2002 / Published online: 31 January 2003 Contribution to the Proceedings of the Symposium on Combined QM/MM Methods at the 222nd National Meeting of the American Chemical Society, 2001 Correspondence to: I. H. Hillier Acknowledgements. We thank EPSRC and BBSRC for support of the research and D.G. Truhlar for the use of the POLYRATE code.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulfonate films

Immobilization of urease and glutamate dehydrogenase enzymes in electrochemically prepared polypyrrole-polyvinyl sulfonate films (PPY-PVS) was carried out using physical adsorption and electrochemical entrapment techniques. Detailed studies on optimum pH, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy of the enzymes in the immobilized state were conducted. The value of the apparent Michaelis-Menten constant was experimentally determined to be 2.5 and 2.7 for physically adsorbed and electrochemically entrapped urease in PPY-PVS films, respectively.     

丙烷选择氧化制丙烯醛MoVTeO/SiO2催化剂结构与性能研究

考察了MoVTeO/SiO₂系列催化剂对丙烷选择氧化制丙烯醛反应的催化性能,结合XRD,Raman和TPD等表征结果研究了催化剂结构、表面性质与催化性能之间的关系.结果表明,以Mo为主要活性组分的催化剂(MoV_0.2Te_0.1/SiO₂)具有较好的催化性能.在V,Te组分存在下,Mo物种的分散度和MoO₃的可还原性能提高.以表面钼酸盐和多钼酸盐类形态存在的高分散Mo物种有助于提高催化活性,而催化剂较弱的表面酸性对丙烯醛的生成有利.     

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 μM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e⁻/H⁺ acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.     

Te掺杂对MoPO/SiO2催化剂上丙烷选择氧化制丙烯醛反应的影响

 考察了Te的添加及Te含量对MoPO/SiO2催化剂催化丙烷选择氧化制丙烯醛反应性能的影响. MoPO/SiO2催化剂对丙烷选择氧化制丙烯醛反应的中间产物丙烯选择性较高,而Te的添加促进了丙烯向丙烯醛的转化. N2吸附、XRD、Raman、XPS、H2-TPR、丙烷脉冲、NH3-TPD和Py-IR等实验结果表明,催化剂添加Te后虽然比表面积有所下降,但单位比表面积上的酸量增加,催化剂低温可还原性得到改善,从而有利于丙烷的转化. Te可能有利于丙烯α-H的脱除和/或烯丙基的插氧反应.     

Selective inhibition of 6-phosphogluconate dehydrogenase from Trypanosoma brucei

A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.     

DFT法研究3-羟基丙烯醛的双键旋转异构反应机理

利用密度泛函理论(DFT)分别在B3LYP/6-31G**和B3LYP/6-311＋＋G**的计算水平上优化了基态3-羟基丙烯醛分子在双键旋转异构反应过程中的平衡态以及过渡态的几何构型，分析了反应过程中键参数的变化，计算了该反应的内禀反应坐标(IRC)，发现在重排反应途径上存在一个四元环骨架的中间体.通过振动分析对平衡态和过渡态进行了确认，并得到了零点能.计算结果表明，基态3-羟基丙烯醛分子的双键旋转异构反应经过两步完成，第一步反应位垒稍高，第二步反应位垒较低，存在着发生重排反应的可能性.     

Coupled hydroperoxide lyase and alcohol dehydrogenase for selective synthesis of aldehyde or alcohol

The main objective of this work was to improve the selective synthesis of a volatile compound: aldehyde or alcohol using a coupled-enzyme system. A novel method of synthesis of C₆-aldehyde or alcohol was carried out in the presence of hydroperoxide lyase (HPLS) activity coupled to alcohol dehydrogenase (ADH) activity. After cleavage of the initial substrate, hydroperoxy fatty acid catalyzed by HPLS, the second enzyme, ADH, can catalyze the reduction of the aldehyde to the corresponding alcohol, or the oxidation of contaminating alcohol into aldehyde, depending on the cofactor present in the medium (oxidized or reduced form). We succeeded in improving the synthesis of one of the products. When coupling HPLS to NADP, the selectivity of hexanal production from 13-hydroperoxy linoleic acid was improved, and hexanol production was reduced 5 to 10 times after 15 min of reaction at 15 °C and pH 7.0. In another experiment, HPLS was coupled to ADH in the presence of NADH. The production of alcohol (hexenols) was then favored especially when using 13-hydroperoxy linolenic acid as substrate at concentrations >15 mM, reaching 95% of the products. Coupling of the enzymatic reactions (cleavage reduction) not only reduced the number of steps but also allowed us to increase the conversion rate of the initial substrate (hydroperoxy fatty acid). Structures of the compounds produced in this work were confirmed using gas chromatography-mass spectroscopy analysis. Each of these products has its own delicately different fresh odor that can be used in various applications.