漆酶催化半醌自由基中间体动力学的模拟研究

基于漆酶催化5,6-二溴-2,3-二氰基氢醌生成半醌自由基的动力学过程建立了相应的动力学模型,通过对实际动力学曲线的模拟,建立了半醌自由基的生成与衰减机理,为进一步研究半醌自由基与其它生物活性物质的相互作用奠定了基础.     

ESR自旋稳定化技术在漆酶化学中的应用

用ESR自旋稳定化技术对十六个儿茶酚类底物的漆酶催化氧化反应进行了ESR追踪,捕获到相应的半醌自由基．结果表明,ESR自旋稳定化技术的运用,开辟了一条在静态条件下用漆酶／O2体系获取高稳态浓度半醌自由基的有效途径．     

漆树漆酶的催化氧化作用（ⅫⅡ）

以５,６－二溴－２,３－二氰基氢醌为底物,在ＰＨ４．５０条件下,用分光光度法考察了ＦｅＳＯ４对漆树漆酶催化氧化反应的影响。结表明,在本文实验条件下ＳＯ４＾２－离子对酶促反应的影响基本可以忽略,而Ｆｅ＾２离子对漆酶的催化氧化反应则有明显的竞争性抑制作用,研究证实,Ｆｅ＾２＋离子的抑制作用是通过它还原酶促反应的产物半醌自由基阴离子来实现的。     

漆树漆酶的催化氧化作用(ⅩⅠⅠⅠ)-Fe2+离子对漆酶催化氧化反应的影响

以5,6-二溴-2,3-二氰基氢醌为底物,在pH4.50条件下,用分光光度法考察了FeSO4对漆树漆酶催化氧化反应的影响.结果表明,在本文实验条件下SO2-4离子对酶促反应的影响基本可以忽略,而Fe2+离子对漆酶的催化氧化反应则有明显的竞争性抑制作用.研究证实,Fe2+离子的抑制作用是通过它还原酶促反应的产物半醌自由基阴离子来实现的.     

漆树漆酶的催化氧化作用 (ⅹⅢ)──Fe~(2+)离子对漆酶催化氧化反应的影响()

以５,６－二溴－２,３－二氰基氢醌为底物,在ｐＨ４．５０条件下,用分光光度法考察了ＦｅＳＯ４对漆树漆酶催化氧化反应的影响．结果表明,在本文实验条件下ＳＯ２－４离子对酶促反应的影响基本可以忽略,而Ｆｅ２＋离子对漆酶的催化氧化反应则有明显的竞争性抑制作用．研究证实,Ｆｅ２＋离子的抑制作用是通过它还原酶促反应的产物半醌自由基阴离子来实现的     

肉桂酸类抗氧化剂结构—活性关系研究

利用邻菲罗啉化学发光法研究了中草药有效成分氯原酸、连翘酯甙等肉桂酸类抗氧化剂对HO·的清除作用。量子化学计算[3-21G、B3LYP/6-31G(d)]对实验结果给予了合理的解释。 计算结果显示抗氧化剂形成半醌式自由基前后的能量之差(ΔE)能较好地衡量其活性的高低;抗氧化剂半醌式自由基与邻位取代基形成分子内氢键或通过共振形成邻苯醌可使内能降低,半醌式自由基更稳定;脱氢酚羟基的对位含有给电子取代基团时,可使半醌式自由基易形成并提高其稳定性。     

醇水介质中漆酶催化氧化漆酚及其类似物

本文作者在醇水介质中对漆酚及其类似物的酶催化氧化反应进行了研究。探讨了混合溶剂中漆酶的行为。结果发现邻苯二酚被烷基取代后更有利于漆酶的催化氧化。反应可能经过两个步骤,先是底物氧化生成黄色苯醌类物质,然后醌类中间物偶联形成多聚酚。     

丝裂霉素C还原代谢机理研究

采用光谱电化学方法,结合循环伏安法、紫外-可见光谱、圆二色光谱等多种手段,提出了丝裂霉素C(MMC)的还原代谢机制。其还原活化为MMC首先从电极上得到一个电子,形成半醌自由基,随后脱去甲氧基形成吲哚半醌自由基,接着环乙亚胺基水解开环,最后吲哚半醌自由基歧化得到吲哚醌和吲哚氢醌。     

