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…     

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。     

重组大肠杆菌合成左旋多巴条件的优化

通过对合成体系各组分对产物形成的影响的研究。分别求得了底物邻苯二酚,丙酮酸及乙酸氨的表观米氏常数,表观底物抑制常数和最适底物浓度,并得出合成左旋多巴的最适反应体系为：1%丙酮酸,1.2%邻苯二酚,2%乙酸铵,0.1?TA,0.2%亚硫酸钠,用氨水调pH至8.0。加入从与合成体系等体积培养液中收获的重组大肠杆菌细胞,于15℃反应16h,L-DOPA的产量为15.36g/L。     

猫豆荚壳中左旋多巴的提取及含量测定

猫豆荚壳中含左旋多巴,测定含量为0.96％,提取收率为0.547％。     

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Citrobacter freundii 48003-3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12h,L-DOPA的量可达到9．5g／L。     

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。     

酪氨酸酚裂解酶的固定化与转化条件的优化

以欧文氏菌(Erwinia herbicola)来源的酪氨酸酚裂解酶的重组大肠埃希菌Escherichia coli BL21为研究对象,研究固定化大肠埃希菌生产L-酪氨酸的条件。以海藻酸钠为载体,采用单因素实验分别考察了载体材料、明胶浓度、反应时间、苯酚浓度和辅助剂(二氧化硅、硅藻土和碳酸钙)等因素对L-酪氨酸生产的影响,发现明胶浓度、反应时间、苯酚和碳酸钙等因素的影响较为显著,进而通过正交实验探索最优条件。结果表明,生产L-酪氨酸的最优条件:载体为4%海藻酸钠与6%明胶的混合载体,苯酚浓度0.08 mol/L,反应时间8 h,于载体中添加0.6%碳酸钙。此条件下,连续反应9次后L-酪氨酸的产量达到64.5 g/L,比优化前提高了451.3%。     

酶法脱蛋白提取大枣多糖工艺的研究

讨论了从提取环磷酸腺苷后的枣汁中提取枣多糖的最佳工艺条件,包括枣汁浓缩4倍,加无水乙醇调枣汁中乙醇体积分数为60%,醇沉5 h;木瓜蛋白酶脱蛋白效果最佳,木瓜蛋白酶液与枣汁的体积比为0.4∶ 1,温度60℃、pH5.0,酶解90 min,蛋白脱除率可达91.8%;AB-8树脂脱色,脱色率为73.64%,多糖得率为94.4%.红外光谱显示,提取的多糖与常规方法提取的多糖成分相同.     

多酶复配合成海藻糖及其分离提取的研究

以大米淀粉为原料,多酶复配制备海藻糖。确定了实验室条件下多酶复配生产海藻糖的最佳条件:以15%(m/V)大米淀粉为底物,催化温度45℃、pH 6. 0、DE值16、α/β-CGTase加量为1. 4U/ml、催化28h后糖化处理12h,海藻糖转化率由双酶法催化的50%提高至73%。在底物浓度为25%(m/V)时,海藻糖产量最高达到182. 5g/L,随后对高浓度海藻糖进行分离提取,分别考察了活性炭脱色、离交分离、浓缩结晶等对海藻糖提取效率的响。     

Purification and characterization of L-DOPA decarboxylase from human kidney

L-DOPA decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-DOPA decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-DOPA decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-DOPA is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-DOPA decarboxylase activity has also been tested.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Development of a multienzyme reactor for dopamine synthesis: II. Reactor engineering and simulation

Aspects of reaction engineering associated with multienzyme reactions have been studied in a system where dopamine is produced from catechol, pyruvate and ammonium by sequential enzymatic reactions catalyzed by tyrosine phenol lyase (TPL) and tyrosine decarboxylase (TDC). Microbial cells containing TPL activity (Erwinia herbicola) and TDC activity (Streptococcus faecalis) were coimmobilized in glutaraldehyde cross-linked porcine gelatin beads with a mean diameter of 2.8 mm for use in the reactions. Measurement of the transport properties in the beads indicate that the gelatin matrix does not significantly increase the diffusion resistance and that dopamine partitions into the matrix (K = 2). A packed-bed reactor containing the coimmobilized cell beads successfully produced dopamine, although with a low conversion. Using computer simultaneous it is shown that separate, sequential TPL and TDC, rather than simultaneous, reactions, would require smaller reactors overall for the same conversion. (c) 1992 John Wiley & Sons, Inc.     

