固定化啤酒酵母细胞催化有机硅酮不对称还原

研究了固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应,系统探讨了振荡速度、底物浓度、固定化细胞浓度、pH值和反应温度对反应速度、产率和产物光学纯度的影响。结果表明,上述因素对固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应均有较显著的影响。振荡速度以150r/min为宜,底物浓度和固定化细胞浓度分别为14mmol/L和0.15g/mL较佳,适宜的pH值为7.3,最佳反应温度为25℃～30℃。在该优化反应条件下,反应最大产率和产物的光学纯度分别高达84.9%和90.2%ee。     

水/有机溶剂双相中固定化啤酒酵母细胞催化有机硅酮不对称还原

研究了水/有机溶剂双相中,固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原成（－）-1-三甲基硅乙醇的反应,系统探讨了振荡速度、有机溶剂疏水性、水相与有机溶剂相体积比、水相pH值和反应温度对反应速度、产率和产物光学纯度的影响.结果表明,上述因素对固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应均有较显著的影响.正己烷为该反应最好的有机溶剂,振荡速度以150 r/min为宜,水相与有机溶剂相体积比以1∶2较佳,适宜的pH值为8,最佳反应温度为25～30℃.在该优化反应条件下,反应最大产率和产物的光学纯度分别高达96.8％和95.7％(ee).     

离子液体对固定化Saccharomyces cerevisiae细胞催化乙酰基三甲基硅烷不对称还原反应的影响

对比研究了C4MIm.BF4-缓冲液混合体系和缓冲液单相体系中固定化面包酵母Saccharomyces cerevisiae细胞催化乙酰基三甲基硅烷不对称还原反应的特性,系统探讨了离子液体C4MIm.BF4对该反应的初速度、最大转化率和产物对映体纯度的影响规律。在各自最优的反应条件下,固定化面包酵母细胞在缓冲液单相体系中催化乙酰基三甲基硅烷不对称还原反应的初速度、最大转化率及产物e.e.值分别为84.8 mmol/(L.h)、99.2%和≥99.9%;而在C4MIm.BF4-缓冲液混合体系中,该反应的初速度、最大转化率及产物e.e.值分别为87.0 mmol/(L.h)、99.0%和≥99.9%。离子液体的存在,提高了固定化面包酵母细胞催化该反应的速度,但降低了固定化酵母细胞的操作稳定性。     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

还原酶催化羰基不对称还原的应用进展

羰基不对称还原作为合成手性醇的重要方法,已成为近年来有机合成的研究热点。与传统化学法相比,利用还原酶催化前手性羰基化合物的不对称还原具有显著优势。介绍了还原酶的来源与形式,对完整细胞还原酶与游离还原酶在手性药物不对称合成中的应用进行了简要综述。     

羰基还原酶产生菌SW2026的产酶条件及其不对称催化还原4＇-氯苯乙酮

以4＇-氯苯乙酮为模型底物,对筛选得到的Candida krusei SW2026的羰基还原酶产酶条件进行研究。结果表明：适宜的发酵培养基组成为甘油50g/L,玉米浆20g／L,KH2PO4 4g／L,MgSO4·7H2O 1．5g／L;适宜的培养条件为温度30℃,初始pH6,摇床转速200r／min,发酵周期48h。在产酶发酵条件下培养的湿细胞对4＇-氯苯乙酮进行不对称还原反应,产物（S）-4＇-氯-α-苯乙醇的产率最高达88．56％,e．e．值稳定在87％左右。     

生物催化不对称还原制备氟代手性醇

由于氟原子的特殊性质，化合物中引入氟原子可显著改变其物理化学性质。因此，氟原子在药物中的应用越来越广。此外，80%药物分子结构属于手性分子。其中，氟代手性醇常见于手性药物结构中，该类结构的合成方法研究具有重要的意义。不对称还原含氟酮是合成此结构的常见方法。与化学还原方法相比，生物催化还原具有对映选择性强、产率高和易于分离纯化等优点。生物催化，特别是酶催化还原含氟酮类化合物成为手性药物合成领域的研究热点。本文从纯化酶催化和全细胞催化两个方面，综述了近年来含氟酮生物催化还原合成氟代手性醇的研究进展，并分析总结了氟代对酮生物催化还原的影响，最后对生物催化还原法未来的发展进行了展望。     

