OPD-H2O2-HRP伏安酶联免疫分析体系酶催化反应的研究

应用电化学分析、高效液相色谱、紫外-可见光谱、红外光谱和核磁共振等技术对邻苯二胺(OPD)-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫分析体系的酶催化反应进行了详细深入的研究.用化学方法制得了HRP酶催化H2O2氧化OPD的产物纯品.伏安法和高效液相色谱实验说明,在所选择的酶催化反应条件下,酶催化反应只生成一种产物;经紫外-可见光谱,红外光谱和13C核磁共振谱鉴定,产物为2,3-二氨基吩嗪.写出了酶催化反应过程,同时对酶催化反应产物的电极还原过程也进行了研究.     

离子液体中的生物催化反应

本文综述了近年来离子液体中生物催化反应的研究进展。离子液体作为新的绿色溶剂,用于生物催化反应具有以下特点:在离子液体中酶有良好的稳定性、选择性和反应活性。离子液体可溶解极性大的反应物, 产物易分离,酶和离子液体可重复使用。对离子液体中的生物催化进行了展望。     

基于短肽自组装与共组装的纳米纤维人工水解酶

将水解酶活性中心催化三联体氨基酸(His/Ser/Asp)引入9-芴亚甲氧羰基苯丙氨酸二肽(Fmoc-FF)双亲短肽序列中,利用短肽的自组装性能,构建了具有对硝基苯酚乙酸酯水解活性的超分子纳米纤维人工水解酶.研究结果表明,形成规则的纳米纤维结构是获得催化活性的必要条件.9-芴亚甲氧羰基(Fmoc)基团间的弱相互作用促使β-折叠二级结构的形成.通过对比天然水解酶的米氏动力学方程、最适催化温度及p H结果可知,所制备的超分子纳米纤维人工水解酶具有与天然酶相似的酶学性质.金属离子Ca2+和Ba2+对人工水解酶活性具有激活作用,而Mg2+,Ni2+,Co2+,Cu2+和Zn2+则抑制酶活性.     

载酶纳米催化体系用于疾病诊疗的研究进展

酶作为一种具有高度特异性和高效性的催化剂, 可在细胞器中通过复杂有序的生化反应调节细胞的代谢过程. 受细胞区隔化结构的启发, 仿生设计纳米酶催化体系、 构筑限域酶催化微环境从而提高酶催化活性的研究为酶催化应用开辟了新思路. 纳米催化体系保留了小尺寸、 大比表面积、 肿瘤部位选择性富集等优势, 在疾病的诊疗方面发挥了巨大的优势. 本文首先总结了天然酶、 模拟酶和级联酶体系的催化机理, 对仿生构筑的纳米酶催化材料的载体体系进行了概述, 介绍了纳米酶催化体系在生物成像方面的应用, 讨论了其在相关代谢类疾病的作用途径, 并对纳米酶催化体系用于生物诊疗的发展前景进行了展望.     

基于模拟酶-天然酶级联反应的双模式传感平台用于生物标志物的超灵敏检测

重要生物标志物例如碱性磷酸酶(ALP)的高灵敏度检测和准确分析对于疾病的早期检测和治疗至关重要.本工作合成了Cu基金属有机框架材料HKUST-1,探索了其类氧化酶性质.通过设计HKUST-1模拟酶与ALP天然酶的级联催化体系,构建了高灵敏度及高选择性的荧光/紫外双模式检测平台,用于生物标志物ALP及焦磷酸根离子(PPi)的检测.利用所设计的级联催化反应对信号的有效放大以及荧光和紫外双信号输出,对ALP的检测限分别低至0.0078和0.039nmol·L^-1.本工作首次开发了基于模拟酶-天然酶级联催化放大的双模式生物分析方法,实现了两种生物标志物的灵敏检测以及酶抑制剂的抑制效率评估,并将其应用到人血清样品中ALP的超灵敏分析,在临床诊断中具有巨大应用潜力.     