漆树漆酶的催化氧化作用(X)──叔丁基儿茶酚类底物的漆酶催化氧化和漆酶的微环境效应

对6个含叔丁基的儿茶酚类底物的离解和漆酶催化氧化反应进行了考察。结果表明:叔丁基的存在,有利于增进对底物的结合,并可以利用漆酶活性中心周围提供的pH环境和质子载体性质,促进底物的离解,使这类底物能在远低于其一级离解的弱酸性环境中进行反应。由于叔丁基的特性和漆酶的微环境效应的共同作用,这些底物在pH=6.0的水溶液环境中都能顺利地氧化成相应邻醌,并具有合成价值。ESR结果证明:这些底物在弱酸性条件下的漆酶催化反应经历了质子转移和单电子氧化步骤。     

某些还原性生物分子对竹红菌甲素敏化产生^1O~2的抑制作用

存在还原性的生物分子如甲硫氨酸、尿酸和 还原性谷胱甘肽时,竹红菌甲素经受单电子还原形成甲素的半醌自由基、在去氧溶液中观察到了 甲素半醌负离子自由基的ESR信号.在充氧的溶液中检测到了超氧负离子自由基.还原剂的存在降低了单重态氧的生成并引起超氧负离子自由基的生成.     

介孔SiO2/Fe3O4中空磁性微球的漆酶固定化

以介孔SiO2/Fe3O4磁性中空微球作为载体,采用物理吸附法对漆酶进行固定化,考察了时间、温度和pH值对漆酶固定化效果的影响,并对固定漆酶的活性及稳定性进行了研究.结果表明,介孔SiO2/Fe3O4磁性中空微球吸附漆酶分子后,介孔材料的比表面积与孔体积均减小.在3 h时复合微球对漆酶的吸附达到平衡,复合微球中介孔SiO2对漆酶的有效固定量为689 mg/g,大大高于纯介孔材料MCM-41的漆酶固定量(319 mg/g).在pH=3～6的条件下,复合微球中固定漆酶仍保持70%以上的相对酶活.当温度不高于60℃时,固定漆酶的相对酶活仍保持65%以上.固定漆酶的pH稳定性和热稳定性都明显优于游离漆酶,固定漆酶的米氏常数为1.05 mmol/L,与游离漆酶相比,固定漆酶与底物的亲和力有所降低.当2,4-二氯苯酚的浓度为10 mg/L时,固定漆酶对其去除率在6 h时达到81.6%,表现出很好的催化活性.     

Enhanced Production of Laccase from Coriolus versicolor NCIM 996 by Nutrient Optimization Using Response Surface Methodology

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.     

A novel water-in-ionic liquid microemulsion and its interfacial effect on the activity of laccase

It is of great significance to develop an appropriate water-in-ionic liquid (W/IL) microemulsion suitable for the expression of the catalytic activity of a given enzyme. In this paper, the phase diagram of a new AOT/Triton X-100/H(2)O/[Bmim][PF(6)] pseudo ternary system is presented. With the aid of nonionic surfactant Triton X-100, AOT could be dissolved in hydrophobic ionic liquid [Bmim][PF(6)], forming a large single phase microemulsion region. The water-in-[Bmim][PF(6)] (W/IL) microemulsion domain was identified electrochemically by using K(3)Fe(CN)(6) as a probe. The existence of W/IL microemulsions was demonstrated spectrophotometrically by using CoCl(2) as a probe. New evidences from the FTIR spectroscopic study, which was first introduced to the W/IL microemulsion by substituting D(2)O for H(2)O to eliminate the spectral interference, demonstrated that there existed bulk water at larger ω(0) values (ω(0) was defined as the molar ratio of water to the total surfactant) in the W/IL microemulsion, which had remained unclear before. In addition to the inorganic salts, biomacromolecule laccase could be solubilized in the W/IL microemulsion. The laccase hosted in the microemulsion exhibited a catalytic activity and the activity could be regulated by the composition of the interfacial membrane.     

新型有机-无机纳米复合粒子的制备及其固定化漆酶研究

制备了四氨基酞菁钴(CoTAPc)-Fe3O4纳米复合粒子, 用红外光谱(IR)、X射线衍射(XRD)、X射线能谱(EDS)、场发射扫描电镜(FEG-SEM)及振动样品磁强计等对其进行了表征. 结果表明, 形成了CoTAPc包覆在Fe3O4纳米粒子表面的纳米复合粒子. 粒子呈现不规则球形, 平均粒径为70 nm, 矫顽力为316.4 A/m, 接近超顺磁性. 以此纳米复合粒子作为载体, 通过交联法固定漆酶, 固定化酶最适反应温度为45 ℃, 最适pH为3; 固定化酶比游离酶具有更好的热稳定性、贮存稳定性及操作稳定性, 且易于分离.     

