自然(非灭菌)环境白腐真菌降解活性艳红染料

采用碳氮摩尔比分别为56/2.2和28/44的液体培养基, 进行了白腐真菌在非灭菌环境条件下对活性艳红染料的脱色实验. 结果显示, 在非灭菌环境下, 限氮液体培养基(C/N 摩尔比为56/2.2)中的白腐真菌对活性艳红具有很高的脱色率, 脱色率最高达到92%; 而限碳液体培养基(C/N摩尔比为28/44)很容易感染葡萄球菌等细菌, 从而使脱色反应停止. 而且, 感染细菌的体系pH变化很大, 脱色第4天时, pH已升高到9.31, 造成这一结果的主要原因是限碳反应体系发生了染菌现象. 因此, 初步确定在非灭菌环境中, 限氮液体培养基可在一定程度上抑制细菌生长, 而限碳液体培养基很容易感染细菌.     

五氯苯酚降解的超声诱导

人为或自然因素会导致挥发性或不挥发有毒有机物存在于饮用水中,这一现象 已成为国际上共同关心的问题。从长期对健康状况来说,即使不能辨别饮用水中的 味道和气味,但只要有十亿分之几毫克的有毒有机物存在,就足以使水不能饮用。 所以,废水处理刻不容缓。同废水处理相关的实验方法中,超声作为一种处理方法 ,早有报道,因为超声化学效应主要是空化,空化是自由基,特别是羟基自由基产 生的根源,而痉基自由基是强烈而非特殊的氧化物,它能迅速同水中化合物发生反 应。作者以五氯苯酚为模拟水样,分别用低频（16 kHz）和高频[（800 ± 1） kHz]以及其组合进行超声降解研究。研究表明复频降解效果最好,最差为低频。在 Fenton类试剂存在下,与Fenton类单独降解效果相比,复频则是它的20.93倍,高 频是它的4.9倍,低频与它几乎无变化。实验表明,频率组合对有机污染物的降解 是一条有效途径,但需要更进一步的研究。     

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.     

人类细胞色素P450 2E1酶在不同乙醇浓度下构象变化的分子动力学模拟

细胞色素P450(CYP) 2E1家族酶是一种具有双重功能的单加氧酶, 能够参与市场上6%药物的代谢而具有重要的作用. 这类酶与酒精的消耗、 糖尿病、 肥胖症以及厌食症等密切相关, 引起了广泛的研究兴趣. 目前尚未见从原子水平上对这种酶在不同乙醇浓度下构象行为的研究. 基于此, 本文研究了花生四烯酸(AA)与CYP2E1复合物结构在不同乙醇浓度下构象与能量变化的特点. 对于在不同乙醇浓度下AA与CYP2E1的复合物结构, 采用分子动力学模拟结合自由能计算的方法进行研究. 分子动力学模拟结果表明, His109和Lys243氨基酸残基对AA与CYP2E1的结合起到了至关重要的作用. 当体系的乙醇浓度较高时, AA的结合能力有所下降, 这种结合能力的下降是由于AA与CYP2E1之间氢键相互作用力的减弱所致. 本研究对于AA与CYP2E1复合物结构在不同乙醇浓度下, AA分子与CYP2E1分子结合能力下降以及CYP2E1的构象变化给出了详细的解释. 本研究工作得到的结论对于实验和理论研究均有重要意义, 可为后续细胞色素P450酶类催化活性的研究提供理论支持.     

Ru掺杂BiOBr空心微球的原位合成及其光催化CO_2还原和有机污染物降解性能研究

采用水热法原位合成了Ru掺杂BiOBr空心微球(Ru/BiOBr)复合光催化剂,并对其进行了XRD、 SEM、 TEM、 EDS、 DRS、 EIS等表征.结果表明,所合成的BiOBr材料是由许多小厚度的交错纳米片自组装而成的,同时Ru纳米颗粒成功负载到BiOBr表面,该复合材料对还原CO_2和降解有机模拟污染物(罗丹明B, RhB)具有良好的光催化性能.当Ru的掺杂量为0.4%时复合材料的光催化活性最佳, 4 h后甲醇产量可达1103μmol/g_(cat),并且60 min内对RhB的降解率达到98%.除此之外,还讨论了复合材料的光催化机理和稳定性.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Intracellular transport and localization of microsomal cytochrome P450

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH₂-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.     

