耐高温重组CotA蛋白的胆红素氧化酶反应动力学及活性鉴定

已知源于枯草芽孢杆菌内生孢子的CotA蛋白具有漆酶和胆红素氧化酶活性。然而,其分离纯化极为困难。本研究对表达与纯化的重组CotA蛋白的胆红素氧化酶特性及氧化还原功能进行鉴定。基因转染及筛选获得了表达CotA的P. pastoris菌株|继而,表达的重组CotA蛋白经DEAE-Sepharose FF 及Sephadex G-75层析分离与纯化,产物得率为25%,纯化产物的酶比活性为 4 U/mg。经SDS-PAGE 和 MALDI-TOF MS 分析显示,其分子质量为65 kD。纯化的CotA蛋白能够催化胆红素氧化,生成胆绿素,且催化反应速率受反应溶液中溶解氧含量的影响,提示纯化的重组CotA具有胆红素氧化酶活性。酶反应进一步证明,CotA的胆红素氧化酶反应最适pH值为pH 8.0,最适温度为60℃。该酶在90℃条件下的半衰期为7 h,提示CotA胆红素氧化酶具有高度的热稳定性。CotA修饰的摄谱仪石墨电极可直接电催化分子氧（O2）还原,具有很好的电流响应。我们的结果表明,重组的CotA蛋白具有耐高温胆红素氧化酶活性。更重要的是,我们的结果还提示重组的CotA蛋白在酶生物燃料电池阴极的制备上具有较好的应用潜能。     

血清胆红素的光敏氧化

竹红菌乙素是一种芘醌类的光敏剂.实验结果表明在可见光的照射下.它能加速血清胆红素的光氧化,氧化速率提高5倍以上.比较在不同溶剂中各种活性氧淬灭剂对胆红素光氧化的抑制作用,指出血清胆红素光敏氧化反应包括自由基氧化反应(Ⅰ型反应)和单态氧氧化反应(Ⅱ型反应)等多重机制.     

嗜碱芽孢杆菌cotA基因克隆表达及部分性质研究

为了获得具有高性能的多铜离子氧化酶,从极端微生物嗜碱芽孢杆菌(Bacillus clausii KSM-K16)中克隆表达了其芽孢外衣蛋白CotA。根据B.clausii KSM-K16的CotA序列及大肠杆菌密码子的偏爱性,设计和合成出该基因全长序列,构建pET28a-cot A重组表达载体,将其转化到Escherichia coli BL21(DE3)并诱导表达出重组蛋白,纯化并研究了CotA部分生化特性。重组CotA约62 kD,表现出蓝绿色并在波长609 nm有铜离子特征吸收峰,能氧化ABTS、SGZ和胆红素等底物;以SGZ为底物,最适pH和温度分别为7.5和90℃;在80℃孵育2 h,能保持70%活性;在pH4-11范围分别孵育1 h,能保持90%活性。结果显示,嗜碱芽孢杆菌CotA是一种具有漆酶和胆红素氧化酶活性的典型多铜离子氧化酶,表现出热和酸碱稳定性。     

胆红素氧化反应的激光表面增强拉曼光谱研究

本文报道了胆红素和胆绿素,以及氧化胆红素的ＳＥＲＳ谱图。通过对其谱图的分析发现,氧化胆红素ＳＥＲＳ谱与胆绿素ＳＥＥＳ谱极其相近。表明胆红素在碱性环境中可被氧化成胆绿素。另外,通过对比胆红素和胆绿素的ＳＥＲＳＳ谱,发现在银胶界面它们的吸附构象不同。     

单胺氧化酶抑制剂在临床方面的应用

单胺氧化酶（monoamine oxidase, MAO）是人体内天然存在的一种酶,催化单胺类物质氧化脱氨反应的酶。人体内含有两种单胺氧化酶：单胺氧化酶A 和单胺氧化酶B。单胺氧化酶A 主要分布在儿茶酚胺能神经元中;单胺氧化酶B 主要分布在5- 羟色胺能神经元、组胺能神经元和神经胶质细胞中,这两种亚型都均可以使单胺类神经递质失活。而单胺氧化酶抑制剂则能够通过抑制单胺氧化酶的对单胺类物质的氧化活性,从而达到减轻或者消除由各种原因引起的单胺类物质减少或单胺氧化酶活性过高导致的疾病。本文主要总结了近几年单胺氧化酶抑制剂在临床上用于治疗帕金森病、抑郁症和幽门螺旋杆菌方面的最新进展。     

单胺氧化酶抑制剂在临床方面的应用

单胺氧化酶(monoamine oxidase,MAO)是人体内天然存在的一种酶,催化单胺类物质氧化脱氨反应的酶。人体内含有两种单胺氧化酶:单胺氧化酶A和单胺氧化酶B。单胺氧化酶A主要分布在儿茶酚胺能神经元中;单胺氧化酶B主要分布在5-羟色胺能神经元、组胺能神经元和神经胶质细胞中,这两种亚型都均可以使单胺类神经递质失活。而单胺氧化酶抑制剂则能够通过抑制单胺氧化酶的对单胺类物质的氧化活性,从而达到减轻或者消除由各种原因引起的单胺类物质减少或单胺氧化酶活性过高导致的疾病。本文主要总结了近几年单胺氧化酶抑制剂在临床上用于治疗帕金森病、抑郁症和幽门螺旋杆菌方面的最新进展。     

