基于可见吸收信号的乳酸脱氢酶光纤传感器

报道一种测定乳酸脱氢酶活力的基于可见吸收信号的光纤生物传感器，在该传感体系中，通过辅酶Ｉ的氧化还原对（ＮＡＤ＾＋／ＮＡＤＨ）将乳酸脱氢酶和心肌黄酶催化的两个脱氢过阳以耦合，第一个脱氢过程对分析对象进行了化学识别，第二个脱氢过程引起可见吸收信号的变化，该传感器对０～４００Ｕ／Ｌ的乳酸脱氢酶有线性响应关系，检测下限为４８ＵＬ，该传感器已用于人体血清中乳酸脱氢酶活力的测定。     

介体型乳酸脱氢酶生物传感器的研制及应用

本报道了能对丙酮酸产生良好响应的电流型乳酸脱氢酶生物传感器。该传感器以耐尔兰Ａ修饰浸石墨电极为基体电极，将酶直接固化在蚕丝蛋白膜上。在ＰＨ７．４的ＮａＨ２ＰＯ４－ＮａＯＨ介质中，当２．４×１０＾－４ｍｏｌ／Ｌ的辅酶Ｉ（ＮＡＤＨ）存在下，该传感器的响应电流与丙酮酸浓度在３．２×１０＾－５ｍｏｌ／Ｌ范围内有良好的线性关系。响应时间为６０ｓ．本讨论了影响传感器响应的各种因素，用此传感器测定了人血清中     

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.     

Synthesis of glutamate by reductive amination of 2-oxoglutarate with the combination of hydrogenase and glutamate dehydrogenase

Glutamate synthesis by reductive amination of 2-oxoglutarate was performed by the combination of NADH regeneration system and glutamate dehydrogenase (GluDH). The conversion of 2-oxoglutamate to glutamate was 98% after 3 h, and the turnover number of NAD⁺was 17.     

醇脱氢酶结构和作用机理研究进展

介绍了醇脱氢酶的种类, 酵母醇脱氢酶和肝醇脱氢酶等两类常用的醇脱氢酶的物理化学性质和活性位点结构. 归纳了对肝醇脱氢酶和酵母醇脱氢酶作用机理的研究, 重点评述了醇脱氢酶催化反应中的两个关键步骤质子转移和氢化物转移过程机理的研究进展.     

醇脱氢酶电极的研制

通过化学交联法将醇脱氢酶(ADH)固定在玻碳电极表面(Φ=5mm),使用N-甲基吩嗪甲基疏酸盐(PMS)和铁氰化钾为介体、间接测定酶促反应中生成的还原辅酶Ⅰ(NADH)。工作电位+300mv(vs.SCE)乙醇测定的线性范围为0.05～1.0mmol/L,响应时间小于20s。如果电极每天测定30次,可以使用两周。并对电极的选择性进行了讨论。     

介体型乙醇生物传感器的研制及应用

本文报道了电流型乙醇生物传感器的研制与应用。该传感器以健合型耐尔蓝修饰浸蜡石墨电极为基体电极，将乙醇脱氢酶及ＮＡＤ＾＋固定在人造丝网上，成为一种无试剂的乙醇生物传感器。在ｐＨ８．８的Ｔｒｉｓ／ＨＣｌ介质中，该传感器的响应电流与乙醇浓度在０．１０－１．０ｍｍｏｌ／ｌ范围内有良好的线性关系。     

Synthesis of alanine and leucine by reductive amination of 2-oxoic acid with combination of hydrogenase and dehydrogenase

Alanine synthesis by reductive amination of pyruvate was performed by the combination of NADH regeneration system and alanine dehydrogenase (AlaDH). The conversion of pyruvate to alanine was 99% after 1 h. Leucine synthesis was also carried out by the combination of NADH regeneration system and leucine dehydrogenase (LeuDH). The conversion of 4-methyl-2-oxovalerate to leucine was 60% after 1.5 h.     

含离子液体［bmim］Cl的反应介质中马肝醇脱氢酶的催化特性

 研究了马肝醇脱氢酶(HLADH)在含离子液体1-丁基-3-甲基咪唑氯酸盐(［bmim］Cl)的反应介质中的催化特性. 以乙醇为底物时,该酶在［bmim］Cl含量≤0.15 g/ml的体系中的活力高于在不含离子液体的体系中的活力; 离子液体浓度过高(＞0.15 g/ml)对酶活性有明显的抑制作用. 反应温度和pH对含离子液体的反应介质中酶活力的影响规律与不含离子液体时的规律相似. 与不含离子液体的反应介质相比, HLADH在含0.05 g/ml ［bmim］Cl的体系中催化乙醇氧化的活化能下降,酶反应的Vmax和Km均升高. 反应体系中低浓度(≤0.1 g/ml)的离子液体能提高酶的热稳定性,但高浓度(＞0.1 g/ml)的离子液体可降低酶的热稳定性. 紫外二阶导数光谱显示,在含不同浓度离子液体的反应介质中酶分子构象的变化有较大的差异.     

Measurement of lactate in whole human blood with near-infrared transmission spectroscopy

We have evaluated the potential of near-infrared spectroscopy (NIRS) as a technique for rapid analysis of lactate in whole blood. To test the NIRS technique, a comparison was made with a standard clinical method using whole blood samples taken from five exercising human subjects at three different stage of exercise. To expand lactate concentration within the physiological range, standard additions method was used to generate 45 unique data points. Spectra were collected over the 2050-2400 nm spectral range with a 1 mm optical path length quartz cell. Reference lactate concentrations in the samples were determined by enzymatic measurements. Estimates and calibration of the lactate concentration with NIRS was made using partial least squares (PLS) regression analysis and leave-N-out cross validation on second derivative spectra. Separate calibrations were determined from each of the subject samples and cumulative PRESS was used to determine the number of PLS factors in the final model. The results from the PLS model presented are generated from the five individual calibration coefficient vectors and provided a correlation coefficient of 0.978 and a standard error of cross validation of 0.65 mmol l⁻¹ between the enzymatic assay and the NIRS technique. To study the parameters that impact the spectra baseline and the correlation between the calculated model and the data, referenced measurements of lactate against baseline spectrum were made for each individual. A correlation coefficient of 0.992 and a standard error of cross validation of 0.21 mmol l⁻¹ were found. The results suggest that NIRS may provide a valuable tool to assess physiological status for both research and clinical needs.