用再生丝素固定过氧化物酶及其传感器在有机相中的研究

用再生丝素固定过氧化物酶及其传感器在有机相中的研究钱江红，刘永成，刘海鹰，于同隐，邓家祺（复旦大学化学系、高分子科学系，上海，２００４３３）关键词再生丝素，生物传感器，过氧化氢酶在非水介质中的催化活性已有研究［１］，生物传感器在有机相中有许多优点：如...     

酶的改造及其催化工程应用

酶作为生物催化剂在食品、饲料、化妆品以及医药等诸多领域逐渐发挥重要作用。但是,酶对外界环境如pH和温度等很敏感,而实际的反应条件和生物体的生理环境差异较大,因此酶在实际应用中不稳定、容易失活,催化效率下降。酶的这一特点大大限制了其工业化应用。目前,定向进化、糖基化以及化学修饰等方法被广泛用于酶分子的改造以提高其稳定性、催化效率以及扩大其底物范围。其中,定向进化通过模拟自然进化机制,在体外改造基因从而获得性能优化的酶突变体,已经成为了酶改造的重要技术。在酶的实际应用过程中,介质工程、固定化以及多酶催化体系构建等技术被广泛用于提高酶的催化效率。其中,多酶催化体系由于其底物通道效应可以显著提高级联酶反应的效率而备受关注。本文首先重点介绍了近年酶应用的现状,然后从酶定向进化、糖基化以及化学修饰的角度总结了酶改造的方法,最后从介质工程、酶固定化以及体外多酶催化体系等方面进一步总结了酶实际应用中的催化工程策略。     

仿硒酶研究进展

硒是人体中必需的微量元素,它与各种疾病和人类健康息息相关。硒在生物体内以硒代半胱氨酸形式表现其生理活性和功能。为了探索硒在硒蛋白中结构和功能关系并可能发展成硒相关的适用药物,人们付出许多努力来发展硒蛋白模拟化学。由于硒酶—谷胱甘肽过氧化物酶（GPX）重要的抗氧化作用以及潜在的药用价值,国际上广泛开展了对它的人工模拟研究。本文对近年来硒酶模拟化学和生物学相关研究进展进行了综述。     

多溴二苯醚的生物代谢机制研究进展

作为一种广泛使用的阻燃剂,多溴二苯醚(PBDEs)及其衍生物羟基化多溴二苯醚(OH-PBDEs)和甲氧基化多溴二苯醚(MeO-PBDEs)等已从环境介质、各种生物体甚至人体内检测出来.但是对于生物体内低溴代PBDEs及其羟基化和甲氧基化衍生物的来源及其是否由PBDEs在生物体内代谢形成等问题,目前仍存在较多争论.由于PBDEs进入生物体后的生物转化、代谢途径与其对生物体的毒性有密切关系,因此是研究者们关注的焦点.本文在对生物体内PBDEs及其代谢物的来源和分布进行分析的基础上,综述了PBDEs在生物体内发生脱溴还原代谢和氧化代谢的研究进展,从体内原位代谢和体外模拟代谢研究两方面探讨了PBDEs的生物代谢机制、代谢途径及还原和氧化代谢中可能涉及的代谢酶,指出进一步深入开展PBDEs的生物积累和代谢研究的方向.     

酶工程与有机合成

酶工程与有机合成袁中一（中国科学院上海生物化学研究所上海２０００３１）关键词酶工程酶催化有机合成光学拆分分类号Ｏ６２９．８１酶工程和酶在非水介质中的催化１．１酶酶，一大类以蛋白质为主体的生物催化剂．具有在常温常压和中性ｐＨ值等温和条件下，高效率地进行...     

模拟酶聚合物在酯水解反应中的催化作用

模拟酶聚合物是近年才发展起来的聚合物科学的新领域。它通过用人工合成的聚合物来模拟生物酶的特性,以便为合成高效的新型催化剂开辟新的途径,同时,在分子生物学的研究方面也可以用这种化学模拟的方法来研究酶的结构和功能之间的关系。存在于一切生物体内的酶是由氨基酸组成的蛋白质大分子化合物,是生物体内进行各种化学反应的有效催化剂。在酶分子上有所谓的     

过氧化物酶模拟及应用研究进展

过氧化物酶是一类在生物体系中广泛存在的酶蛋白,本文从过氧化物酶催化反应的模拟作用,包括酶模拟、酶催化反应介质模拟以及酶催化反应氢供体底物的模拟3个方面评述过氧化物酶模拟作用的研究进展,并简要介绍在分析化学及免疫分析中的应用。     