生物酶HRP催化H~2O~2氧化间苯二胺反应的研究

应用电化学分析,高效液相色谱(HPLC),紫外-可见光谱(UV-vis),红外光谱(IR)和核磁共振(NMR)等技术对辣根过氧化物酶(HRP)催化H~2O~2氧化间苯二胺(MPD)的反应进行了研究。伏安法和高效液相色谱实验说明,在所选择的酶催化反应条件下,酶催化反应生成一种产物。用化学方法制得了HRP酶催化H~2O~2氧化MPD的产物纯品。经UV-vis,IR和^1HNMR谱鉴定,产物为2,7-二氨基吩嗪。写出了酶催化反应过程,同时对酶催化反应产物的电极还原过程也进行了研究。     

含三元环腈的生物转化反应与应用*

腈的对映选择性生物转化反应是合成高光学活性羧酸及其酰胺衍生物的有力手段。 本文介绍了在红球菌AJ270催化下,一系列含三元环结构的腈,包括环丙腈、环氧丙腈和氮杂环丙腈的生物转化反应。提出了腈水合酶和酰胺水解酶酶活中心的假设,即腈水合酶可能位于比较疏散的立体空间环境中,而酰胺水解酶则处于相对深埋的位置且空间大小有限。另外本文还探讨了腈的生物催化反应在天然产物及生物活性分子合成中的应用。     

细菌漆酶的功能与催化特性

正漆酶(laccase)(EC 1.10.3.2)是一类含铜的多酚氧化酶(copper-containing polyphenol oxidase),属于蓝色多铜氧化酶(blue multi-copper oxidase,MCO)家族,能催化多种酚类、芳胺类化合物的氧化,同时将分子氧还原成水.漆酶突出的催化特性是它的底物具有广泛性、催化反应具有复杂性,生     

光合细菌羰基还原酶不对称还原苯乙酮的研究

从本实验室筛选得到的类球红杆菌(Rhodobacter sphaeroides)中,通过超声破碎、硫胺沉淀、DEAE-Sepha-dexA-25阴离子交换层析分离得到一种较纯的依赖NADPH的羰基还原酶.对其进行SDS-PAGE电泳分析,显示一条带,测得其相对分子量约为37kD.建立了羰基还原酶催化苯乙酮反应体系并对其进行优化,得出该酶催化苯乙酮的最适反应pH值为8,最适温度为37℃,在pH值为7～9之间比较稳定,其热稳定性较低.该酶对苯乙酮的米氏常数Km和最大反应速率Vmax分别为0.26mmol·L-1和2.4μmol·min-1·mg-1,最佳反应时间为24h·催化苯乙酮的主要产物为(S)-苯乙醇,其产率为58.5%,ee值可达到99%以上,是很有前景的生物催化剂之一.对酶催化与辅酶再生体系相结合进行了初步的研究,提出了氢化酶再生辅酶与菌绿素再生辅酶体系,为今后的酶催化工业生产奠定了基础.     

酶催化反应在立体有机合成中的应用

讨论了水解酶、交换酶以及氧化还原酶催化的位置、对映和潜手性立体专一的反应，包括酶催化的醇或酯的水解反应、酯交换反应、含羰基的化合物及活泼双键的还原反应、ＳＰ￣３杂化的碳及烯的氧化反应（包括Ｂａｙｅｒ－Ｖｉｌｌｉｎｇｅｒ氧化反应）。回顾了酶立体选择性地合成某些重要目标结构的生物催化方法，讨论了酶催化反应的生物合成前景。参考文献１８篇。     

Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide

Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.     

Immobilized respiratory chain activities fromEscherichia coli utilized to measure d and l-lactate,succinate, l-malate, 3-glycerophosphate,pyruvate, or NAD(P)H

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.     

Hydrogen Evolution from L-Lactate and L-Malate with the Combination of Lactate Dehydrogenase or Malate Dehydrogenase and Hydrogenase from A. Eutrophus H16

Hydrogen evolution system from L-lactate and L-malate consisting of lactate dehydrogenase or malate dehydrogenase and hydrogenase from cell free extracts of Alcaligenes eutrophus H16 was established. When the solution containing L-lactate, lactate dehydrogenase, NAD and hydrogenase was incubated at 30°C hydrogen evolution was observed. Similarly, the hydrogen evolution was also observed from the L-malate incubated at 30°C.     

1-乙酰基-3-(2-羟基-4,6-二甲氧基苯基)-5-芳基-2-吡唑啉的合成及Cu2+荧光探针行为

设计合成了6个1-乙酰基-3-(2-羟基-4,6二甲氧基苯基)-5-芳基-2-吡唑啉化合物4a～4f.测试了它们的紫外光谱和荧光光谱,研究了其对铜离子的选择性识别作用.结果表明,化合物4f作为铜离子荧光探针,受常见离子干扰较小,对于铜离子有着较高的选择性和较低的检出限.     

