枯草杆菌漆酶重要功能位点的保守性与可变性分析

在对比分析天然枯草杆菌漆酶(CAR)的一级结构与三维空间结构的基础上,锁定了CAR的底物结合通道和铜离子结合位点,设计并构建了CAR的4种突变体.对比分析了野生型酶与4种突变体酶的性质及催化功能,结果表明,该漆酶分子中位于第二保守区的H155位点是漆酶催化所必需的,H155即使被其同义氨基酸(如Arg)替换,也会因氧化还原电势的降低而降低漆酶的催化活力,同时还会导致漆酶的热稳定性和酸碱稳定性降低,以及最适p H值酸移;位于非保守区的A158位点与I224位点的保守性对漆酶的催化是有利的,而S210G突变增强了漆酶的稳定性与催化功能,这可能与S210G突变增强了漆酶的底物通道上一段富含Pro片段的柔性有关.此外,A158/R155H突变(CAHE)可能导致漆酶不耐冷,在低温(50℃)时引起酶活明显降低.所研究的漆酶在2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)介体存在下,对靛蓝类染料(如靛蓝胭脂红)和三苯甲烷类染料(如结晶紫)的脱色率均高于对偶氮类染料(如甲基红和刚果红)和蒽醌类染料(如活性亮蓝),说明该漆酶对染料有一定的选择性.     

微量热法研究单底物酶促反应的热力学和动力学性质及过渡态的分析

用LKB-2107型微量热系统, 测定了漆酶催化氧化3, 4-二羟基苯甲醛、邻甲氧基酚、邻苯三酚、3, 4, 5-三羟基苯甲酸反应的热谱图, 利用热谱图计算了米氏常数(Km)、反应速率常数(k2)和热力学参数(ΔrHm, ΔG0, ΔT^≠, Ea, ΔST^≠)。并应用过渡态理论对其催化过程进行了分析。结果表明: 稳定过渡态结构有利于酶促反应, 酶-底物在反应物时相互作用仅仅是降低酶的催化效率。提出两种可能提高酶催化效率的方法。由活化熵(ΔST)<0得出酶-底物在过渡态的结构较酶-底物复合物的结构更为有序。     

过渡金属离子对漆酶催化活性的影响

以2,2＿连氮＿双(3＿乙基苯并噻唑＿6＿磺酸)(ABTS)为底物,在pH＝4.5的条件下,用分光光度法考察了5种金属离子(Fe2+、Ca2+、Mg2+、Mn2+、Cu2+)对漆酶催化活性的影响。实验发现Fe2+对漆酶的催化活性显示出抑制作用,并进一步探讨了其抑制特征、抑制位点和作用方式。结果表明,Fe2+对漆酶催化活性抑制属竞争性抑制过程,抑制作用是通过还原ABTS·来实现的。     

聚乙二醇基(PEG)水凝胶包埋漆酶研究

漆酶(Laccase)在有机污染物降解以及废水处理等环境治理领域有着杰出的作用，但在极端温度和pH下容易失活，限制了其应用。因此，设计具有较好保护作用的漆酶固定化载体，以扩大其在环境污染防治领域的应用具有重要的研究意义。采用紫外光照聚合法将漆酶固定在水凝胶(PEG)上，然后与戊二醛(GA)交联，得到了固定化漆酶水凝胶微球Lac/particles。以2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)为底物，对包埋后的漆酶的活性进行了考察。研究结果表明，在凝胶的保护下，Lac的pH耐受性、温度耐受性以及储存稳定性等方面均比游离漆酶有着不同程度的增强。     

基于固定化电子媒介体的漆酶电极的制备与应用

采用一种新方法引入媒介体,即将媒介体2,2-连氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)固定在多壁碳纳米管(MWCNTs)上,将壳聚糖与漆酶共同修饰在玻碳电极表面,制备了漆酶电极。实验发现,ABTS能够被牢固地吸附在多壁碳纳米管上,且吸附了ABTS的多壁碳纳米管在水中的分散性能大为改善。由于ABTS的存在,该漆酶电极能够更加有效地催化氧气还原,使氧气的还原电位从-0.1 V升至0.6 V。研究了修饰在多壁碳纳米管上的ABTS的电化学行为,并讨论了碳纳米管修饰量、pH值及温度对漆酶电极催化性能的影响。该方法简单,制备的漆酶电极对氧气还原的催化效率高,有望应用于植入型生物燃料电池。     

水／有机体系中漆酶酶促催化2,6-二甲氧基苯酚及产物表征

作为酶促催化底物之一的2,6-二甲氧基苯酚（DMP）经常用于测定漆酶活性．然而到目前为止,其酶促产物结构却仍不清楚．由此,研究了漆树漆酶对其催化氧化反应．在水,有机溶剂体系中,催化得到一种产物3,3′,5,5′-四甲氧基-1,1′-联苯-4-4′-二酚（TMBP）,对其用红外、核磁和色谱一质谱等系统测定,并简要探讨了酶促反应机理．     