A four-electron O(2)-electroreduction biocatalyst superior to platinum and a biofuel cell operating at 0.88 V

O2 was electroreduced to water, at a true-surface-area-based current density of 0.5 mA cm-2, at 37 degrees C and at pH 5 on a "wired" laccase bioelectrocatalyst-coated carbon fiber cathode. The polarization (potential vs the reversible potential of the O2 /H2O half-cell in the same electrolyte) of the cathode was only -0.07 V, approximately one-fifth of the -0.37 V polarization of a smooth platinum fiber cathode, operating in its optimal electrolyte, 0.5 M H2SO4. The bioelectrocatalyst was formed by "wiring" laccase to carbon through an electron conducting redox hydrogel, its redox functions tethered through long and flexible spacers to its cross-linked and hydrated polymer. Incorporation of the tethers increased the apparent electron diffusion coefficient 100-fold to (7.6 +/- 0.3) x 10-7 cm 2 s-1. A miniature single-compartment glucose-O2 biofuel cell made with the novel cathode operated optimally at 0.88 V, the highest operating voltage for a compartmentless miniature fuel cell.     

A membrane-less enzymatic fuel cell with layer-by-layer assembly of redox polymer and enzyme over graphite electrodes

Layer-by-layer (LBL) assembly of alternate osmium redox polymers and glucose oxidase, at anode, and laccase, at cathode, using graphite electrodes form a membrane-less glucose/O(2) enzymatic fuel cell providing a power density of 103 μW cm(-2) at pH 5.5.     

蛋黄-壳结构Fe3O4@SiO2@PMO磁性微球的制备及对漆酶的固定化

以蛋黄-壳结构的Fe₃O₄@SiO₂@PMO磁性微球作为载体, 采用交联法对漆酶进行固定, 考察了戊二醛浓度等对固定效果的影响, 并对固定后漆酶的活性进行了研究. 结果表明, 蛋黄-壳结构的磁性微球负载漆酶仅需6 h, 磁性微球对漆酶的固载量高达475 mg/g. 固定后漆酶的稳定性显著提高, 在pH=2.5~4.5的强酸性条件下, 固定后漆酶酶活仍可保持70%以上, 即使温度升高至60 ℃, 固定后漆酶的相对酶活仍保持65%以上. 这说明漆酶经所合成的材料固定后, 其耐酸和耐热能力都明显优于游离漆酶. 包覆的Fe₃O₄粒子使得材料很容易经磁铁分离法回收, 固定后漆酶经磁分离循环使用10次后仍然能保留85%的酶活, 具有良好的可操作性和稳定性, 有效降低了漆酶的使用成本.     

CuTAPc-Fe3O4纳米复合粒子及其漆酶固定化研究

漆酶的固定化研究对基于漆酶催化的光纤生物传感器具有十分重要的意义，制备了四氨基酞菁铜(CuTAPc)-Fe3O4纳米复合粒子，并用红外(IR)、场发射扫描电镜(FEG—SEM)、X射线衍射(XRD)、能谱、粒径仪等对其进行了表征．结果表明形成了以CuTAPc包覆在Fe3O4纳米粒子表面的纳米复合粒子，粒子呈现不规则球形，且分布均匀，粒子平均粒径在50nm左右，用此纳米复合粒子通过戊二醛交联法固定了漆酶，固定后的酶比游离酶具有更好的贮存稳定性及操作稳定性，这为研制高性能的光纤生物传感器打下了较好的基础。     

Fuel‐Free Bio‐photoelectrochemical Cells Based on a Water/Oxygen Circulation System with a Ni:FeOOH/BiVO4 Photoanode

A bio‐photoelectrochemical cell (BPEC) based on a fuel‐free self‐circulation water–oxygen–water system was fabricated. It consists of Ni:FeOOH modified n‐type bismuth vanadate (BiVO₄) photoanode and laccase catalyzed biocathode. In this BPEC, irradiation of the photoanode generates photocurrent for photo‐oxidation of water to oxygen, which is reduced to water again at the laccase biocathode. Of note, the by‐products of two electrode reactions could continue to be reacted, which means the H₂O and O₂ molecules are retained in an infinite loop of water–oxygen–water without any sacrificial chemical components. As a result, the assembled fuel‐free BPEC exhibits good performance with an open‐circuit potential of 0.97 V and a maximum power density of 205 μW cm^?2 at 0.44 V. This BPEC based on a self‐circulation system offers a fuel‐free model to enhance multiple energy conversion and application in reality.