双层固定化酵母催化还原4-氯-乙酰乙酸乙酯的研究

以海藻酸钠为内层载体,利用机械强度良好的聚乙烯醇(PVA)改性海藻酸钠混合物作为外层载体,双层固定化高活性酵母,得到固载的生物催化剂,用于由磷酸缓冲溶液和环己烷组成的双相体系,对4-氯-乙酰乙酸乙酯(COBE,ethyl 4-cloro-3-oxobutyrate)进行不对称催化加氢合成(S)-4-氯-3-羟基丁酸乙酯[(S)-CHBE],ethyl[(S)-4-chloro-3-hydro butanoate].在最适条件下,双层固定化酵母的不对称催化还原反应优于游离细胞和单层固定化细胞.在底物COBE的最佳浓度和进料速率分别为170mmol/L,125mL/h的连续操作条件下,最高(S)-CHBE转化率为94.2%,最高对映体过量值为98%.与游离细胞催化COBE相比,反应转化率提高3%,最佳底物浓度比游离细胞增加1倍;与单层固定化细胞催化还原相比,对映体过量值提高8%.连续性实验表明,双层固定化酵母细胞在连续使用5d,其转化率和对映选择性没有明显下降.     

近平滑假丝酵母细胞催化乙酰基三甲基硅烷不对称还原反应

 采用近平滑假丝酵母细胞用于催化乙酰基三甲基硅烷不对称还原反应, 可高选择性地生成 (R)-1-三甲基硅乙醇. 结果表明, 固定化于海藻酸钙的细胞催化该反应的产物收率比游离细胞的高. 不同辅底物对该反应的影响显著, 以葡萄糖为辅底物时, 反应的初速率较快, 产物收率较高. 该反应的最适条件为: 辅底物 (葡萄糖) 浓度 110 mmol/L, 振荡速度 180 r/min, 缓冲液 pH 值 6.0, 反应温度 30 oC, 底物浓度 20 mmol/L. 在此反应条件下反应的初速率、产物收率和产物的 ee 值分别为 11.4 μmol/h, 96.5% 和 99.9%.     

水相中酵母细胞催化4-氯乙酰乙酸乙酯不对称还原反应

 利用活性酵母细胞催化4-氯乙酰乙酸乙酯(COBE)的不对称还原可以直接合成具有光学活性的4-氯-3-羟基丁酸乙酯(CHBE). 实验发现,在水相体系中主要生成D-(S)-型产物,COBE的转化率及CHBE的收率和光学选择性都比较高. 考察了底物和产物的浓度、辅助底物的种类和浓度、体系的pH和温度以及菌体培养条件等因素对反应的影响. 结果表明,在较低COBE浓度下R型产物占优,在高浓度下主要是S型产物; 较高的反应温度有利于S型产物的生成,pH≈8.0时可获得较高的S型产物选择性; 产物CHBE对COBE的还原反应具有一定的抑制作用. 在酵母催化COBE还原的同时需要一定的辅助底物来再生辅酶NAD(P)H,利用乙醇、异丙醇和仲丁醇作辅助底物可获得较高的立体选择性. 对数生长期和厌氧条件培养的细胞对生成S型产物较有利.     

固定化细胞酶法拆分N-乙酰-D,L-3-甲氧基丙氨酸

利用氨基酰化酶固定化细胞酶法拆分了N-乙酰-D,L-3-甲氧基丙氨酸. 考察了温度、pH值、底物浓度、金属离子和拆分时间对酶促反应的影响. 确定了氨基酰化酶固定化细胞手性拆分N-乙酰-D,L-3-甲氧基丙氨酸的最佳工艺条件为pH=7.0, 反应温度50 ℃及底物浓度500 mmol/L. 10^-4 mol/L的Co²⁺和Mg²⁺对氨基酰化酶有显著激活作用, Cu²⁺和Zn²⁺对酶促反应有明显抑制作用. 在最佳条件下, 氨基酰化酶固定化细胞对N-乙酰-L-3-甲氧基丙氨酸的摩尔转化率达96%.     

培养基碳源对固定化酵母细胞催化乙酰基三甲基硅烷不对称还原反应的影响

 尽管含芳香基等较大取代基团的底物能被生物转化为高对映体纯手性有机硅醇,但生物催化其他底物还原反应的产物收率及对映体选择性均较低. 作者筛选到一株能高效催化羰基/羟基不对称转化的酵母菌株,探讨了用固定化酵母细胞高效催化乙酰基三甲基硅烷不对称还原制1-三甲基硅乙醇的可能性,系统研究了培养基碳源及其浓度对该反应的影响. 发现在水/正己烷两相体系中,固定化酵母细胞能催化乙酰基三甲基硅烷不对称还原成1-三甲基硅乙醇. 可通过调节培养基碳源的种类及其浓度在一定程度上控制反应的产物收率及对映体选择性. 以最适碳源(3.0%麦芽汁)培养的酵母细胞催化该反应的产物收率和对映体过量值分别为91.3%和72.8%,远高于文献报道值.     