Purification and properties of bilirubin oxidase fromMyrothecium verrucaria

Bilirubin oxidase was purified from a culture filtrate of Myrothecium verrucaria Mv 2, 1089 by DEAE-cellulose and Sephadex G-100 column chromatographies. The purified enzyme had a specific activity of 30 U/mg protein and showed a single band on polyacrylamide gel electrophoresis. Some of the general properties of this bilirubin oxidase were as follows: the optimum pH for the enzyme reaction was 7.5 and the optimum temperature was 50 degrees C. The enzyme was stable at pH ranging from 9.0 to 9.5. The mol wt was calculated to be 61,900-62,700 by SDS-PAGE and gel-filtration technique. The apparent Km value of the bilirubin oxidase was calculated to be 9.4 x 10(-5) mol/L. The enzyme activity was greatly reduced by incubation of bilirubin oxidase with Fe2+, Hg+, NaN3, NH+4, and Zn2+. The enzyme reaction was inhibited in the presence of Ca2+, Hg+, Zn2+, Fe2+, and BSA.     

脱氢酶电化学生物传感器的研究进展

自然界中超过400种脱氢酶使用辅酶-烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)作为生物催化反应中氢和电子的传递体,因此烟酰胺型辅酶的电化学氧化对构筑此类脱氢酶电化学生物传感器具有重要的意义.本文介绍了还原型辅酶在人工电子媒介体存在下的电化学氧化,以及脱氢酶电化学生物传感器的设计和应用.     

Synthesis of NAD analogs to develop bioorthogonal redox system

Three new nicotinamide adenine dinucleotide(NAD) analogs were synthesized,and their characteristics as cofactors for Escherichia coli malic enzyme(ME) and its double mutant ME L310R/Q401C were analyzed.Each pair of the NAD analog and the double mutant showed good orthogonality to the natural pair of NAD and ME in terms of catalyzing oxidative decarboxylation of L-malic acid.Results indicated that molecular interactions between redox enzyme and cofactor could be further explored to generate new bioorthogonal redox systems.     

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD⁺) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker??s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD⁺ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker??s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.     

NAD(P)+-NAD(P)H models. 90. Stereoselection controlled by electronic effect of a carbonyl group in oxidation of NAD(P)H analog

4-Monodeuterated NAD(P)H model compounds (1,4,6,7-tetrahydro-1,6,11-trimethyl-5-oxo-5H-benzo[c]pyrido[2,3-e]az epin; 11Me-MMPAH) have been oxidized with a series of p-benzoquinone and its derivatives in the presence of Mg2+. The models have an axial chirality with respect to the orientation of carbonyl dipole, the dihedral angle of which is larger than 55 degrees out of the plane of dihydropyridine ring. Without Mg2+, the anti- (with respect to the carbonyl dipole) hydrogen is 3 to 32 times more reactive than the corresponding syn-hydrogen, whereas, when Mg2+ is present in the system, the selectivity is shifted toward the syn-preferency. Mg2+ plays the role of a Lewis acid catalyst to control the stereochemistry at the same time as it catalyzes the reaction.     

Reversible,electrochemical interconversion of NADH and NAD+ by the catalytic (Ilambda) subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I)

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain and catalyzes the oxidation of beta-NADH by ubiquinone, coupled to transmembrane proton translocation. It contains a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur (FeS) clusters, and at least one ubiquinone binding site. Little is known about the mechanism of coupled electron-proton transfer in complex I. This communication demonstrates how the catalytic fragment of complex I, subcomplex Ilambda, can be adsorbed onto a pyrolytic graphite edge electrode to catalyze the interconversion of NADH and NAD+, with the electrode as the electron acceptor or donor. NADH oxidation and NAD+ reduction are completely reversible and occur without the application of an overpotential. The potential of zero current denotes the potential of the NAD+/NADH redox couple, and the dependence of ENAD+ on pH, and on the NADH:NAD+ ratio, is in accordance with the Nernst equation. The catalytic potential of the enzyme, Ecat, is close to one of the two reduction potentials of the active site FMN and to the potential of a nearby [2Fe - 2S] cluster; therefore, either one or both of these redox couples is suggested to be important in controlling NADH oxidation by complex I.