D-氨基酸氧化酶的研究进展

D-氨基酸氧化酶(DAAO)是一种以黄素腺嘌呤(FAD)为辅基的典型的黄素蛋白酶类.DAAO可氧化D-氨基酸生成相应的酮酸和氨.介绍了D-氨基酸氧化酶的研究现状,包括在自然界的分布、生理功能、生物学特性、催化机理、基因克隆和序列分析以及基因表达.     

Identification and Characterization of Citrus Peel Uronic Acid Oxidase

Uronic acid-rich plant materials such as pectin are potential renewable feedstocks for the chemical industry. Uronic acid oxidase activity was first reported in extracts of citrus leaves, and was subsequently found to be widely distributed in plants, with the highest activity detected in extracts of the pectin-rich citrus peel. Herein we report the identification of the primary sequence of uronic acid oxidase from extracts of the peel of Citrus sinensis, by partial purification and protein mass spectrometry. Activity of the enzyme, a member of the berberine bridge family, was confirmed by recombinant expression in Pichia pastoris. Biochemical characterization of the recombinant enzyme is reported. Our findings facilitate further investigation of the biological function of uronic acid oxidation in the economically important orange fruit, and also provide a basis for the development of a catalyst for bioconversion of uronic acids.     

多酚氧化酶催化单宁的氧化聚合反应研究

研究了多酚氧化酶催化单宁的氧化聚合反应情况 ,探索了反应条件及 7种化合物对酶活性的抑制效果 ,对产物进行了红外光谱和紫外光谱测试 ,相对粘度法测定了产物的分子量。结果表明 ,反应的最佳条件是pH为 5 .2 9,温度为 3 0℃ ,在所用抑制剂中 ,硫酸铜的抑制作用最强 ,与单宁酸的IR图相比 ,产物的 -OH吸收带变窄 ,在 12 60cm-1处吸收峰增强 ,而紫外吸收无变化。产物平均分子量为 3 5 41,说明多酚氧化酶能催化单宁的氧化聚合     

氨基G酸及2—萘胺—3,6,8—三磺酸的合成研究

本文以Ｇ盐为原料,经加氨化反应合成了氨基Ｇ酸,并以氨基Ｇ酸为原料,经发烟硫酸磺化反应合成了２－萘胺－３,６,８－三磺酸,并对影响反应的因素做了探讨。研究表明,随反应温度、反应时间、氨水浓度、氨比、催化剂用量的提高,氨基Ｇ酸收率增大。用４０％￣６０％发烟硫酸,反应温度为１３０℃,氨基Ｇ酸／ＳＯ３＝１：７．５￣１：８．５时,２－萘胺－３,６,８－三磺酸产品收率约为９０％。     

固定化D-氨基酸氧化酶转化头孢菌素C为戊二酰基-7-氨基头孢霉烷酸

部分纯化的变异三角酵母D-氨基酸氧化酶(DAO)在碱性条件下变性过氧化氢酶,再与大孔聚甲基丙烯酸缩水甘油酯高聚物共价交联。与游离酶相比,固定化酶的最适反应温度升高,最适pH范围变宽,对温度和pH的稳定性都有明显的提高。固定化DAO在搅拌反应器中催化头孢霉素C(CPC)转化为戊二酰基-7-氨基头孢霉烷酸(Gl-7-ACA)。以0.03g/mL的CPC为底物,在温度25℃、pH7.2条件下,产物的得率>93%,副产物得率<5%。经过110批反应后,固定化酶保持64%的初始活力。     

Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

Age-Dependent Effects of Jasmonic Acid Treatment and Wind Exposure on Foliar Oxidase Activity and Insect Resistance in Tomato

Jasmonic acid (JA) treatment of tomato plants induces several defense-related oxidative enzymes and increases pest resistance in a manner thought to simulate natural insect wounding. In a full-factorial greenhouse experiment, we examined the independent and interactive effects of plant age and exposure to wind-induced mechanical stress (MS), on the ability of JA to induce defense in tomato. In general, treatment of 4-, 6-, and 8-week-old tomato plants with 1 mM JA resulted in the induction of peroxidase and polyphenol oxidase activity and reduced the relative growth rate of first-instar Manduca sexta larvae fed treated leaves, in accordance with other studies. Peroxidase activity increased with plant age and was induced by JA most strongly in older plants. In contrast, polyphenol oxidase activity did not change with plant age and was induced by JA most strongly in young plants. While relative growth rates of M. sexta were lower on older plants overall, JA reduced growth rates most strongly in young plants, in which JA treatment enhanced polyphenol oxidase activity by more than 70%. MS enhanced the activity of peroxidase, but substantially reduced the activity of polyphenol oxidase; the latter most intensely on older plants. M. sexta tended to grow more slowly on MS-treated plants, although this effect was not significant. Thus, reduced polyphenol oxidase activity in MS-treated plants did not lead to an increase in growth rate of M. sexta, possibly because peroxidase activity was still elevated in MS-treated plants. Significant interactions between JA and MS and three-way interactions were not detected for any variable, although the inductive effects of both JA and MS interacted in complex ways with plant age. Our results indicate that resistance traits in tomato are differentially affected by JA and wind exposure and differ in their relative contribution to defense as plants age.     

