构建高表达酪氨酸酚裂解酶重组大肠杆菌合成左旋多巴

以PCR技术扩增得到弗氏柠檬酸细菌ATCC 8090中酪氨酸酚裂解酶的结构基因tpl,与表达载体pQE30连接后构建质粒pQTPL,并转化到E. coli M15中进行表达,在加入0.2mmol/L的IPTG、18℃表达14h的诱导条件下,酪氨酸酚裂解酶比活为208u/mg.50ml摇瓶中E.coli M15(pQTPL)合成左旋多巴产量达55g/L.     

溴酚衍生物的合成及其蛋白酪氨酸磷脂酶1B抑制活性研究

目的合成具有新型结构的系列溴酚衍生物,测试其体外蛋白酪氨酸磷脂酶1B(PTP1B)抑制活性并初步探讨其构效关系。方法以香草醛或3,4-二羟基苯甲醛为原料,经溴化、还原、醚化等反应制得目标化合物。借助重组人源PTP1B蛋白水解底物p NPP的方法,测定目标物对PTP1B的抑制活性。结果合成了18个溴酚衍生物,其结构经EI-MS、ESI-MS、~1H-NMR、~(13)C-NMR谱确证。结论目标化合物的PTP1B抑制活性与化合物中溴原子的数目、位置以及烷基链的长度有关;化合物6c体外PTP1B抑制活性最好(IC50=0.572μmol·L~(-1))。     

色氨酸酶的催化功能及其在L-色氨酸酶法合成中的应用

色氨酸酶是一种依赖磷酸吡哆醛的多功能酶,由相同的4个亚基组成,每个亚基含有一个磷酸吡哆醛结合位点,催化活性需要NH4+或K+存在。它不仅能催化L-色氨酸的分解,而且能催化一系列α,β-消去反应和β-取代反应。其催化机制主要包括外部醛亚胺的形成和醌型中间物的生成。在高浓度底物条件下,色氨酸酶能催化丙酮酸、吲哚和氨合成L-色氨酸。通过β-取代反应,色氨酸酶催化L-丝氨酸、L-半胱氨酸合成L-色氨酸,这为酶法工业化生产L-色氨酸提供了重要途径。此外,色氨酸酶在制备硫醇类香料化合物中也发挥重要作用。     

酪氨酸转氨酶法制备L-高苯丙氨酸

目的为抗高血压新药的共同中间体L-高苯丙氨酸的制备奠定基础。方法构建工程菌E.coli BL21(DE3)pET 30a-tyrB,乳糖诱导表达酪氨酸转氨酶;利用酪氨酸转氨酶对底物2-氧-4-苯基丁酸进行转化;利用HPLC、MS、1H-NMR进行产物检测与结构鉴定。结果构建了工程菌E.coliBL21(DE3)pET 30a-tyrB,乳糖诱导酪氨酸转氨酶表达,制备得到L-高苯丙氨酸。结论利用转氨酶催化法可以高效制备L-高苯丙氨酸。     

苯二醛缩氨基硫脲的合成及其酪氨酸酶抑制活性研究

目的 合成苯二醛单缩和二缩氨基硫脲类化合物,并初步研究其抑制酪氨酸酶的活性和作用机制。方法 以5种苯二醛和氨基硫脲为原料,通过缩合反应合成9个目标化合物;采用蘑菇酪氨酸酶多巴速率氧化法和酶抑制动力学实验,测定目标化合物对酪氨酸酶的抑制活性和作用机制;选择化合物3a和4a进行抑制机制和抑制动力学研究。结果 目标化合物的结构经¹H-NMR、¹³C-NMR及MS确证;所有化合物抑制酪氨酸酶的活性均优于对照药物曲酸;苯二醛二缩氨基硫脲3a~3d的活性明显强于相应的单缩氨基硫脲4a~4d;化合物3a和4a对酪氨酸酶的抑制作用均表现为混合型可逆抑制作用。结论 苯二醛二缩氨基硫脲类化合物具有优异的抑制酪氨酸酶的活性,值得进一步深入研究。     

