戊烷与颗粒型甲烷单加氧酶的相互作用研究

颗粒型甲烷单加氧酶(Particulate methane monooxygenase, PMMO)是一个与细胞膜结合的金属酶, 能将烷烃生物催化为醇. 研究PMMO与烷烃的结合模式及催化机制将有利于设计合成一个新的模拟酶, 进而有效地利用烷烃作为新能源. 用分子对接方法获得了PMMO单体与一系列烷烃的结合模式, 并对PMMO单体和PMMO-戊烷复合物进行了6 ns的分子动力学模拟, 最后对复合物进行了构象成簇及结合能分析. 结果表明, 戊烷结合到靠近Zn^2＋的疏水口袋中, 该口袋由pmoA亚基的M45～W60和R190～T193以及pmoC亚基的Q161三个片段组成. 动力学结果表明, 与PMMO单体比, PMMO-戊烷复合物保持着相近的运动模式, 但幅度更明显, 另外, 戊烷在疏水口袋中的大幅度运动对于PMMO发挥催化作用是必须的. 结合能计算揭示疏水相互作用是戊烷与PMMO稳定识别的主要驱动力, 所有模拟结果与实验数据吻合较好.     

聚苯胺用作乙醇脱氢反应中的电子传递介质

脱氢酶（ADH）在电化学氧化还原反应中是很重要的一种酶,但它在催化有机分子脱氢反应时需烟酰胺腺嘌呤二核苷酸（NAD＋）参与,后者从底物接收电子生成还原形NADH.脱氢酶电极是根据NADH的电化学氧化产生的阳极电流构成的[1－3].然而NADH与裸体炭电极和铂金电极之间的直接电子传递是非常困难的,往往需一个相当高的过电位[4].另一个问题是生成物易将电极玷污[5,6].克服这些问题的方法是使用均相电子传递介质,例如在底物溶液中加入Meldola蓝、Nile蓝A和NMP＋甲替硫酸盐等[7－9],及复相电子传递介质,例如将镍六氰基高铁酸盐固…     

溶胶-凝胶固定化多酶催化二氧化碳转化为甲醇反应初探

 为了探索温室气体CO2的固定和利用的新途径，以正硅酸乙酯为\r\n前驱体，用改进的溶胶－凝胶法对甲酸脱氢酶、甲醛脱氢酶和乙醇脱氢\r\n酶进行了包埋共固定化，并以包埋的三种酶为催化剂，以还原型烟酰胺\r\n腺嘌呤二核苷酸（NADH）为电子供体，在低温低压下将CO2转化为甲醇\r\n．初步研究了反应温度、pH值、酶含量及NADH用量对甲醇收率的影响．\r\n实验结果表明，在37℃和pH7．0的条件下，甲醇的收率可达92．4％．\r\n由于酶空间构型的微小变化和空间位阻效应的存在，与液相酶反应结果\r\n相比，包埋后的酶活性略有降低．     

甲烷氧化菌素催化纳米金合成

甲烷氧化菌素(methanobactin,mb)是具有过氧化氢还原酶活性的荧光肽.从甲基弯菌Methylosinus trichospo-rium IMV3011限铜培养介质中分离mb,采用紫外可见全波长扫描法观察mb催化对苯二酚还原氯金酸合成纳米金的作用和影响,当mb/氯金酸/对苯二酚反应液中mb的浓度分别是2.5×10-5mol/L、5.0×10-5mol/L和1.0×10-4mol/L时,形成的纳米金溶液的特征峰分别是561.5 nm(OD561=0.158)、548.0 nm(OD5 48=0.426)、536.5 nm(OD5 36=0.541),特征峰波长减小,对应的吸光值增大,表明mb能够催化对苯二酚还原氯金酸合成纳米金,并且可以通过调控mb的浓度控制纳米金的合成量及粒径大小.     

辅酶NADH与色氨酸共振能量转移的荧光动力学研究

采用时间相关单光子计数技术, 结合紫外-可见吸收光谱和稳态荧光光谱, 对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究. 单体色氨酸、 牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明, 只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移. 进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道, 时间分辨荧光光谱和衰减相关光谱(DAS)证实, 蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件. DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移, 同时给出了NADH和色氨酸之间的能量转移效率, 为研究NADH和蛋白之间的相互作用提供了新思路.     

甲烷氧化细菌催化二氧化碳生物合成甲醇的研究

甲烷氧化细菌中包含的甲烷单加氧酶(MMO)、甲醇脱氢酶(ADH)、甲醛脱氢酶(FaldDH)、甲酸脱氢酶(FateDH)经过一系列反应能够把甲烷深度氧化生成二氧化碳，并生成一定的能量物质．把二氧化碳还原为甲醇是一个需要能量的过程，目前还没有已知的有机体在温和条件下完成这一反应．研究发现，甲基弯菌Methylosi-nus trichosporium IMV 3011可以催化二氧化碳生物转化生成甲醇．在休眠的悬浮细胞中充人二氧化碳后，反应一段时间在反应液中检测到了甲醇．二氧化碳转化成甲醇是一个需要能量推动的反应，为了补充反应所消耗的能量．反应一段时间后需要用甲烷进行再生，以恢复细胞中的还原当量NADH．我们进行了反应再生的交替连续批式反应，甲醇积累量能够维持在一个比较稳定的水平．理论上，反应不会增加温室效应，这是一个有效的、环境友好的、可恢复的反应过程．     