面包酵母不对称还原酮基泛解酸内酯

目的:利用市售面包酵母作催化剂,对酮基泛解酸内酯不对称还原进行了研究,并考察了底物浓度、辅因子、环糊精添加量和pH值对不对称还原反应的影响.方法:以市售面包酵母作催化剂,酮基泛解酸内酯作为底物在摇瓶中进行催化,用手性色谱柱对催化产物进行了检测.结果:发现反应条件为底物浓度低于2.56-mg/ml,辅因子选用蔗糖,β-环糊精添加量为16.7mg/ml,pH 4～5适合于酮基泛解酸内酯的不对称还原.在如上优化的催化条件下,产物得率大于45%,对映体过量率(e.e.)大于90%.结论:以酵母细胞作催化剂进行酮基泛解酸内酯不对称还原工艺具有生产光学纯泛解酸内酯的潜力.     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Kinetics of Inhibition of Horse Liver Alcohol Dehydrogenase byp-Methylbenzyl Hydroperoxide

Abstract

The complex kinetic behaviour of p-methylbenzyl hydroperoxide in its inhibitory action on horse liver alcohol dehydrogenase was examined. The kinetic patterns are markedly different at very low (<10^?8 M) and high (> 10^?7 M) hydroperoxide concentrations. In both cases very low inhibition constants (4nM and 14nM, respectively) were found. A possihle mechanistic model based on these results is discussed.     

Kinetics of Ethanol Oxidation Catalyzed by Yeast Alcohol Dehydrogenase in Aqueous Solutions of Sodium Dodecylsulfate

A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of beta-NAD(+) which reaches its maximum value (7,100 +/- 1,700 microM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 +/- 1 mM for 15 mM SDS and 50 +/- 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).     

Kinetic Modeling of Acetophenone Reduction Catalyzed by Alcohol Dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis> sp.

NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (k_d=0.0141 min⁻¹) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

NAD+-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

An NAD⁺-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K _m and V _max for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K _m and V _max for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH₂-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

牛肝辅酶Ⅱ依赖性视黄醇脱氢酶cDNA的克隆及组织表达

迄今为止的研究证明 ,维生素A亦称视黄醇(retinol)的生理功能是通过其两步氧化代谢产物视黄醛与视黄酸 (亦称维甲酸 )来完成的 .视黄醛通过其光学异构体 1 1 顺式视黄醛与视觉细胞内的视蛋白 (opsin)结合组成视色素 .感光时 ,1 1 顺式视黄醛转变成全反式视黄醛从视蛋白脱落 ,这一过程同时传导到大脑产生视觉[1 ] .全反式维甲酸 (all transretinoicacid)则通过与其在核内受体 (RARα ,β ,γ)结合调节基因的转录来发挥其许多重要的生理功能 ,包括正常胚胎的发育 ,形态、神经系统的形成 ,成体动物的生长、发育、繁殖等 ,并通过调解组织及…     

葛花鸢尾苷的酸水解及其对乙醇脱氢酶激活作用的研究

本文对葛花提取物鸢尾苷经酸解法得到鸢尾苷元的水解工艺及鸢尾苷和鸢尾苷元对乙醇脱氢酶的激活作用进行了研究.结果表明,葛花鸢尾苷酸水解的最佳条件为:固液比1∶1 (mg/mL),甲醇浓度50％,反应温度80℃,反应时间5h,盐酸浓度4％,此条件下,鸢尾苷水解率可达99％以上.鸢尾苷和鸢尾苷元对乙醇脱氢酶均有一定的激活作用,其激活率分别为127.35％和132.35％.酸水解提高了葛花异黄酮类物质的生物利用率.     

香水莲花总提取物对小鼠急性酒精肝损伤的实验研究

目的:观察香水莲花提取物对酒精所致小鼠急性化学损伤的保护作用。方法:雄性昆明种小鼠70只,随机分为正常对照组、急性酒精肝损伤模型组、水飞蓟素阳性对照组(46.7 mg/kg)以及香水莲花低、中、高剂量组(120、180、240 mg/kg)共6组,检测肝组织甘油三酯(TG)、丙二醛(MDA)和还原型谷胱甘肽(GSH)含量,称量体重和肝脏重量。结果:香水莲花总提取物能够抑制急性酒精肝损伤小鼠肝组织的TG、MDA含量升高(P0.05),增加GSH含量(P0.05)。结论:香水莲花总提取物对急性酒精肝损伤有明显的保护作用。