重组大肠杆菌全细胞用于苯乙酮酸的绿色生物合成

苯乙酮酸是化学合成中重要的合成砌块,可用于合成多种药物中间体,探索苯乙酮酸的绿色合成工艺具有重要的意义。以包含D-扁桃酸脱氢酶Lh DMDH编码基因的重组大肠杆菌全细胞为催化剂,考察了它在无辅酶和辅底物添加的条件下对D-扁桃酸生物转化的效果,并对催化产物进行了纯化和鉴定。结果表明,本研究成功实现了在无辅酶和辅底物添加条件下苯乙酮酸的生物合成,产物的得率和纯度分别为45%和99%左右。成果也为外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成奠定了基础。     

生物不对称合成R-(-)-扁桃酸的影响因素

研究了酵母细胞(Saccharomyces cerevisiae)生物不对称还原苯乙酮酸合成R-(-)-扁桃酸的过程中,环境因子对底物的转化效率和产物对映体过量值的影响. 结果表明,高浓度的底物苯乙酮酸对酵母细胞的催化还原活性具有较显著的抑制效应. pH 6.5、温度32℃、严格厌氧为较适宜条件,底物苯乙酮酸的转化率和产物扁桃酸的得率分别可达97.0%和96.1%,R-(-)-扁桃酸的对映体过量值(ee)为95.1%.     

化工中间体苯乙酮酸的合成初探

概述化工中间体苯乙酮酸的合成方法及用途,介绍由苯乙酮经扁桃酸合成苯乙酮酸的情况。     

包括辅酶原位再生的偶联酶反应联产1,3-二羟基丙酮和2-氨基丁酸

将甘油脱氢酶(GDH)和亮氨酸脱氢酶(LDH)偶联,以甘油和2-丁酮酸为底物,同时合成1,3-二羟基丙酮(DHA)和2-氨基丁酸,并实现辅酶NAD+的循环再生. 考察了底物和辅酶NAD+浓度对产物产率和辅酶总转换数(TTN)的影响,结果表明,当NAD+初始浓度为0.1 mmol/L时,TTN值可达5110;底物甘油和2-丁酮酸的转化率分别为1.4%和2.6%. 通过分析各反应底物和产物对GDH和LDH反应初速率的影响,认为产物DHA和2-氨基丁酸对GDH和LDH的强烈抑制效应是造成反应转化率低的主要原因. 因此,采取有效的产物去除措施将是提高偶联反应效率的关键.     

提高不对称还原合成(R)-扁桃酸的效率

以葡萄酒酵母Saccharomyces ellipsoideus为催化剂,对提高不对称还原苯乙酮酸生产(R)-扁桃酸效率进行了研究,分别利用了分批补料、添加吸附树脂的方法降低底物对反应过程的抑制作用,提高反应的底物浓度。并且通过对吸附树脂吸附性能的考察,筛选出了一种对底物有较快吸附速率和较大吸附量的树脂NKAⅡ,可以显著降低底物对反应过程的抑制,提高反应的底物浓度。最终发现将两种方法结合,在添加吸附树脂的体系中进行分批补料是最高效的方法,将底物浓度从33.31 mmol/L提高到166.53 mmol/L,并且产率达到了91.6%,e.e.值为100%,而且菌体重复使用性能很好。     

酵母耦合原位分离技术不对称合成(R)-扁桃酸甲酯

以酿酒酵母(Saccharomyces cerevisiae AS2.1392)全细胞为催化剂不对称还原苯甲酰甲酸甲酯合成(R)-扁桃酸甲酯,该催化剂催化速度快、操作稳定性好。研究了底物和产物浓度对反应初速度的影响,建立了底物和产物抑制模型,并采取分批加入底物和添加树脂的方式解除底物和产物抑制。通过考察不同树脂对底物和产物的吸附量以及对生物还原反应的影响,筛选出了一种较适合的大孔吸附树脂NKA-Ⅱ。在优化的树脂加入量和加入模式下,当底物浓度为180mmol/L时,产物产率由35.0%提高到71.2%,对映体过量值(ee)保持在95%左右。     