Spectroscopic and Electrochemical Characterization of Cytochrome P450st‐DDAB Films on a Plastic‐Formed Carbon Electrode

We have studied the characterization of thermophilic cytochrome P450 (P450st)‐didodecyldimethylammonium bromide (DDAB) films by using UV‐vis absorption, resonance Raman spectroscopy, and electrochemical methods. The observed Raman spectrum indicated near‐native conformation of the heme iron in DDAB film on the surface of a glass slide, while on the surface of a plastic‐formed carbon (PFC) electrode, the conformation of P450st‐DDAB was very similar to that of heme‐DDAB film, suggesting the release of heme from P450st in DDAB films on PFC electrodes. When NaBr was added as salt to the casting solution, the result of Raman spectrum indicated near‐native conformation of P450st in DDAB film even on the PFC electrode, but no redox potential of P450st which has near native structure was observed. This study suggests the essential experimental conditions when working with heme protein‐DDAB films as, in some cases, heme iron from proteins is released on the surface of the electrode.     

Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X

Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs. One challenge is to identify the respective drug metabolites and to compare substrate specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in E. coli. The substrate scope of the variants was determined using active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili peppers – was oxidized most efficiently (4.36?μM/min) in a whole cell mediated biotransformation. The products were isolated, purified and their structures elucidated by 1D and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain. Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.     

Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450cam‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide

The hydroperoxo iron(III) intermediate P450_camFe^III–OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450_cam, could be transiently observed in the peroxo‐shunt oxidation of the substrate‐free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450_camFe^III–OOH species as a function of varying reaction conditions, such as pH, and concentration of H₂O₂, camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate‐free form of P450_cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid–base catalyst and is mainly governed by the ability of H₂O₂ to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$ . Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic O?O bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)‐camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe^III–OOH intermediate in the oxidation of the natural substrate of P450_cam.     

Chemo‐ and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase

Hydroquinone (HQ) is produced commercially from benzene by multi‐step Hock‐type processes with equivalent amounts of acetone as side‐product. We describe an efficient biocatalytic alternative using the cytochrome P450‐BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi‐rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one‐pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450‐BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti‐inflammatory and anti‐bacterial activities.     

高分子共混物中Friedel-Crafts烷基化反应原位增容与催化降解机理

采用GPC方法对共混组分的分子量、接枝率和降解度等数据进行分析, 发现AlCl₃催化剂对PS/POE共混物的作用表现在原位增容和催化降解两个方面. 结合Friedel-Crafts烷基化反应的特点细化反应过程, 发现PS/POE共混物在AlCl₃作用下发生接枝反应时, 有大量碳正离子出现, 而碳正离子周围的电子重排会导致分子链断裂, 从而引起共混组分出现大幅度降解. PS/POE/AlCl₃共混物中原位接枝与催化降解是一对竞争性反应, 同时存在, 并相互竞争.     

Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films

NADPH‐cytochrome P450 reductase (CPR) serves as electron donor for cytochrome P450 catalyzed monooxygenase reactions utilizing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as electron transfer cofactors. Here, stable films of human and rabbit CPRs with didodecyldimethylammonium bromide (DDAB), dimyristoylphosphatidyl choline (DMPC), and poly(diallyldimethylammonium) (PDDA) were made on pyrolytic graphite (PG) electrodes for comparative structural and electrochemical studies. CD and UV‐VIS absorbance spectra suggested that near native CPR conformation is retained in PDDA films, and some conformational changes occur in DMPC or DDAB films. Cyclic voltammetry of these films gave quasireversible pairs of peaks at average formal potential ?0.246±0.008 V vs. NHE. In human CPR‐DDAB (H‐CPR‐DDAB), a second pair of peaks at +0.317 V vs. NHE was found that depended strongly on identity of buffer and salt. Excepting H‐CPR in DDAB, films showed similar voltammetry, formal potentials, and k_s values. While CPR‐PDDA films had near native CPR structures, electrochemical parameters did not differ significantly from CPR‐DMPC films. The relative independence of film voltammetry from the influence of film materials for CPRs is in contrast with heme iron proteins that, while retaining near native structures, have formal potentials that depend significantly on identity of the film material.