左旋多巴的酶法合成

左旋多巴（3,4－dihydrox－ylphenylalanine、L－DOPA）不但是治疗常见老年病——帕金森病的主要药物,它还有其它许多医疗用途。随着我国人们医疗保健水平的提高和人口老龄化程度的加剧,左旋多巴的需求量也将增加。酶法合成特别是微生物酶法合成左旋多巴以其良好的经济性受到了广泛重视。主旋多巴属于手性药物,其对映作右旋多巴可引起毒性反应[1],故医学上需控制它的旋光度,这就会给化学合成法生产左旋多巴带来麻烦。1990年,西北工业大学的孙晓莉[2]用可避免DL拆旋操作的不对称合成法合成主旋多巴,但工艺非常复杂。左旋多巴也可…     

嗜麦芽假单胞茵生产L-DOPA发酵条件的研究

<正> 前言左旋多巴(L-dopa)是一种在医药卫生,保健美容等诸多方面有着显著功效的氨基酸,它的生产很早就引起了人们的重视,人们通过化学合成和生物合成两条途径来获得 L-DOPA,二者相比,后者更加简单、方便。国内有人从植物中提取L-DOPA,但由于受到原料来源限制,产量小。与从植物中提取多巴相比,利用微生物来发酵生产 L-DOPA 有着生产周期短、易培养、占地面积小等诸多优点,利用微生物生产多巴是一种经济且有前途的方法。     

Citrobacter freundii休止细胞催化合成L-多巴

以在L 酪氨酸诱导下高效表达酪氨酸酚解酶的菌株Citrobacterfreundii 4 80 0 3 3的休止细胞为生物催化剂 ,以邻苯二酚、丙酮酸钠、醋酸铵为前体 ,选择性合成L DOPA。研究了反应温度、pH和前体浓度等对合成L DOPA的影响。最优反应条件下 ,反应 1 2h ,L DOPA的量可达到 9 5g/L。     

厚壳贻贝Mytilus coruscus足丝盘及足丝的多巴分析

多巴(3,4-1-dihydroxyphenylalanine,DOPA)是贻贝足丝粘附蛋白中的一种特殊的氨基酸,由酪氨酸经羟化后生成,与贻贝足丝粘附蛋白的强粘附性能具有直接联系.目前,已鉴定的多种贻贝足丝蛋白序列中均发现有不同含量的DOPA存在.蛋白中DOPA的定量检测对于了解DOPA在蛋白粘附中的作用以及粘附蛋白的...     

异源表达多巴脱羧酶促进大肠杆菌从头合成多巴胺

多巴胺是多种天然抗氧化药物生物合成的前体物质,在人体内作为神经递质调控中枢神经系统的多种生理功能,常用于多种类型休克的临床治疗。目前,通过微生物合成技术已经实现了多巴胺的从头合成,但是合成效率很低。针对该问题,在左旋多巴 (l-DOPA) 大肠杆菌工程菌基础上,利用不同拷贝数质粒表达野猪Sus scrofa来源的多巴脱羧酶基因Ssddc,实现了葡萄糖到多巴胺的生产。为了进一步提高多巴胺合成效率,从100个候选基因中筛选出5个多巴脱羧酶基因进行测试,其中来源于人Homo sapiens多巴脱羧酶基因Hsddc的工程菌摇瓶发酵的多巴胺产量最高,达到3.33 g/L;而来源于果蝇Drosophila melanogaster多巴脱羧酶基因Dmddc的工程菌摇瓶发酵的左旋多巴残余量最低,仅有0.02 g/L;这两株工程菌分批补料发酵表明,多巴胺的产量可以分别达到13.3 g/L和16.2 g/L,左旋多巴残余量分别是0.45 g/L和0.23 g/L。将多巴脱羧酶基因Dmddc和Ssddc分别整合到基因组上,获得遗传稳定的工程菌,在分批补料发酵条件下,多巴胺产量最高达到17.7 g/L,是目前国内外报道的最高产量。     