铜绿假单胞菌对映选择性降解制备R-扁桃酸

以外消旋扁桃酸为唯一碳源,从土壤中筛选得到一株选择性降解S-扁桃酸的铜绿假单胞菌,可用于选择性降解S-扁桃酸制备R-扁桃酸。考察了温度、pH值、底物浓度等因素对S-扁桃酸降解的影响,并对发酵培养基成分及转化条件进行了优化。结果表明,在35℃、pH值8.0、底物浓度131.4 mmol.L-1的优化条件下,68 h发酵完成,R-扁桃酸收率为48.2%、光学纯度为99.2%。     

D-氨基酸氧化酶的分子改造及应用研究进展

D-氨基酸氧化酶是一类含有黄素腺嘌呤二核苷酸的氧化还原酶,能够催化D-氨基酸氧化脱氢,生成相应的α-酮酸、过氧化氢和氨。该类酶在自然界中分布广泛,主要来源于真核生物和少数原核生物。作为一种经典的生物催化剂,D-氨基酸氧化酶具有反应条件温和、底物谱广泛、对映体选择性好等特点,在合成医药、农药和精细化学品等方面具有重要的应用价值。本文综述了D-氨基酸氧化酶的基本蛋白结构特征及其催化机制,重点介绍了D-氨基酸氧化酶底物特异性和热稳定性分子改造的策略和代表性成果以及该类酶在生物催化中的应用,例如制备7-氨基头孢烷酸、手性氨基酸、胺类化合物和α-酮酸。最后探讨了D-氨基酸氧化酶目前在生物催化应用过程中存在的问题。后续的研究可围绕新酶的挖掘与改造展开工作。基于对映体选择性和底物识别的分子机制,理性设计酶的催化性能,并以挖掘或改造获得的D-氨基酸氧化酶作为新酶元件,用于构建功能化学品生物合成新途径。     

扁桃酸的酶促光学拆分

对在非水体系中,利用脂肪酶催化水解扁桃酸乙酯外消旋混合物拆分R(-)-扁桃酸进行了初步的研究.筛选出脂肪酶N435作为催化剂,叔丁醇作为溶剂.确定了最适的反应条件：脂肪酶N435浓度为2.5 g·L^-1,RS-扁桃酸乙酯浓度为0.25 mol·L^-1,水∶RS-扁桃酸乙酯的摩尔比为5∶1,反应温度为40℃,摇床转速为200 r·min^-1,反应时间为24 h.在此条件下,R(-)-扁桃酸乙酯的转化率为41.6%,对映体过量百分率达84.0%.考察了底物R(-)-扁桃酸乙酯和产物R(-)-扁桃酸对反应的抑制作用,在此基础上运用顺序机制和拟稳态法,建立了反应的动力学模型,模拟计算结果和实验结果吻合较好.     

α-酮异己酸的生物合成研究进展

α-酮异己酸是重要有机酸、药用氨基酸合成前体、新陈代谢调节因子和治疗药物,其生物合成路径条件温和,环境友好。本文对α-酮异己酸的生理功能和体内代谢机理进行了归纳,并着重对其生物合成路径的研究进展进行了综述。现有研究表明,α-酮异己酸可用葡萄糖为底物,通过代谢工程改造的谷氨酸棒杆菌或大肠杆菌工程菌发酵合成,但产物浓度较低;或以L-亮氨酸为底物,经氨基酸转氨酶、氧化酶、脱氨酶、重组工程菌或全细胞催化转化合成,产物浓度较高。α-酮异己酸高产菌株的筛选、利用代谢工程方法对菌株进行改造以构建高效工程菌、发酵与分离提取工艺优化等问题,是今后需要研究的重点。     

烟草叶片组织块悬浮培养合成辅酶Q_(10)的研究

以新鲜烟草叶片制得的组织块为研究对象,建立了一种烟叶组织块悬浮培养合成辅酶Q10的方法。考察了培养温度、pH值、培养时间对辅酶Q10合成的影响。另外,采用均匀设计法对培养液中烟酸、盐酸硫胺素、半胱氨酸、对羟基苯甲酸、硫酸镁质量浓度进行了优化。在最适的培养条件下,烟草叶片组织块中辅酶Q10的质量分数达到208.09μg/g,比烟草叶片中的初始质量分数提高了18.7倍。文中建立的烟草叶片组织块悬浮培养合成辅酶Q10的方法具有周期短、操作简单的特点,是一种高效生产辅酶Q10的方法。     