E.coli L-天冬酰胺酶结构与功能及其纯化工艺

综述近年来有关大肠杆菌来源的L-天冬酰胺酶的结构与功能、纯化工艺以及酶的修饰的研究进展。L-天冬酰胺酶具有抗肿瘤活性,目前临床上已将其用于儿童急性淋巴细胞白血病的治疗,是治疗此类白血病的重要药物。     

固定化酰化酶在青霉素裂解反应中的影响因素分析

在酶促脱酰法裂解青霉素工业钾盐生产药用中间体6-氨基青霉烷酸(即6-APA)的反应杌理中,在不同反应物浓度、pH及温度下将直接影响酰化酶的活性及其使用寿命。本文通过多组实验进行比较分析摸索出酰化酶的最适宜的工艺条件以达到延长酰化酶的使用寿命,降低6-APA的生产成本的目的。     

几种因素在L-天冬酰胺酶化学修饰中对酶活力及抗原性的影响

为了寻求在较好地保持酶活力的同时解除L-天冬酰胺酶抗原性的方法,采用不同分子量的乙酸酐、右旋糖酐和单甲氧基聚乙二醇,作为修饰剂和不同的修饰方法对该酶进行了化学修饰。结果表明在保持酶活性和降低抗原性方面,大分子修饰剂右旋糖酐、单甲氧基聚乙二醇优于小分子乙酸酐,底物保护修饰优于直接修饰;活化PEG,优于活化PEG₁。在底物保护下的PEG,修饰酶其抗原性完全解除的同时,酶活力保持在30%以上。     

异柠檬酸裂解酶的制备及其抑制剂筛选模型的建立

目的 制备结核分枝杆菌异柠檬酸裂解酶(isocitrate lyase,ICL)并建立ICL抑制剂筛选模型.方法 通过四步层析法或金属螯合层析法纯化大肠埃希菌BL21(DE3)表达的ICL,并以异柠檬酸为底物,用纯化的ICL裂解舁柠檬酸,生成乙醛酸可与苯肼反应生成苯腙,苯腙在324nm波长下产生光吸收峰,测定酶促反应体系在324nm波长下光吸收检测异柠檬酸裂解酶的活性,根据待测样品对酶活性的抑制程度,筛选异柠檬酸裂解酶抑制剂.结果 用金属螯和层析法得到纯度为90%、比活力为2.8U/mg的ICL,建立并优化ICL抑制剂筛选模型,模型的信噪比(S/N)远大于3,变异系数(CV)远小于10%.结论 通过金属螯合层析法获得了特异性高、稳定性好的ICL抑制剂筛选模型,该模型可有效的应用于异柠檬酸裂解酶抑制剂的高通量筛选.     

丹皮酚衍生物的合成及其抗肿瘤细胞增殖研究

以间苯二酚为原料, 经过酰化、醚化反应合成了15个丹皮酚及其衍生物, 以HeLa、MCF-7细胞为靶细胞, 采用MTT法进行了初步的体外抗肿瘤活性研究。结果表明, 4-位甲氧基是丹皮酚抗肿瘤活性的增效官能 团, 酮羰基侧链为丹皮酚抗肿瘤活性的必需官能团, 与羰基相连侧链的碳原子数小于4时, 随着侧链的延长其抗肿瘤细胞增殖活性会不断加强, 优选出的化合物2d对HeLa和MCF-7细胞株具有较显著的抗增殖活性, IC₅₀值分别为2.67和4.74 μmol·L⁻¹, 这为丹皮酚抗肿瘤活性构效关系的研究打下了基础, 值得进一步研究。     

Antithyroid drug carbimazole and its analogues: synthesis and inhibition of peroxidase-catalyzed iodination of L-tyrosine

Synthesis and biological activity of the antithyroid drug carbimazole (CBZ) and its analogues are described. The introduction of an ethoxycarbonyl group in methimazole and its selenium analogue not only prevents the oxidation to the corresponding disulfide and diselenide but also reduces the zwitterionic character. A structure-activity correlation in a series of CBZ analogues suggests that the presence of a methyl substituent in CBZ and related compounds is important for their antithyroid activity.     