人源多巴脱羧酶的表达、纯化以及高通量抑制剂筛选模型的建立

帕金森病是一种常见的神经系统退行性疾病。多巴脱羧酶（DDC）是帕金森病研究的靶点蛋白之一,但是目前没有高通量的测活模型。因此,需要构建一种高通量多巴脱羧酶抑制剂的筛选模型,用于发现新型抑制剂。采用克隆表达纯化得到多巴脱羧酶和用于酶偶联反应的磷酸烯醇式丙酮酸羧化酶（PEPC）。基于一系列酶联反应将CO2固定,检测其含量,从而测定多巴脱羧酶的活性。结果得到人源多巴脱羧酶和磷酸烯醇式丙酮酸羧化酶的体外纯酶,建立了一种高通量筛选模型,并且从70个天然化合物中,筛选得到2个多巴脱羧酶的抑制剂。成功构建了一种基于体外纯酶高通量多巴脱羧酶抑制剂的筛选模型。     

海南龙血树DNA提取与ISSR反应体系的优化

目的:从海南龙血树叶片巾提取出高质量的总DNA,建立与优化海南龙血树ISSR的反应体系.方法:采用4种DNA提取方法,提取海南龙血树叶片中的总DNA,并对DNA进行紫外和电泳检测.采用改良CTAB法提取了基因组DNA模板,对海南龙血树ISSR-PCR反应体系中各个主要影响因子进行了优化和筛选.结果:改良CTAB法提取的DNA A260/A260在1.7～1.9之间,纯度高、杂质少、DNA完整性好.根据PCR产物的琼脂精凝胶检测结果,由试验得到的最佳反应体系为:60ng模板DNA,1.5mmol/L Mg2+,0.25mmol/L dNTPs,1.0μmol/L引物,1U Taq酶,总体积为20μl.结论:改良CTAB法可以从海南龙血树叶片中提取高质最DNA,该反应体系适用于应用ISSR标记开展海南龙血树DNA指纹、遗传多样性等研究.     

棉花高质量DNA的提取及SRAP反应体系的优化

建立一种简便、快速、经济、有效的gDNA提取方法,并以得到高质量的gDNA为模板摸索棉花SRAP反应体系的最优条件。在提取棉花DNA之前对样品进行预除酚处理,采用CTAB法并加以改进,摸索了一种简便、快速、经济、有效的gDNA提取方法。结果表明预处理除酚法提取天然彩色棉的gDNA为白色,OD260 nm/OD280 nm平均值达到1.900,OD260 nm/OD230 nm平均值1.659,琼脂糖电泳检测表明所提取的DNA较完整,RNA含量少;通过对重要参数进行摸索和优化试验,建立一套稳定可靠、扩增效果好、可重复性强的适用于棉花的SRAP反应体系：25μL的反应体系中,模板DNA量30ng、2.5mmolm/L Mg2＋浓度、0.8μmol/L的上下游引物、200μmol/L的dNTPs以及Taq酶1U。     

左旋多巴/ 卡比多巴联合恩他卡朋治疗帕金森病的临床研究

目的:观察左旋多巴/卡比多巴联合恩他卡朋(levodopa/carbidopa combined with entacapone,LC+E)治疗帕金森病(Parkinson's disease,PD)的临床效果。方法:选择我院2013年1月~2014年6月收治的112例PD患者,随机分为两组。其中对照组52例采用左旋多巴/卡比多巴(LC)治疗,观察组60例采用左旋多巴/卡比多巴联合恩他卡朋(LC+E)治疗。观察并比较两组治疗前后帕金森病评分量表(Unified Parkinson's Disease Rating Scale,UPDRS)的评分变化情况。结果:与治疗前比较,治疗后两组UPDRS-II日常生活能力评分,UPDRS-III运动能力评分显著下降,而UPDRS-VI SCHWABENGLAND日常活动能力评分显著上升,差异有统计学意义(P0.05);观察组各项变化情况比对照组明显,差异有统计学意义(P0.05)。两组UPDRS-I精神、行为、情绪和Hoehn与Yahr分级均无显著改善,差异无统计学意义(P0.05)。结论:左旋多巴/卡比多巴联合恩他卡朋可明显缓解PD症状,疗效优于左旋多巴/卡比多巴治疗,且安全性高,值得临床推广。     