Network formation by aza‐Michael addition of primary amines to vinyl end groups of enzymatically synthesized poly(glycerol adipate)

A highly efficient approach for the synthesis of polyester‐based networks via aza‐Michael addition of primary amines to α,β‐unsaturated (vinyl) end groups of poly(glycerol adipate) (PGA) was achieved. By acylation of PGA with 6‐(Fmoc‐amino)hexanoic acid side chains via Steglich esterification, protected amine‐functionalized PGA was obtained. This was followed by the removal of fluorenylmethyloxycarbonyl (Fmoc) protecting groups and the synthesis of PGA‐based networks under catalyst‐free conditions. The successful conjugate addition of primary amines to vinyl end groups and network formation were confirmed using ¹³C magic angle spinning NMR and Fourier transform infrared spectroscopy. Network heterogeneity and defects were quantitatively investigated using ¹H double‐quantum NMR spectroscopy. Finally, a hydrogel was prepared with potential biomedical applications.     

悬浮聚合法制备聚乙醇酸的工艺

利用悬浮聚合的方法,选用甲基硅油作为体系的分散介质,MOA-3作为分散剂,乙醇酸为单体,辛酸亚锡为催化剂,合成出了特性黏度高达0.845 dL/g的聚乙醇酸（PGA）,采用红外、核磁、DSC、XRD等方法表征了聚乙醇酸的结构和性质。通过一系列单因素实验探讨了聚合温度、反应时间、催化剂用量等对聚乙醇酸特性黏度的影响规律。经过正交实验得出各因素对特性黏度影响的大小,并得出最佳的制备工艺为：温度194 ℃,反应时间113 h,催化剂用量0.02%。     

辅酶类物质对长春花细胞合成长春新碱的调控作用

添加5种不同浓度的辅酶类物质,根据单因素实验设计正交实验,研究其对抗肿瘤生物碱长春新碱合成的调控作用,并探讨其作用机理. 结果表明,单独添加每种辅酶类物质对长春新碱的合成均有促进作用,确定其最佳添加浓度为(mmol/L): DL-硫辛酸1.0, VB1 1.5, FMN 3.0, ATP 3.0,赖氨酸3.0,该条件下长春新碱合成量(细胞干重)为0.217 mg/g,为首次在长春花细胞培养中成功合成长春新碱.     

Degradation profiles of poly(methylene oxide) and poly(glycolic acid) for inflow control devices in oil wells

The viability of the application of hydrolysable polymers, such as poly(methylene oxide) (PMO) and poly(glycolic acid) (PGA), in formation treatments in oil wells, was studied; we examined their addition to inflow control devices (ICDs), which are instruments used to control the oil‐flow profile along production intervals. Thereby, the structural and morphological changes of PMO and PGA samples exposed to common chemicals, such as hydrochloric acid (HCl) and sodium chloride (NaCl) solutions and xylene, used in the procedure were evaluated under conditions similar to those observed in southeastern Brazilian offshore oil fields (temperature range = 50–130 °C, 41.4 MPa). The aim of this study was to verify whether the polymers hydrolyzed and could be further removed from the ICDs. Techniques including high‐pressure liquid chromatography, ion chromatography, ¹³C‐NMR, differential scanning calorimetry, scanning electron microscopy, and optical microscopy were applied in this study. Xylene did not produce expressive effects in the samples. PMO was significantly affected by 15% HCl at 75 °C but was not completely dissolved after 24 h of exposure; this counter indicates its use. The PGA hydrolysis profile in NaCl brine was suitable for application around 75 °C. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43786.     