Review of L-tyrosine confirming its safe human use as an adjuvant

Although there is a long history of exposure to allergy vaccines containing L-tyrosine, there has been no central publication reviewing its adjuvant properties in animal and human studies together with an assessment of its safe use. This paper summarizes a range of investigational data (unpublished) available to the authors as well as published literature reports. An array of in vitro and in vivo studies showed that L-tyrosine has ideal adjuvant properties, comprising a high adsorptive power for proteins, enhancement of IgG antibody induction with no stimulatory effect on IgE antibody level and action as a short-term depot adjuvant, delaying the bioavailability of allergenic materials rather than directly influencing immunocompetent cells. A series of preclinical safety investigations comprised single-dose parenteral studies in the mouse and rat, repeat-dose parenteral toxicity studies over 28 days in the rat and dog (up to 25 mg kg(-1) day(-1)) plus genotoxicity and local tolerance studies. No signs of toxicity or genotoxicity were seen; repeat-dose toxicity studies showed expected white cell and spleen weight immunostimulatory effects; local-dose site reactions were also seen and were confirmed in local tolerance studies. Findings from a range of clinical studies using allergy vaccines containing L-tyrosine reflected the lack of toxicity seen in animal work and showed evidence of enhanced immunostimulatory activity. Local injection site reactions (a common response to any form of clinical vaccination) in these studies were likely to be due to the presence of L-tyrosine per se. The lack of findings of toxicological concern found during this review supports the hypothesis that L-tyrosine is a safe adjuvant for human use.     

Chemical and enzymatic synthesis of tritium labelled coenzymes

Details of the synthesis of tritium labelled co enzymes‐nicotinamide adenine dinucleotide and coenzyme A by isotope exchange and enzymatic reactions are reported. It was established, that among the investigated chemical reactions, the most effective is solid state isotope exchange with gaseous tritium. This method was used to produce [³H] nicotinamide adenine dinucleotide (111 Ci/mmol), [³H] coenzyme A (3.9 Ci/mmol) and D ‐[G‐³H] pantothenic acid (43 Ci/mmol). It was shown that most of the tritium in the labeled nicotinamide adenine dinucleotide and coenzyme A was localized in the nicotinamide (98%) and adenine (89%) sites, respectively. For synthesis of coenzymes labelled with tritium at other sites we developed enzymatic methods which used labelled precursors. Optimum conditions for enzymatic synthesis of [adenine‐³H] nicotinamide adenine dinucleotide from [2,8‐³H] ATP and [pantothenate‐³H] coenzyme A from D ‐[G‐³H] pantothenic acid were determined. The tritium labelled acetyl coenzyme A was synthesized by acetylation of labelled coenzyme A with acetic anhydride. The methods chosen allow one to produce tritium labelled coenzymes at high specific activity. Copyright © 2003 John Wiley & Sons, Ltd.     

β-内酰胺抗生素的酶法合成研究进展

β-内酰胺抗生素的酶法合成是21世纪β-内酰胺抗生素发展的必然趋势之一,本文对近年来β-内酰胺抗生素合成研究、产品的分离纯化、酶反应器研究进行概述。     

HPLC determination of 4-hydroxy-anethole trithione in plasma via enzymatic hydrolysis and its application to bioequivalence study