细胞色素P450酶电化学生物传感器的构建及其药物代谢应用

药物代谢过程是药物在体内产生药效和毒性的主要过程,发展廉价、方便、快速、高通量的体外药物代谢研究方法对新药的开发和设计、给药的方法和剂量、临床药物的检测等都有重要的指导意义. 细胞色素P450酶（CYP450酶）在药物的I相反应中起到关键作用,以电极代替辅酶NADPH提供CYP450酶催化反应过程中需要的两个电子,构建CYP450酶电化学生物传感器可实现药物的初步筛选. 大量研究表明,CYP450酶在电极表面合适的固定方法与电极材料可有效提高传感器的检测性能. 本文主要综述近年来CYP450酶电化学生物传感器的构建及其在药物代谢研究方面的应用,并展望其研发前景.     

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了南27个氨基酸残基组成的酸性多肽（AP）的一级结构。选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节（CG）多肽与蛋白质的组成与分布，发现CG中含有AP酶解的二聚体短肽。这些短肽序列分别为2（NKDEEQRELLKAISNLL）、2（NKDEEQRELLKAISNL）、2（SGVSLLTSNKDEEQREL）和2（LTSNKDEEQRE LL），均以L-R（氨基酸残基）方式断裂。以AP为探针，结合MALDI-TOF质谱分析技术，发现CG中含有超微量的L-R多肽内切酶，其分子量为78218.25Da。本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能。     

一种基于检测酶反应中间体而测定漆酶活性的动力学分析法

在反相胶束（AOT／n-octane）介质中，漆酶（laccase）－邻苯二胺（o-phenylenediamine,OPDA）本系的酶催化反应呈现“超级活性”，并且酶催化反应中间体与产物2，3－二氨基吩嗪（2，3－diaminophemazine,DAP）的吸收光谱能很好的剥离分开。通过理论，表明酶催化反应中间体生成的初始速率与漆酶活性存在定量关系，采用初始速率法测定中间体的浓度，建立了一种基于检测酶催化反应中间体而测定漆酶活性的动力学分析新方法。该方法由于在反相胶束介质中采用初始速率法进行测定，具有较高的灵敏度和选择性。测定漆酶活性的线性范围为0－2.5U；检出限为0.033U。     

Biocatalysis made to order

Recombinant DNA technology is now being explored to engineer enzyme molecules. It has many far-reaching applications in biocatalytic processes of enzyme engineering. The facts have pursued certain important industrial, biomedical, and environmental problems. These current excitements are mainly focused on the basis of gene cloning and in vitro mutagenesis for overproduction and redesigning of enzymes, as well as their probable implications in industry, antibiotic research, and waste degradation.     

Magneto-Mechanical Model of an Enzyme

It has always been difficult to clearly explain to biochemistry students how enzymes work. The idea of enzyme-substrate stereospecificity, active site, coupled reactions, inhibitor or activator effects, mono- and bisubstrate reactions or any other aspect related to the mechanism of action of enzymes are in general abstract and difficult concepts for students taking introductory courses in biochemistry. On the other hand, students are more familiar with mechanical or magnetic objects, and normally they have no problem understanding how they work. Accordingly, a magneto-mechanical model is proposed as a didactic resource to show in class how enzymes can catalyze biochemical reactions. This model is also helpful for introducing the concept of coupled reactions and many other structural and mechanistic aspects of enzyme reactions.     

Blood appearance of rat alkaline phosphatase originating from the duodenum in vitro

The major source of rat serum alkaline phosphatase (ALP) is well known to be from the intestinal enzyme, but it is still unclear whether it is from the duodenal or the ileal enzyme. The organic origin was investigated by means of two-dimensional electrophoresis. Major isoelectric points and molecular masses for activities of duodenal enzyme treated with both phosphatidylinositol-specific phospholipase C and neuraminidase were identified apparently with those of the major serum enzyme. In organ culture, the normal duodenal enzyme was released in the highest amounts to the culture medium. These results indicate that the major source of serum ALP in adult rats is basically from the duodenal enzyme. On the other hand, lectin affinity chromatography for ALPs showed that the ALP in the medium from culture duodenum and liver had the same complex-type sugar chain as with the ALP in the duodenal tissue. Although the duodenal ALP induced by glucosamine in vitro had the hybrid-type chain, sugar chains of the induced ALP in the culture medium were of the complex type, indicating that medial ALPs possessing the same sugar chain as the native duodenal enzyme, complex type, are mainly released from their tissues in normal conditions.     