大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu²⁺的改性大尺寸SiO₂大孔材料作为载体, 考察了时间、pH和给酶量对漆酶固定化效果的影响, 并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡, pH为4.5、漆酶与载体比例为5 mg·g^-1时固定化效果最好, 酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽, 固定化后, 漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K_m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性, 与底物反应反复操作10批次后剩余酶活为72.7%。     

偶联酶催化分光光度法测定黄嘌呤

研究了以黄嘌呤氧化酶-辣根过氧化物酶-苯酚-4-氨基安替比林反应为显色体系测定不同样液中黄嘌呤浓度的新方法。确定该测定方法的最佳反应条件为:黄嘌呤氧化酶(XO)0·32U/mL,辣根过氧化物酶(HRP)7·0U/mL,4-氨基安替比林(AAP)1mmol/L,苯酚(PA)6mmol/L溶于100mmol/LTris-HCl缓冲液(pH8·4);反应温度为37℃,保温时间为20min;检测波长为508nm。本方法测定黄嘌呤浓度的线性范围为0·2～10·0mmol/L,线性关系良好(R=0·9978),检测限为0·05mmol/L。方法操作简单易行,测定结果准确可靠,可有效应用于普通实验室和常规临床血液生化检测。     

模拟酶催化反应用于环境样品中苯酚的测定

以氯化血红素(Hemin)作为过氧化物模拟酶,4-氨基安替比林(4-AAP)和苯酚(Phen)为氢供体底物,通过模拟酶催化反应活化H2O2显色反应原理,建立了酚类化合物的测定方法。结果表明,在pH 11.2条件下,苯酚浓度在0~7.38×10-6mol/L范围内与吸光度有良好的线性关系,苯酚的表观摩尔吸光系数为1.73×105L.mol-1.cm-1。以污水为样品,采用加入回收法测定了酚类化合物的含量,结果满意。     

漆树漆酶的催化氧化作用——Ⅳ.二茂铁及其衍生物的酶促氧化

漆酶是含铜单电子氧化酶,主要催化邻、对苯二酚(胺)类底物氧化,它对不溶于水的二茂铁(Fc-H)及其衍生物(Fc-R)的催化氧化未见报道。本文考察了漆树漆酶在磷酸缓冲液-二乙二醇丁醚(DGBE)中对Fc-H和15个Fc-R的催化氧化反应。     

喜树碱的电还原特性及应用研究

The voltammetric behavior of camptothecin （CPT） in Britton-Robinson （B-R） buffer solutions （pH 2.09-9.07） was studied by the means of linear sweep voltammetry （LSV）, cyclic voltarnmetry （CV） and normal pulse voltammetry （NPV） at a hanging mercury drop electrode. In different pH range of B-R buffer solutions, CPT could cause three reduction waves. In B-R buffer solutions （pH 2.09-5.46）, wave P1 yielded by CPT was a two-electron wave. Between pH 6.01 and 9.07, CPT could yield two reduction waves P2 and P3. In addition, the pure CPT obtained from camptotheca acumina grown only in China was determined by NPV, and a linear response was observed in the range of 2.0 × 10^-3-4.0 × 10^-2 mmol·L^-1 with a 0.9991 correlation coefficient and a 8.0 × 1^-4 mmol·L^-1 detection limit for CPT.     

Thermokinetic Studies on the Activation of Arginase by Glycine

The activation of bovine liver arginase, which catalyzes the hydrolysis of L‐arginine to L‐ornithine and urea, by glycine was studied by thermokinetic methods at 37°C in 40 mmol·L^?1 sodium barbiturate‐HCl buffer solution (pH 9.4). Results of this experiment indicate that an appropriate concentration of glycine can enhance the activity of arginase, and the relative activation rate reached its maximum value, 74%, when the concentration of glycine in reaction system was 1 mmol·L^?1 and the initial concentration of arginine was 5 mmol·L^?1. With the increase of substrate concentration, the relative activation rate decreased in a definite glycine concentration. Michealis constant K_m of reaction decreased from 5.53 to 3.31 mmol·L^?1 and inhibition constant of product L‐ornithine K_p increased from 1.18 to 3.73 mmol·L^?1 when glycine concentration was 1 mmol·L^?1. For these reasons one possible activation mechanism of arginase by glycine was suggested that the activation effect results from the competition of glycine and arginine to enzyme activity position. When one or two of the activity positions of arginase are occupied by glycine, it is propitious for the enzyme to complex with substrate and obstruct L‐ornithine from combining with enzyme, and when all of the activity positions are occupied by glycine, the activation effect vanishs and the inhibition effect appears.     