菠萝SRAP反应体系的建立及优化

目的:建立一种适合菠萝基因扩增的 SRAP 反应体系.方法:用改良 CTAB 法提取菠萝 DNA,对扩增结果影响重要的反应组分 Taq 酶、Mg2 、随机引物及 dNTPs 进行单因素体系优化,以确定最佳菠萝 SRA P反应体系.结果:用这种方法建立的菠萝SRAP 反应体系为:20μL 反应体系中含1×PCR buffer,2.5mmol/L Mg2 、1.2U TaqDNA 聚合酶、0.2mmol/L dNTPs、0.3umol/L随机引物、20ng DNA 模板.结论:用引物Me4-Em4 组合对供试菠萝 19 个品种进行扩增,结果扩增条带清晰、丰富、重复性好,此 SRAP反应体系适合菠萝基因型扩增.     

Purification and characterization of 3,4-dihydroxyphenylalanine oxidative deaminase from Rhodobacter sphaeroides OU5

An enzyme involved in the catabolism of 3,4-dihydroxyphenylalanine (DOPA) was isolated from Rhodobacter sphaeroides OU5. The enzyme catalyzes the formation of 3,4-dihydroxyphenylpyruvic acid (DOPP) and ammonia from DOPA. Formation of ammonia by DOPA oxidative deaminase was O2 dependent and the enzyme isolated to its homogeneity has 100% affinity for DOPA. DOPA oxidative deaminase is functional at low concentrations of the substrate (< 100 micromol.L(-1)) and is independent of NADH. The molecular mass of the purified enzyme is approximately 190 kDa and the enzyme could be a pentamer of 54, 42, 34, 25, and 23 kDa subunits as determined by SDS-PAGE.     

Mycobacterium leprae and phenoloxidase activity.

Our earlier studies indicated that the enzyme o-diphenoloxidase was absent in Mycobacterium leprae separated from depromatous human tissues. At that time the bacilli were not available from any other source. The existence or absence of this enzyme in M. leprae recovered from infected armadillo tissues were reinvestigated. The intact cells which were metabolically active, failed to oxidize DOPA. Likewise, DOPA and its derivatives were not oxidized by the enzymatically active cell-free preparations from M. leprae. Upon incubation of DOPA for more than 2 h with whole cell suspensions or particulate fractions, there was no development of colour with an absorption maximum of 540 nm as has been reported for an intermediate of DOPA oxidation. However, DOPA and several phenolic compounds were very actively oxidized by mushroom tyrosinase. The results suggested that M. leprae is deficient in o-diphenoloxidase, and this enzyme is not an intrinsic characteristic of this mycobacterium.     

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave‐dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L‐tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L‐DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L‐DOPA and dopamine to DOPA‐melanin and dopamine‐melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave‐adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave‐adapted insects and teleosts.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Purification and properties of aromatic L-amino acid decarboxylase (4.1.1.28) of rat brain]

L-aromatic aminoacid decarboxylase has been purified more than thousand times from homogenates of rat brain, in several steps : centrifugation, DEAE-cellulose, CM cellulose, hydroxylapatite, DEAE sephadex. Its properties have been studied, most of them on an intermediate fraction of the purification, because of the instability of the purified enzyme in spite of the addition of different stabilizing agents : the enzyme decarboxylates 5-hydroxytryptophan (5 HTP) and DOPA in a ratio constant throughout the purification but does not decarboxylate tryptophan, tyrosine, histidine at a measurable rate. Optimum pH, Km, Vm, have been measured with 5 HTP and DOPA as substrates. The enzyme has a molecular weight of 115.000, an apparent isoelectric point of 6,4-6,5. It is inhibited by serotonin, dopamine, some cations : Cu++, Fe++, Ni++ by N-ethylmaleimide, sodium dodecylsulfate. Some pyridoxal-5 phosphate (PLP) remains strongly bound to the enzyme. For relatively weak concentrations of substrate, the enzyme is inhibited by an excess of PLP ; for weak concentrations of PLP, the enzyme in inhibited by an excess of substrate, particularly of DOPA. We also observe a spontaneous decarboxylation of the substrates that reaches a plateau and is enhanced by high concentrations of PLP, by serotonin, dopamine, Cu++ and reduced by mercaptoethanol and the presence of crude or boiled homogenates. Several possible explanations of the spontaneous decarboxylation and of the enzymic inhibitions by an excess of PLP and by the substrates are given.     

