Expression, purification and characterization of the soluble CuA domain of cytochrome c oxidase ofParacoccus versutus

The key subunit Ⅱ of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (Cu_A) domain. The Cu_A domain of Paracoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cu_A domain in pET11d vector was expressed in E. coli BL21 (DE3). The results showed that the Cu_A domain was expressed mostly in inclusion bodies and the Cu_A domain protein synthesized in E. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dialyzed and recombined with Cu⁺/Cu²⁺ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band in electrophoresis, was obtained. The UV-visible absorption spectrum of Cu_A domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 nm respectively, which can be assigned to the charge transfer and the interactions of obitals of Cu—S and Cu——Cu in the mixed-valence binuclear metal center (Cu₂S₂R₂). The far-UV CD spectrum indicated that this domain is predominantly in β-sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.     

纤维素固定化葡萄糖氧化酶的研究

用高碘酸钠（NaIO4)溶液将棉纤维素氧化成氧化纤维素,并将其作为载体使葡萄糖氧化酶能够固定化。考察了溶液的pH值、氧化时间、温度和氧化剂浓度等氧化条件对氧化纤维素醛基含量的影响,优化了氧化条件,研究了固定化葡萄糖氧化酶的活性,借助傅里叶红外光谱和酸－碱滴定法等手段,探讨了葡萄糖氧化酶的固定化机理。研究结果表明,纤维素固定化葡萄糖氧化酶具有较高的生物活性。     

一类素数阶完全幻方构造的优化性与自动生成

通过对一类素数阶 (N≥ 3)幻方算法的改进得到一类素数阶完全幻方的两种构造方法 ,并比较它们的特点。根据算法 ( )用 C语言自动生成任意素数阶 (N≥ 5 )完全幻方。     

具不变集的平面三次系统极限环的不存在性

用定性分析的方法,证明了具有两条三次代数曲线解ｙ＾２＝（ａｘ＾３＋ｂｘ）＾２的平面三次系统无级限环,但可以有奇闭轨。     

高能重离子辐照对氧化物高温超导体的临界电流密度？…

运用层状高温超导体的临界电流密度理论模型,对１８０ＭｅＶ的Ｃｕ＾１１＋辐照高温超导体Ｙ－１２３和Ｂｉ－２２１２的临界电流密度的影响做了分析,并对试验结果进行了拟合。拟合结果与试验结果符合得较好,这表明辐照产生的柱状缺陷作为主要的钉扎中心,具有较高的钉扎能,且柱状缺陷在直径上与磁通涡旋有相近的尺寸。     

理想调节网络与电力负荷聚类特征

讨论了理想调节网络对三相电力负荷中的无功与三相不平衡进行完全调节的基本原理,给出了应用理想调节电纳与模糊c均值聚类算法提取负荷聚类特征的方法,聚类特征可用于用户电力调节系统的优化设计。工程实例表明了该方法的正确性与有效性。     

橡椀单宁降解酶特性的研究

借助于没食子酸丙酯为模型化合物作为底物,对一株能抵抗橡椀单宁(鞣花类单宁)生物毒性并能有效降解这种单宁的菌株的降解特性进行了研究。结果表明这株菌不仅具有单宁酶的活性,而且具有多酚氧化酶（PPO）的活性。通过对不同底物对内胞霉PPO酶促反应速度的影响研究证实橡椀单宁的降解不仅与单宁酶有关还与多酚氧化酶有关。     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

Polyphenol oxidase (PPO) is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO, a metalloenzyme oxidase, catalyzes the oxidation of ο-diphenol to ο-quinone. The present paper de- scribes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate. The results show that benzaldehyde, p- hydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was esti- mated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analy- ses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, p- hydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyano- benzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other com- pounds. These results provide a basis for developing PPO inhibition-based pesticides.     

公路项目综合评价方法研究

基于公路建设项目与社会、经济、环境协调发展的角度考虑,并结合中国国情,作者建立了公路建设项目各指标协调发展的三级评价指标体系即目标层指标、准则层指标和指标层指标.运用模糊数学理论,作者建立了NPV、Nd、IRR、BCR的隶属函数.运用集值统计方法对社会效益指标体系和环境效益指标体系中的七个指标进行了无量纲化处理,得到无量纲矩阵.通过对定量与定性指标的评判,得到单因素评价矩阵.运用AHP法,对若干备选方案作相对评价和综合评价,确定各因素的权重,排列出各方案的优劣次序.根据最大隶属原则,求得了公路项目各指标协