TiO2光催化分解酸性红G和活性艳红K-2G的研究

研究了在紫外光的照射下,由TiO2催化分解染料酸性红G和活性艳红K-2G的行为,并且探讨了TiO2的添加量、染料结构、染料的初始浓度以及溶液pH值等因素对上述2种染料光催化分解的影响.实验结果表明,当TiO2的添加量为3g·L-1时,2种染料的催化分解率最大;在碱性条件下(pH＝8.5)的分解效果好于酸性条件(pH＝5.5).     

Ascorbate Oxidase Mimetic Activity of Copper(II) Oxide Nanoparticles

Although oxidase mimetic nanozymes have been widely investigated, specific biological molecules have rarely been explored as substrates, particularly in the case of ascorbate oxidase (AAO) mimetic nanozymes. Herein, we demonstrate for the first time that copper(II) oxide nanoparticles (CuO NPs) catalyze the oxidation of ascorbic acid (AA) by dissolved O₂ (as a green oxidant) to form dehydroascorbic acid (DHAA), thus functioning as a new kind of AAO mimic. Under neutral conditions, the Michaelis–Menten constant of CuO NPs (0.1302 mm ) is similar to that of AAO (0.0840 mm ). Furthermore, the robustness of CuO NPs is greater than that of AAO, thus making them suitable for applications under various conditions. As a demonstration, a fluorescence AA sensor based on the AAO mimetic activity of CuO NPs was developed. To obtain a fluorescent product, o-phenylenediamine (OPDA) was used to react with the DHAA produced by the oxidation of AA catalyzed by CuO NPs. The developed sensor was cost-effective and easy to fabricate and exhibited high selectivity/sensitivity with a wide linear range (1.25×10⁻⁶ to 1.125×10⁻⁴ m ) and a low detection limit (3.2×10⁻⁸ m ). The results are expected to aid in expanding the applicability of oxidase mimetic nanozymes in a variety of fields such as biology, medicine, and detection science.     

过氧化氢对D-氨基酸氧化酶转化头孢菌素C为戊二酰-7-氨基头孢烯酸反应的影响

在D-氨基酸氧化酶D1AAO转化头孢菌素C(CPC)为戊二酰基-7-氨基头孢烯酸(Gl-7-ACA)的反应中,所生成的过氧化氢同时与CPC和Gl-7-ACA发生的氧化副反应是造成目标产物收率损失的关键因素之一.通过联用溶氧电极和过氧化氢电极,测定了DAAO转化CPC为Gl-7-ACA的反应体系内溶氧浓度与相应的过氧化氢浓度的变化曲线,测知反应体系中过氧化氢浓度积累最高可达5 mmol·L-1.模拟该反应条件下过氧化氢的氧化反应可知,在过氧化氢浓度较低时,溶液的酸性可加速该氧化反应.在中性条件下,过氧化氢浓度为5 mmol·L-1时,反应1h后,CPC的氧化损失为2%,Gl-7-ACA的氧化损失为3%.通过改进氧气分布器、搅拌转速和并调控氧气流量以控制溶液中溶解氧的浓度,来适度地降低物系中过氧化氢积累浓度,使反应收率提高了2.2%.     

酶偶联分光光度法测定胞内黄嘌呤氧化酶活性

建立了新的酶反应体系显色分光光度法检测节杆菌胞内黄嘌吟氧化酶的活性。考察了温度、pH值和底物浓度对检测体系的影响,确定了黄嘌呤氧化酶活性检测的最优条件,获得了良好的检测线性关系。此检测方法操作简便,结果准确可靠,可作为普通实验室和临床上测定黄嘌呤氧化酶活性的有效生化手段。     

金橙G共振光散射法测定脱氧核糖核酸

研究了生物染色荆金橙G（OG）与脱氧棱糖核酸（DNA）相互作用的共振光散射光谱。结果发现,DNA对OG的共振光散射有增强效应,且在特定波长（358nm）下,其增强值（△I）与加入DNA的浓度呈良好的线性关系,据此建立了一种定量测定DNA的共振光散射法。该方法线性范围为0．053-1．20μ·mL^-1,检出限为0．0159μ·mL^-1,回收率为98．6％-102．3％。该方法简便、快速,用于合成样品中DNA的测定,结果令人满意。     