A simple, selective and reproducible high-performance liquid chromatographic (HPLC) method via enzymatic hydrolysis of glucuronide conjugates of 4-hydroxy-anethole trithione (ATX) was established for simultaneous determination of ATX. Human plasma samples were hydrolyzed by beta-glucuronidase and followed by subsequent extraction with cyclohexane-isopropanol (95:5, v/v) using mifepristone as the internal standard. Chromatography was carried out on a reverse phase C(18) column (250 mm x 4.6 mm, 5 microm) and kept at 30 degrees C, with UV detection set at 346 nm. The mobile phase consisted of a mixture of methanol and water (75:25, v/v), at a flow rate of 1 ml/min. It was validated and proved to be linear in the range of 20-1500 ng/ml, with the regression equation Y = 0.0016C-0.0069, r=0.9992. And the limit of quantification (LOQ) concentration in plasma was 20 ng/ml. The absolute recoveries of ATX at three concentrations were 32.04, 38.95 and 44.06% and the relative recoveries were 104.80, 102.53 and 107.04%, which showed that the analytical method was sensible, accurate and reproducible. The method was utilized on a double-blind, randomized, single dose, two period, and crossover bioequivalence study of ATT tablets produced by different companies in 20 healthy male Chinese subjects, with a washout between every two periods. Blood samples were collected over each period of 10h and various pharmacokinetic parameters were determined. Natural log-transformed values were compared by analysis of variance followed by classical 90% confidence interval for C(max), AUC(0-t) and AUC(0-infinity) and was found to be within the range, which indicated that the two products were bioequivalence.     

头孢氨苄酶法缩合工艺的研究进展

综述了青霉素乙酰化固定化酶和头孢氨苄酶法缩合工艺的研究进展;探讨了头孢氨苄酶法缩合工艺中酶催化缩合反应的影响因素、产品的分离与纯化以及过量原料的回收和酶反应器的选择等问题.结果认为头孢氨苄酶缩合反应的适宜条件为侧链与7-氨基脱乙酰氧基头孢烷酸(7-ADCA)的投料比率为11～21,反应底物的浓度为300～600mmol·L-,反应温度在5～35℃之间,7-ADCA的转化率可以达到93%以上.     

EGFR酪氨酸激酶及其抑制剂的研究进展

表皮生长因子受体(EGFR)酪氨酸激酶是细胞外信号传递到细胞内的重要枢纽,它在信号传导、细胞增殖、分化以及各种调节机制中发挥重要作用,并在多种癌细胞中过度表达。许多研究表明,抑制EGFR酪氨酸激酶活性,可抑制肿瘤生长。本文对EGFR酪氨酸激酶及其几种小分子抑制剂在肿瘤治疗中的研究进展作一综述。     

酶法合成阿莫西林过程中蛋白残留检测

摘要：目的 探究阿莫西林生产合成过程中蛋白残留量,确定最适生产条件。方法 通过单一变量法,研究不同条件酶法合成阿莫西林过程中蛋白残留量。结果 随着母核侧链摩尔比降低,合成阿莫西林过程中蛋白残留量却有所提高,当比例达到1:1.15后,蛋白含量趋于稳定。生产温度越低、反应时间越长,蛋白质残留量越高,当温度达到15℃时蛋白含量最低,随着温度升高蛋白含量逐渐升高。磁力搅拌反应过程中蛋白残留量(2189.01mg/L)远远大于机械搅拌(177.50mg/L)。反应批次对蛋白残留量影响较小,其残留量稳定在180~190mg/L左右。结论 生产过程中采用15℃,1:1的母核侧链摩尔比,机械搅拌的方式合成的阿莫西林蛋白残留量最少。     

新型手性配体的合成及其在沙美特罗关键中间体合成中的应用

目的合成沙美特罗的关键手性中间体。方法设计合成新型手性配体单磺酰化1，2-双-（3，5-二甲基苯基）乙二胺，通过与金属铑配合生成手性催化剂，利用该催化剂催化沙美特罗的关键手性中间体的不对称氢转移反应。结果与结论新型手性配体具有良好的催化活性和对映体选择性，催化合成得到的沙美特罗中间体的对映体选择性的ee％值为91％。