Influence of sodium dodecyl sulfate on the phase transition of thermoresponsive hyperbranched polymer in water

The influence of sodium dodecyl sulfate(SDS) on the cloud point temperature(Tcp) of the aqueous solution of thermoresponsive hyperbranched polyethylenimine derivative HPEI-IBAm was studied systematically. When p H was below 8.5, HPEI-IBAm was positively-charged. Initially, the Tcp of HPEI-IBAm decreased significantly, followed by an obvious increase with the increase of SDS concentration. The lower the p H was, the higher the SDS concentration was required to achieve the minimum Tcp. When p H was above 8.5, HPEI-IBAm was neutral and raising the SDS concentration led to the gradual increase of Tcp. Compared to linear poly(N-isopropyl acrylamide)(PNIPAm), the Tcp of the current hyperbranched HPEI-IBAm was more sensitive to SDS. The thermoresponsive HPEI-IBAm/SDS complex was used as host to accommodate the non-polar pyrene in water. The lowest SDS concentration for effectively enhancing the solubility of pyrene in water was around 6.4 mmol·L~(-1). When HPEI-IBAm was present, the SDS concentration threshhold was decreased to about 0.31 mmol·L~(-1). Fluorescence technique with pyrene as the hydrophobic probe demonstrated that the SDS concentration of 7.2 mmol·L~(-1) was required to form the hydrophobic domain to accommodate pyrene guests without HPEI-IBAm, while only 0.2 mmol·L~(-1) of SDS was required in the presence of HPEI-IBAm.     

Spectral Studies on the Interaction between Mercuric Ion and ApoCopC

The interaction between mercuric ion and apoCopC in the absence or presence of cupric ion was investigated through difference UV spectra in Hepes buffer （10 mmol·L^-1） at pH 7.4. The results suggest that mercuric ion can bind to C- and N-terminal binding sites of apoCopC, and the conditional binding constants were calculated to be kN=（6.79± 1.12）× 10^6 mol^-1·L and kc=（3.06±0.05）× 10^5 mol^-1·L. Using urea as a chemical agent, the conformational stabilities of apoCopC and HgN^2＋ -CopC-Hgc^2＋ were monitored by fluorescence spectrum in Hepes buffer （50 mmol·L^-1） at pH 7.4. The free energy of stabilization is （14.69±0.85） and （16.66±0.55） kJ.mol^-1, respectively. HgN^2＋ -CopC-Hgc^2＋ is more stable than apoCopC.     

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.     

镁合金表面原位生长有机膦酸盐薄膜及耐蚀行为

利用超声辅助的浸渍涂布方法,在AZ91D镁合金表面原位生长了氨基二乙酸亚甲基膦酸镁铝薄膜,其中包含具有纳米尺度的变形六边形板状颗粒。塔菲尔极化曲线、电化学阻抗谱测量表明,修饰后的AZ91D镁合金的耐腐蚀行为依赖于浸渍所用的膦酸浓度、pH值、温度及时间等。在3.5%NaCl溶液中,未修饰的AZ91D其腐蚀活化能是18.1 kJ.mol-1,而在1.5 mmol.L-1膦酸溶液中pH值分别是1.7、11.5时浸渍涂布有机膦酸盐薄膜后,AZ91D的腐蚀活化能分别是27.9、37.8 kJ.mol-1。     

Determination of phenolic acids using Trametes versicolor laccase

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0 μM caffeic acid, 0.05-0.2 μM ferulic acid, 2.0-14.0 μM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0 μM caffeic acid, 2.0-10.0 μM ferulic acid, 4.0-30.0 μM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.     

云芝漆酶在N,N'-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸基元上的固定及其修饰玻碳电极电化学行为

以N,N′-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸作为固定漆酶的载体,以共价偶联法固定云芝漆酶并测定了固定基元的酶固定量和固定漆酶的比活力。 还研究了固定漆酶热稳定性、重复使用性以及固定漆酶催化2,6-二甲氧基苯酚（DMP）氧化的酶动力学参数。 实验结果表明,这种交联聚合物基元通过共价偶联法固定漆酶的量和固定漆酶的比活力分别可达26.37 mg/g和1.202 U/mg;在交联聚合物基元上固定的漆酶在50 ℃下放置2 h后仍然保持初始活力的83％,重复使用10次后仍保持初始活力的80％以上;交联聚合物固定漆酶催化DMP氧化的表观速率常数k_cat可达1090 min^-1,以固定漆酶的BIS交联聚甲基丙烯酸功能化碳纳米管修饰的玻碳电极在pH=4.4磷酸盐缓冲液中氧还原发生在+724 mV(vs.SCE)。     

链酚胺型配体锌(Ⅱ)配合物模拟碳酸酐酶研究

合成了酚胺型链状配体,N,N'-二(2-羟基苄基)丙二胺(H~2L)及其Zn(Ⅱ)配合物(ZnL),通过元素分析、IR和^1HNRM等手段进行了表征。采用pH电位滴定法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,测定了配体的质子化常数以及配体与金属离子Zn(Ⅱ)配位反应平衡常数。讨论了配体与金属离子Zn(Ⅱ)的配位情况,得到了配位酚羟基的解离常数。运用分光光度法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,在pH=5.5～9.0(50mmol·dm^-^3缓冲溶液)范围内,研究了配合物作为碳酸酐酶模拟物催化对-硝基苯酚乙酸酯(NA)水解动力学,得到了NA酯水解的配合物催化速率常数k~N~P。实验结果表明,ZnHL^+的配位酚羟基的解离常数pK~a为6.83;催化速率常数k~N~P与pH之间不存在Sigmoidal型曲线关系,而是在pH值中性附近有最大值,ZnHL^+对NA酯水解有很好的催化效果,并且采取双重催化机理,是碳酸酐酶很好的模拟物。