The effects of lidocaine and hypoxia on phospholipid biosynthesis in the isolated hamster heart

In this study, the effects of lidocaine and hypoxia on the biosynthesis of phospholipids in the hamster heart were examined. hamster hearts were perfused with [1,3-³H]glycerol under normal and hypoxic conditions, and in the absence or presence of 0.5 mg/mL lidocaine. After perfusion, the radioactivity incorporated into the various phospholipid fractions was determined. With the exception of phosphatidylcholine, the synthesis of phospholipids was generally stimulated by lidocaine perfusion. In contrast, hypoxia caused a general decrease in phospholipid biosynthesis which was partially restored by lidocaine. ATP and CTP levels were severely reduced under hypoxic conditions, but their levels were not restored by lidocaine treatment. The activities of enzymes for phospholipid synthesis were determined under the various perfusion conditions. The activity of phosphatidic acid phosphatase was elevated by lidocaine and decreased by hypoxic treatment. The activity of CTP:phosphatidic acid cytidylyltransferase was increased under hypoxia, with or without lidocaine. Despite the reduction in phosphatidylcholine biosynthesis, no change in the activity of cytidine diphosphocholine (CDPcholine): diacylglycerol cholinephosphotransferase was detected following lidocaine or hypoxic perfusion. However, enzyme activity was inhibited by the presence of lidocaine in the assay mixture. Our results indicate that the reduction in phospholipid biosynthesis under hypoxic conditions was caused mainly by diminishing high-energy nucleotide levels. The enhancement of phospholipid biosynthesis by lidocaine appeared to be mediated in part by modulation of enzyme activities.     

Synthesis and characterization of pH-sensitive and biodegradable hydrogels prepared by γ irradiation using microbial poly(γ-glutamic acid) and poly(ϵ-lysine)

Poly(γ-glutamic acid) (PGA) and poly(?-lysine) (PL) solutions were used as components to prepare mixed hydrogels by γ irradiation. A PGA and PL mixed solution was crosslinked to form a hydrogel with specific water content (weight of absorbed water/weight of dry gel) of 10–100 when the 5 wt % solution of mixed polymer was exposed to γ radiation of 87 kGy dosage under N₂ atmosphere. The specific water content increased with increasing PGA content of the PGA/PL mixed gel. The influence of pH and salt concentration on equilibrium swelling was studied. A characteristic pH-sensitive swelling behavior was obtained using compositional changes of PGA and PL in the gel. PGA/PL 50/50 wt % mixed gel swelling in acid (pH < 4.0) and alkaline (pH > 6.0) conditions and was deswelled between pH 4.0 and 6.0 due to the ionic composition changes of the gel network. With an increase in the ratio of PGA to PL, the hydrogels showed increasing sensitivity to salt solutions (NaCl, Na₂SO₄, and CaCl₂). In addition, degradation of PGA/PL gel by protease produced from Aspergillus oryzae was investigated at 40°C and pH 7.0. PL gel was degraded completely within 2 days. An increase in the ratio of PAG in the PGA/PL mixed gel led to a decrease in the degree of degradation as expected. Some subtle degradation changes were found in the 50/50 and 80/20 wt % (PGA/PL) gels that were degraded by only 3.5 and 3.8% by protease, respectively. © 1995 John Wiley & Sons, Inc.     

由糠醛制备1,1,2-乙烷三羧酸

提出了一种合成 1,1,2 乙烷三羧酸的新方法。以糠醛为原料 ,先制得顺丁烯二酸 ,用顺丁烯二酸与三氯锗烷在酸性条件下加成 ,然后水解得到 1,1,2 乙烷三羧酸 ,收率为 71%。     

生物降解材料聚乙醇酸及乙醇酸共聚物的新发展

介绍了生物降解性材料聚乙醇酸及乙醇酸共聚物的发展现状,综述了聚乙醇酸的合成、乙交酯的纯化、乙醇酸聚合催化剂的选择、乙醇酸聚合物的降解机理、乙醇酸均聚和共聚改性及乙醇酸共聚物在生物医学领域和生态学领域的应用,并讨论了乙醇酸共聚物的发展前景。     

辅酶Q0合成工艺改进

以对甲酚为起始原料,经溴化、甲氧基化、羟甲基化和氧化反应得到了辅酶Q0。重点研究了溴化反应和氧化反应。使用Br2-H2O2作溴化剂,有效利用了溴化反应产生的HBr。在等体积甲酸和多聚磷酸溶剂中,用磷钼酸催化过氧化脲氧化3,4,5-三甲氧基甲苯,以79.6％收率得到目标产物。以对甲酚计,反应总收率71.7％,产品含量99.2％。