Development of a multienzyme reactor for dopamine synthesis: I. enzymology and kinetics

The enzymology and kinetics of tyrosine phenol lyase (TPL) from Erwinia herbicola, and tyrosine decarboxylase (TDC) from Streptococcus faecalis have been investigated for potential use in a coimmobilized multienzyme biocatalytic system for the production of dopamine. In this multienzyme biotransformation using whole cells optimized for each of the respective enzymes, TPL catalyzes the production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) from catechol, pyruvate, and ammonium, and this is subsequently decarboxylated by TDC to produce dopamine. Performing the reactions simultaneously, thereby removing L-dopa, is one option for overcoming the TPL equilibrium constraints. The enzymes have different optimal pH values, so the reaction kinetics at a compromise pH of 7.1, where both enzymes could be operated simultaneously, were investigated. For the concentration range investigated, TPL followed pseudo-first-order kinetics with respect to catechol, pyruvate, and ammonium. TDC exhibited significant product inhibition as well as inhibition by combinations of catechol and pyruvate.     

弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从弗氏柠檬酸细菌（Citrobacter freundii)中扩增得到含有酪氨酸酚解酶基因的DNA片段,定向连续到质粒pUC118上,得到重组质粒pTPL,将此重组质粒转化到受体菌E.colXL-1-Blue MRF′中,通过蓝白斑鉴定挑出阳性菌株。从此阳性菌株中提取质粒pTPL并将此质粒转入到E.coliJM109中,用E.coliJM109（pTPL)制备高活性的酪氨酸酚解酶。对质粒稳定性的研究表明,E.coliJM109（pTPL)在无选择压力下37℃连续培养50代以上,质粒丢失率仅有15％,说明质粒基本稳定。     

L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions

There is evidence that L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor-mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as regulate the melanocyte functions through the activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate-induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate an older theory proposing that receptors for amino acid-derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates.     

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.     

Pleiotrophin receptor RPTP-zeta/beta expression is up-regulated by L-DOPA in striatal medium spiny neurons of parkinsonian rats

L-DOPA is still the drug of choice to treat Parkinson's disease although adverse side effects appear after several years of treatment. These are thought to be the consequence of plastic re-arrangements of the nigrostriatal connections, such as sprouting of the dopaminergic terminals or post-synaptic changes. Pleiotrophin, a trophic factor that we have shown to be up-regulated in the striatum of parkinsonian rats after long-term L-DOPA treatment may play a role in these plastic changes. To determine whether one of the three known pleiotrophin receptors [N-syndecan, receptor protein tyrosine phosphatase type zeta beta (RPTP-zeta/beta) and anaplastic lymphoma kinase] might be implicated in these putative plastic effects, we quantified their expression levels by real-time RT-PCR in the striatum and mesencephalon of rats with partial lesions of the nigrostriatal pathway undergoing L-DOPA treatment. Both pleiotrophin and RPTP-zeta/beta expression was up-regulated in the striatum but not in the mesencephalon of lesioned rats and RPTP-zeta/beta expression was even further increased by L-DOPA. The levels of the RPTP-zeta/beta protein were also increased in the striatum of L-DOPA-treated lesioned rats. Immunofluorescence labeling showed the protein to be constitutively expressed in striatal medium spiny neurons, which are innervated by both the corticostriatal glutamatergic and nigrostriatal dopaminergic systems. RPTP-zeta/beta might therefore be implicated in the plastic changes triggered by L-DOPA treatment and might merit further study as a potential candidate for Parkinon's disease therapy.