The Intracellular Location of Catechol Oxidase: in the Olive Fruit

Catechol oxidase in young olive fruits was located on the innerface of chloroplast thylakoids and in the mitochondria. Thiswas demonstrated by observing the oxidation products of 3,4-dihydroxyphenyl-alanine(DOPA) with the electron microscope in fruit segments prefixedwith glutaraldehyde. Diethyl dithiocarbamate (DIECA), an inhibitorof the enzyme, prevented the accumulation of oxidation productsof DOPA. Olea europaea L, olive fruits, catechol oxidase, ultrastructure     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

Effect of electrostatic interactions on phase stability of cubic phases of membranes of monoolein/dioleoylphosphatidic acid mixtures.

To elucidate effects of electrostatic interactions resulting from surface charges on structures and phase stability of cubic phases of lipid membranes, membranes of 1-monoolein (MO) and dioleoylphosphatidic acid (DOPA) (DOPA/MO membrane) mixtures have been investigated by small-angle x-ray scattering method. As increasing DOPA concentration in the DOPA/MO membrane at 30 wt% lipid concentration, a phase transition from Q(224) to Q(229) phase occurred at 0.6 mol% DOPA, and at and above 25 mol% DOPA, DOPA/MO membranes were in the L(alpha) phase. As NaCl concentration in the bulk phase increased, for 10% DOPA/90% MO membrane in excess water, a Q(229) to Q(224) phase transition occurred at 60 mM NaCl, and then a Q(224) to H(II) phase transition occurred at 1.2 M NaCl. Similarly, for 30% DOPA/70% MO membrane in excess water, at low NaCl concentrations it was in the L(alpha) phase, but at and above 0.50 M NaCl it was in the Q(224) phase, and then at 0.65 M NaCl a Q(224) to H(II) phase transition occurred. These results indicate that the electrostatic interactions in the membrane interface make the Q(229) phase more stable than the Q(224) phase, and that, at larger electrostatic interactions, the L(alpha) phase is more stable than the cubic phases (Q(224) and Q(229)). We have found that the addition of tetradecane to the MO membrane induced a Q(224)-to-H(II) phase transition and also that to the 30% DOPA/70% MO membrane induced an L(alpha)-to-H(II) phase transition. By using these membranes, the effect of the electrostatic interactions resulting from the membrane surface charge (DOPA) on the spontaneous curvature of the monolayer membrane has been investigated. The increase in DOPA concentration in the DOPA/MO membrane reduced the absolute value of spontaneous curvature of the membrane. In the 30% DOPA/70% MO membrane, the absolute value of spontaneous curvature of the membrane increased with an increase in NaCl concentration. On the basis of these new results, the phase stability of DOPA/MO membranes can be reasonably explained by the spontaneous curvature of the monolayer membrane and a curvature elastic energy of the membrane.     

The great DOPA mystery: the source and significance of DOPA in phase I melanogenesis.

One of the important characteristics of tyrosinase is the autocatalytic nature of the oxidation of natural monohydric phenol substrates, such as tyrosine. In vitro tyrosinase exhibits a lag phase in which the maximum velocity of oxidation is attained after a period of induction. This acceleration contrasts with the kinetics of dihydric phenol oxidation which exhibit conventional Michaelis-Menten kinetics. It has been known for half a century that DOPA is a co-factor in the oxidation of tyrosine and addition of a small amount of catechol reduces the length of the lag period. The significance of DOPA is in this action, and DOPA is known to be formed in phase I melanogenesis. Until recently there has been controversy regarding the source of the DOPA in the in vitro reaction system. Most investigators have favoured a mechanism based on the generation of DOPA by a direct hydroxylation of tyrosine. However, recent evidence has suggested that DOPA is indirectly derived by reduction of dopaquinone. In this communication the evidence for the indirect mechanism derived from the use of analogue substrates is reviewed.