便携式血糖仪检测前列腺特异性抗原

基于抗原-抗体特异性识别机理,以单克隆抗体Ab_1修饰的Fe_3O_4磁珠(MB-Ab_1)为反应平台,联合了多克隆抗体Ab_2和乙醇脱氢酶(ADH)修饰的金纳米颗粒(AuNPs)作为信标(AuNPs-Ab_2/ADH),便携式血糖仪(PGM)为检测手段,构建一种前列腺特异性抗原(PSA)检测方法。当PSA存在时,信标AuNPs-Ab_2/ADH通过抗原-抗体特异性识别被捕获到探针MB-Ab_1上,标记的ADH在烟酰胺腺嘌呤二核苷酸(NAD~+)的辅助下将乙醇催化为乙醛的同时,氧化态NAD~+被还原为还原态NADH。而PGM对NADH具有剂量依赖性,故可间接指示ADH的含量,进而用于PSA的检测。在最优条件下,PGM响应信号与PSA浓度的对数呈良好线性关系,线性范围为0.1～50.0 ng/mL,检出限为30 pg/mL。实际血清中PSA的加标回收率为83.3%～114.5%,相对标准偏差为0.05%～0.20%。     

钯/碳纳米纤维复合材料修饰电极同时检测邻苯二酚和对苯二酚

利用电纺丝技术制得钯/碳纳米纤维复合材料(Pd/CNFs),并将其用于修饰玻碳电极Pd/CNF-GCE/CME.Pd/CNF-GCE/CME对邻苯二酚和对苯二酚的氧化还原反应具有较高的电催化活性,显著提高了二者电化学反应的可逆性.考察了支持电解质的酸度对邻苯二酚和对苯二酚电化学响应的影响,选用0.1 mol/L PBS(pH 8.0)作为支持电解质.用微分脉冲伏安(DPV)法对邻苯二酚和对苯二酚进行选择性检测:当混合溶液中存在50 μmol/L对苯二酚时,邻苯二酚的氧化峰电流与其浓度在1～90 μmol/L范围内呈线性关系,检出限为0.3 μmol/L(S/N=3);当存在50 μmol/L邻苯二酚时,对苯二酚的氧化峰电流与其浓度在2～100 μmol/L范围内呈线性关系,检出限为1.0 μmol/L.另外,此修饰电极具有较好的重现性和较强的抗干扰能力.将此修饰电极用于模拟水样中邻苯二酚和对苯二酚的测定,结果令人满意.     

光控光合菌生物催化苯乙酮不对称还原的反应机理

以苯乙酮作为模型底物,通过制备类球红杆菌(Rhodobacter sphaeroides)的载色体和分离纯化的胞内氧化还原酶混合液,构建了以类球红杆菌全细胞为催化剂、氧化还原酶为催化剂以及载色体与氧化还原酶偶合三种不对称还原反应体系,并通过向反应体系中加入最适氢供体乙酸钠和电子供体硫代硫酸钠提高产物的转化收率.通过检测目标产物的收率、对映体过量(ee)值和光学构型,分析了光控不对称还原的生物催化机理,发现光照可以改变胞内(S)-氧化还原酶和(R)-氧化还原酶的活性,从而产生不同构型的产物,加入电子供体和氢供体后,反应收率和ee值提高的原因是由于分别补充了细菌叶绿素分子Bchl失去的电子和NADPH再生所需的活性氢.     

金属卟啉MTPPS对NADH氧化的催化作用

中位-四(对-磺基苯基)卟啉的铁、锰配合物(FeTPPS和MnTPPS)用作NADH氧化的催化剂,均相溶液中的反应动力学用紫外可见光谱测定。结果表明,在除氧的中性溶液中FeTPPS和MnTPPS降低了NADH在玻碳电极上氧化的过电位,过程用EC再生机理解释。在氧饱和的溶液中MTPPS起电子转移中介体的作用促进NADH氧化,其还原态被O~2氧化而生。测得FeTPPS和NADH反应的速率常数为3.3mol^-^1.L.s^-^1,而MnTPPS和NADH反应的速率常数约为FeTPPS的1/2。讨论了MTPPS作为NADH仿生氧化催化剂的前景。     

The role of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of metal ions on particulate methane monooxygenase (pMMO) was studied. The pMMO activity in the membranes was partially inhibited by ethylenediaminetetraacetic acid (EDTA), but remained more than 70% of the as-isolated membranes. The activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, copper ion stimulated the activity, indicating that copper was needed for the activity. When duroquinol and dioxygen were introduced to the EDTA-treated membranes, the electron spin resonance signal of copper did not change, suggesting that the copper cluster did not play as an electron transport and may have another function, such as active site of pMMO or regulator of the activity. On the other hand, the iron signal (g=5.98) decreased by the addition of duroquinol, dioxygen and acetylene, showing an iron atom is contained in the active site of pMMO.     