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.     

Creating novel enzymes by applied molecular evolution

Recent molecular techniques have made it feasible to simulate evolutionary processes and apply in vitro selection to evolve enzymes with novel properties that may have potential benefits for medical and industrial applications. The characterization of such mutants has also provided new insights into how molecular structure determines enzyme function.     

Phage display of enzymes and in vitro selection for catalytic activity

Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes. The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography. The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.     

Unveiling the Potential of Innovative Gold(I) and Silver(I) Selenourea Complexes as Anticancer Agents Targeting TrxR and Cellular Redox Homeostasis

A series of NHC-based selenourea Ag(I) and Au(I) complexes were evaluated for their anticancer potential in vitro, on 2D and 3D human cancer cell systems. All NHC-based selenourea complexes possess an outstanding cytotoxic potency, which was comparable or even better than that of the reference metallodrug auranofin, and were also able to overcome both platinum-based and multi-drug resistances. Intriguingly, their cytotoxic potency did not correlate with solution stability, partition coefficient or cellular uptake. On the other hand, mechanistic studies in cancer cells revealed their ability to strongly and selectively inhibit the redox-regulating enzyme Thioredoxin Reductase (TrxR), being even more effective than auranofin, a well-known TrxR inhibitor, without affecting other redox enzymes such as Glutathione Reductase (GR). The inhibition of TrxR in H157 human cancer cells caused, in turn, the disruption of cellular thiol-redox homeostasis and of mitochondria pathophysiology, ultimately leading to cancer cell death through apoptosis.     

Influence of In Vitro Human Digestion Simulation on the Phenolics Contents and Biological Activities of the Aqueous Extracts from Turkish Cistus Species

Oxidative stress is one of the significant precursors of various metabolic diseases such as diabetes, Parkinson’s disease, cardiovascular diseases, cancer, etc. Various scientific reports have indicated that secondary plant metabolites play an important role in preventing oxidative stress and its harmful effects. In this respect, this study was planned to investigate the phenolic profile and antioxidant and antidiabetic potentials of the aqueous extracts from Turkish Cistus species by employing in vitro methods. In vitro digestion simulation procedure was applied to all extracts to estimate the bioavailability of their phenolic contents. Total phenolic, flavonoid, phenolic acid and proanthocyanidin contents were determined for all phases of digestion. In addition, changes in the quantity of the assigned marker flavonoids (tiliroside, hyperoside and quercitrin) were monitored by High-Performance Thin Layer Chromatography (HPTLC) analysis. The antioxidant activity potentials of the extracts were studied by various methods to reveal their detailed activity profiles. On the other hand, in vitro α-amylase and α-glucosidase enzymes and advanced-glycation end product (AGE) inhibitory activities of the extracts were determined to evaluate the antidiabetic potentials of extracts. The results showed that aqueous extracts obtained from the aerial parts of Turkish Cistus species have rich phenolic contents and potential antioxidant and antidiabetic activities; however, their bioactivity profiles and marker flavonoid concentrations might significantly be affected by human digestion. The results exhibited that total phenolic contents, antioxidant activities and diabetes-related enzyme inhibitions of the bioavailable samples were lower than non-digested samples in all extracts.     

Fluorescence quenching study on the interaction of bases of nucleic acid with electron-accepting sensitizer and serum albumin containing electron-rich tryptophan residue

Static and dynamic fluorescence quenchings of electron-accepting sensitizers including positive charged heterocyclics and neutral cyanoaromatics by bases of nucleic acid (NB) have been investigated. It was found that NB could act as effective electron donors to quench the fluorescence of electron-accepting sensitizers. The quenchings by diffusion-controlled rate coincide well with the static and dynamic Stern-Volmer correlation. On the other hand, the diffusion-controlled fluorescence quenchings of the tryptophan (TRP) residue in the protein enzymes, HSA and BSA, by electron-accepting NB reveal that photochemical dual-damage of protein enzyme and thymine (THM) occur upon u.v.-irradiation, which is characteristic of excitation wavelength-dependence. Therefore, the results illustrate that the lesion interactions of NB with electron-deficient sensitizers or electron-rich TRP fluorophore-containing protein enzymes originate mainly from PET-initiated processes in both cases.     

RNA-cleaving DNA enzymes with altered regio- or enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.