Role of NADPH Oxidase and Xanthine Oxidase in Mediating Inducible VT/VF and Triggered Activity in a Canine Model of Myocardial Ischemia

Background: Ventricular tachycardia or fibrillation (VT/VF) of focal origin due to triggered activity (TA) from delayed afterdepolarizations (DADs) is reproducibly inducible after anterior coronary artery occlusion. Both VT/VF and TA can be blocked by reducing reactive oxygen species (ROS). We tested the hypothesis that inhibition of NADPH oxidase and xanthine oxidase would block VT/VF. Methods: 69 dogs received apocynin (APO), 4 mg/kg intraveneously (IV), oxypurinol (OXY), 4 mg/kg IV, or both APO and OXY (BOTH) agents, or saline 3 h after coronary occlusion. Endocardium from ischemic sites (3-D mapping) was sampled for Rac1 (GTP-binding protein in membrane NADPH oxidase) activation or standard microelectrode techniques. Results (mean ± SE, * p < 0.05): VT/VF originating from ischemic zones was blocked by APO in 6/10 *, OXY in 4/9 *, BOTH in 5/8 * or saline in 1/27; 11/16 VT/VFs blocked were focal. In isolated myocardium, TA was blocked by APO (10⁻⁶ M) or OXY (10⁻⁸ M). Rac1 levels in ischemic endocardium were decreased by APO or OXY. Conclusion: APO and OXY suppressed focal VT/VF due to DADs, but the combination of the drugs was not more effective than either alone. Both drugs inhibited ischemic Rac1 with inhibition by OXY suggesting ROS-induced ROS. The inability to totally prevent VT/VF suggests that other mechanisms also contribute to ischemic VT.     

从甘草中提甘草酸不同提取方法的比较

对几种不同的从甘草中提取甘草酸的提取方法进行了实验比较。结果表明,微波辅助提取法与热回流法、索氏（Soxhlet）提取法、室温提取法等传统方法相比,具有提取高效、快速、完全及节省时间、溶剂和能 耗等优点,是一种适于从甘草中快速提取甘草酸的新方法。     

Molecular Mechanisms of Succinimide Formation from Aspartic Acid Residues Catalyzed by Two Water Molecules in the Aqueous Phase

Aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. The formation of isomerized Asp residues is considered to be associated with various age-related diseases, such as cataracts and Alzheimer’s disease. In the present paper, we describe the reaction pathway of Suc residue formation from Asp residues catalyzed by two water molecules using the B3LYP/6-31+G(d,p) level of theory. Single-point energies were calculated using the MP2/6-311+G(d,p) level of theory. For these calculations, we used a model compound in which an Asp residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. In the aqueous phase, Suc residue formation from an Asp residue was roughly divided into three steps, namely, iminolization, cyclization, and dehydration, with the activation energy estimated to be 109 kJ mol⁻¹. Some optimized geometries and reaction modes in the aqueous phase were observed that differed from those in the gas phase.     

香草酸对酪氨酸酶催化活性的抑制作用

采用酶动力学方法研究了香草酸对酪氨酸酶单酚酶和二酚酶活力的抑制效应。结果表明,香草酸对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,导致单酚酶活力和二酚酶活力下降50%的香草酸浓度(IC50)约分别为1.3 mmol/L和2.6 mmol/L。香草酸能明显延长单酚酶的迟滞时间,2 mmol/L香草酸能使迟滞时间由1.1 m in延长至4.7 m in。香草酸对二酚酶的抑制作用表现为混合型抑制,对游离酶的抑制常数(KI)和对酶-底物络合物的抑制常数(KIS)分别为1.76 mmol/L和8.57 mmol/L。     

阿魏酸衍生物的设计、合成及除草活性

[目的]以天然产物阿魏酸为先导设计合成具有较强除草活性的衍生物.[方法]运用农药分子合理设计原理设计合成阿魏酸酰胺类似物,采用小杯法测定除草活性.[结果]合成了11个新型的阿魏酸衍生物;小杯法测定显示,在400 mg/L质量浓度下,化合物8f和8k的除草活性较好;半抑制浓度测定结果表明:8f对马唐根和茎的IC50值为分...     

青霉素G亚砜的合成

以青霉素G钾盐为原料,采用15%左右低浓度过氧乙酸为氧化剂合成青霉素G亚砜,反应时间2～2.5h,n(过氧乙酸):n(青霉素G钾盐)=(1.1～1.2):1.0,反应温度和结晶温度在0～5℃,总收率可达96%以上。所得青霉素G亚砜可直接从溶液中结晶析出,解决了产物与反应体系的分离。产品纯度较高。