Efficient photocatalytic hydrogen evolution without an electron mediator using a simple electron donor-acceptor dyad

A highly efficient photocatalytic hydrogen evolution system without an electron mediator such as methyl viologen (MV(2+)) has been constructed using 9-mesityl-10-methylacridinium ion (Acr(+)-Mes), poly(N-vinyl-2-pyrrolidone)-protected platinum nanoclusters (Pt-PVP) and NADH (beta-nicotinamide adenine dinucleotide, reduced form) as the photocatalyst, hydrogen evolution catalyst and electron donor, respectively. The photocatalyst (Acr(+)-Mes) undergoes photoinduced electron transfer (ET) from the Mes moiety to the singlet excited state of the Acr(+) moiety to produce an extremely long-lived ET state, which is capable of oxidizing NADH and reducing Pt-PVP, leading to efficient hydrogen evolution. The hydrogen evolution efficiency is 300 times higher than that in the presence of MV(2+) because of the much faster reduction rate of Pt-PVP by Acr(*)-Mes compared with that by MV(*+). When the electron donor (NADH) is replaced by ethanol in the presence of an alcohol dehydrogenase (ADH), NADH is regenerated during the photocatalytic hydrogen evolution.     

大肠杆菌异源表达pMMO活性中心pmoB及生物催化研究

颗粒甲烷单加氧酶（pMMO）是甲烷氧化菌中催化甲烷氧化生成甲醇的一种酶．Methylococcus capsulatus IMV 3021的pMMO活性位点是pmoB亚基,该亚基是一种可溶性蛋白．我们研究将pmoB亚基进行异源表达及生物催化活性的验证．当培养基中烟酰胺腺嘌呤二核苷酸（NADH）浓度为5 mmol／L时,可以观察到异源表达pmoB亚基具有催化甲烷氧化成甲醇活性,生成甲醇浓度为1．04 mmol／L．研究pMMO活性对于开发能直接将甲烷转化成甲醇的新型、环保催化剂有非常重要意义．     

Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus bath: evidence for a Diiron center

Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the well-characterized soluble MMO contains a bis-mu-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the alpha subunit (pmoB) of the alphabetagamma trimer and a site at the interface of the betagamma subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1-2 iron atoms per alphabetagamma show the highest catalytic activity. We have employed M?ssbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per alphabetagamma of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the "Zn site" that accommodates the diiron center in active pMMO.     

Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.     

甲烷氧化菌素-铜配合物催化过氧化氢氧化对苯二酚

为了探讨甲烷氧化菌素(Mb)-铜配合物(Mb-Cu)模拟过氧化物酶的可行性, 利用HP20大孔树脂、 Supelco LC-C₁₈固相萃取和固定化金属亲和层析从甲基弯菌IMV3011中分离纯化得到Mb. 铬天青比色法显示Mb具有铜亲和性. 通过液相色谱-飞行时间质谱联用仪、 紫外光谱和荧光光谱对Mb结构进行了表征. 使用Mb-Cu配合物作为过氧化物酶模拟物, 利用紫外-可见分光光度法研究了Mb-Cu催化过氧化氢氧化对苯二酚的动力学. 考察了体系温度、 Mb-Cu添加量及过氧化氢浓度对催化反应的影响, 发现Mb-Cu符合生物催化剂条件影响的一般规律, 但比生物酶具有更高的热稳定性. 研究结果表明, Mb-Cu可作为催化氧化对苯二酚的过氧化物酶模拟酶.     

Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport.

A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.     

Role of iron and copper in particulate methane monooxygenase of Methylosinus trichosporium OB3b

The role of iron and copper in particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b is described, and an overview of the enzyme's properties is presented. The pMMO from M. trichosporium OB3b was solubilized in the detergent n-dodecyl--D-maltoside and purified by chromatographic techniques. The enzyme consists of 0.9 iron atoms and 12.8 copper atoms per molecule. The iron site in pMMO may be mononuclear non-heme iron. Copper exists as either copper ion coupled to four nitrogen atoms and/or trinuclear copper cluster wherein copper ions are ferromagnetically coupled.     

X-ray crystallographic analysis of 9-cis-rhodopsin, a model analogue visual pigment

Recent progress in high-resolution structural study of rhodopsin has been enabled by a novel selective extraction procedure with rod photoreceptor cells. In this study, we applied the method for rapid and efficient preparation of a purified analogue pigment using bovine rod outer segment membranes with 9-cis-retinal. After complete bleaching of the membranes and subsequent regeneration with the exogenous retinal, 9-cis-rhodopsin is selectively extracted from the membranes using combination of zinc and heptylthioglucoside. The solubilized sample, even with a small amount of contaminating retinal oximes, is shown to be pure enough for three-dimensional crystallization. The X-ray diffraction from 9-cis-rhodopsin crystals was examined and the electron density map at 2.9 angstroms resolution in the chromophore region can be fitted well with the model of 9-cis-retinal Schiff base.