HPLC法测定安宫牛黄栓中体外培育牛黄含量

目的:用HPLC法测定安宫牛黄栓中体外培育牛黄含量。方法:样品经二氯甲烷超声提取后,采用HPLC法测定安宫牛黄栓中胆红素的含量,使用C18色谱柱,乙腈—1%醋酸溶液(95:5)为流动相,检测波长450nm。结果:胆红素线性范围0.0638～1.276μg,平均回收率99.89%。结论:样品处理方法合理,方法学考察符合定量要求,可用于安宫牛黄栓中胆红素的含量测定。     

微生态制剂MMA对新生儿高间接胆红素血症的改善作用

本研究使用微生态制剂ＭＭＡ对２０例新生儿高间接胆红素血症患者进行了口服或十二指肠内灌注治疗。结果发现ＭＭＡ对新生儿高间接胆红素血症有显著改善作用：治疗组血清总胆红素值明显下降,十二指肠液直接胆红素增多,这些与对照组常规治疗法比较均有显著性差异（Ｐ＜００５）,说明微生态制制ＭＭＡ能阻止肠道直接胆红素转变为间接胆红素,减少胆红素的肠肝循环,从而减轻高间接胆红素血症     

草鱼胆绿素IXa的纯化与光学特性

制备胆绿素,有人根据Bonnet and McDonagh方法1,先把毗咤血红素和抗坏血酸偶联氧化为四种胆绿素异构体的二甲酷,再按O'Carra and Colleran方法2用薄层层析进一步纯化各种异构体.也有人根据Lemberg法用结晶胆红素氧化;再分离胆绿素.研究表明,胆绿素是鱼类、两栖类、爬行类和鸟类动物体中血红素之分解终产物及排泄物3,4.因此,可以利用这些动物的胆汁和血液等分离提纯胆绿素,再把胆绿素转变为胆红素.这是广开材料来源,制备更多名贵药用胆红素的途径之一.本文报告用硅胶柱分离纯化淡水草鱼胆汁中的胆绿素IXa及其光学特性.       

产胆红素氧化酶菌种筛选

选出一株产胆红素氧化酶的菌种露湿漆斑菌—Myrotheciumr oriclum,简称MTR89—403。此菌产酶最适培养基:40—80％的马铃薯浸出液100ml、葡萄糖1g,pH6.0。培养条件:500ml三角瓶装100ml培养基,在27℃、200rpm旋转摇床培养84h,得到的最高酶活为1300u/L,比活为12.3u/mg蛋白。     

Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 μg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50–55°C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.     

Bilirubin dehydrogenase,an enzyme in Aspergillus ochraceus IB-3 useful for diagnostic measurement of bilirubin

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 microM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.     

Bilirubin Dehydrogenase,an Enzyme in Aspergillus ochraceus IB-3 Useful for Diagnostic Measurement of Bilirubin

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 μM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.     

Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both M. verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of decolorization; more than 98% decolorization efficiency was achieved after 7 days at 26°C. Additionally, the crude bilirubin oxidase can efficiently decolorize indigo carmine at 30°C~50°C, pH 5.5~9.5 with dye concentrations of 50 mg l(-1)~200 mg l(-1). Bilirubin oxidase was purified and visualized as a single band on native polyacrylamide gel electrophoresis (PAGE). Several enzymatic properties of the purified enzyme were investigated. Moreover, the identity of the purified bilirubin oxidase (BOD) was confirmed by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). These results demonstrate that the purified bilirubin oxidase in M. verrucaria strain has potential application in dye effluent decolorization.     

胆红素氧化酶产酶菌株的分离及最佳产酶条件的研究

A bilirubin oxidase (EC 1.3.3.5) producing strain, Mv 2.1089, was isolated from several strains of Myrothecium verrucaria by dilution method. The optimum conditions of enzyme production were investigated and the results were as follows: the suitable medium was cultured at 25 degrees C on a rotating shaker glucose and peptone, at pH 6.0. The strain was cultured at 25 degrees C on a rotating shaker (150 r/min) for 96 h. Bilirubin oxidase with 0.5-1.5 u/ml was obtained in the culture medium.     

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.     

Characterization of alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in comparison with bilirubin oxidase

An enzyme showing alkaliphilic laccase activity was purified from the culture supernatant of Myrothecium verrucaria 24G-4. The enzyme was highly stable under alkaline conditions, showed an optimum reaction pH of 9.0 for 4-aminoantipyrine/phenol coupling, and decolorized synthetic dyes under alkaline conditions. It showed structural and catalytic similarities with bilirubin oxidase, but preferably oxidized phenolic compounds. The enzyme catalyzed veratryl alcohol oxidation at pH 9.0 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a mediator, suggesting that the laccase mediator system functioned well under alkaline conditions.     

Existence of aa 3-Type Ubiquinol Oxidase as a Terminal Oxidase in Sulfite Oxidation of Acidithiobacillus thiooxidans

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an α-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa ₃-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 °C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A₁ and myxothiazol, which are inhibitors of mitochondrial bc ₁ complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.