有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

在有机溶剂/缓冲液双相体系中, 利用固定化醋酸杆菌Acetobacter sp.CCTCC M209061细胞高对映体选择性地催化1-(4-甲氧基)-苯基乙醇(MOPE)的不对称氧化反应, 成功地拆分外消旋MOPE得到对映体纯(S)-MOPE. 与游离细胞相比, 固定化细胞催化反应速度有所降低, 但其稳定性(包括操作稳定性、 热稳定性和储藏稳定性)明显提高. 固定化细胞连续使用10批次(每批次12 h)后, 仍能保留其初始催化活性的58%以上, 而游离细胞仅保留约20%的相对活性. 在所考察的不同有机溶剂中, 正己烷不仅能较好地溶解底物, 而且对细胞的生物相容性相对较好, 因而提高了反应底物浓度、 反应初速度、 对映体回收率及残留底物e.e.值, 是反应体系中最适宜的有机相. 该反应的最适宜正己烷体积分数为60%, 辅底物为50 mmol/L丙酮, 底物浓度为40 mmol/L, 缓冲液pH=6.5, 反应温度为30 ℃; 在此条件下, 反应初速度为80.4 μmol/min, 反应12 h后, 对映体回收率和残留底物e.e.值分别为51.0%和99.9%, 明显好于水单相反应体系.     

CTAB透性化酵母细胞生物催化合成(S)-(+)-3-羟基丁酸乙酯

 利用十六烷基三甲基溴化铵(CTAB)透性化啤酒酵母细胞中高活性的脱氢酶,借助于辅助底物乙醇和葡萄糖,对3-羰基丁酸乙酯(EOB)不对称还原合成(S)-(+)-3-羟基丁酸乙酯((S)-(+)-3-EHB)进行了研究. 结果表明,CTAB透性化酵母细胞中的醇脱氢酶和6-磷酸葡萄糖脱氢酶的活性分别比未经处理的酵母细胞高482倍和6.5倍. 在相同条件下,CTAB透性化细胞对EOB的还原比未经处理的酵母细胞快. 细胞浓度对反应有明显影响,当透性化酵母细胞浓度＜90 mg/ml时,(S)-(+)-3-EHB的产率和对映体过量值都较低; 当酵母细胞浓度≥90 mg/ml,在最佳进料速率、最适温度和pH条件下,振摇速度为125 r/min时, (S)-(+)-3-EHB的浓度达到最大值314 mmol/L. 在反应开始的6 h内,(S)-(+)-3-EHB的产率可达94%,对映体过量值≥98%,但24 h后产率和对映体过量值分别降低到85%和91%.     

亲水性离子液体[EMIM]BF4对固定化β-葡萄糖醛酸苷酶催化活性的影响

以壳聚糖为固定化载体,采用吸附交联法制备固定化β-葡萄糖醛酸苷酶;以1-乙基-3-甲基咪唑四氟硼酸盐([EMIM]BF4)/缓冲溶液均相体系为介质,研究了亲水性离子液体[EMIM]BF4对固定化酶生物催化甘草酸(GL)合成单葡萄糖醛酸基甘草次酸(GAMG)的影响.实验结果表明,当均相体系中[EMIM]BF4的体积分数为16%,pH为5.4,反应温度为50℃及摇床转速为200 r/min时,酶活力达到最高,并且明显优于纯缓冲溶液体系中的最高酶活.重复利用性实验结果表明,与纯缓冲液介质体系相比,固定化酶在含亲水性离子液体[EMIM]BF4的均相介质中表现出较好的操作稳定性.表观动力学参数和活化能数据表明,亲水性离子液体[EMIM]BF4在催化体系中能够增强酶和底物GL的亲和力,有效稳定酶-底物的过渡态,并降低反应活化能,而使固定化β-葡萄糖醛酸苷酶表现出较高的催化活性.     

活性酵母细胞不对称催化苯乙酮还原及树脂吸附对反应的促进作用

 以苯乙酮为模型底物,研究了水相体系中酵母细胞催化前手性芳香酮不对称还原生成相应手性醇的反应特性. 实验发现,酵母细胞催化苯乙酮不对称还原的产物以(S)-α-苯乙醇为主,反应的立体选择性很高,(S)-α-苯乙醇的对映体过量值可达99%左右. 在pH为7～8, 酵母细胞用量为0.2 g/ml的条件下能获得较高的产物收率(可达35%左右). 酵母细胞能选择性地氧化(S)-α-苯乙醇,而留下(R)-α-苯乙醇. 在反应体系中加入合适的吸附树脂,可以降低底物和产物对细胞的毒害作用,显著提高反应底物的初始浓度,从而提高产物收率.     

重组大肠杆菌细胞不对称还原4-氯乙酰乙酸乙酯合成(R)-(+)-4-氯-3-羟基丁酸乙酯

 从赭色掷孢酵母(Sporobolomyces salmonicolor ZJU0105)中克隆出NADPH依赖型醛基还原酶基因,构建了重组大肠杆菌E.coli BL21(pET28-ALR0105), 该工程菌可以高效地表达醛基还原酶. 将重组细胞用于催化4-氯乙酰乙酸乙酯不对称还原,合成出具有光学活性的(R)-(+)-4-氯-3-羟基丁酸乙酯. 实验发现,在加入适量辅酶及辅酶再生酶的条件下,利用重组细胞催化还原反应可以获得比使用赭色掷孢酵母更高的转化率、产率和ee值,得到了几乎是光学纯的(R)-(+)-型产物,从而解决了酵母细胞催化此类反应ee值较低的问题. 考察了辅酶及共底物的添加、底物和产物的浓度、pH值、温度以及菌体密度等因素对还原反应的影响. 结果表明,不对称还原反应必须在辅酶NADPH和辅酶再生酶系及共底物葡萄糖的参与下进行; 底物和高浓度的产物对还原反应有一定的抑制作用; 当pH＞6.0时,反应的转化率及产率都显著降低; 高密度重组细胞可以减小底物的抑制作用.     

2-(4-羟基苯基)-3,3,3-三氟丙酸乙酯的合成研究

报道了以苯酚和三氟丙酮酸乙酯为起始原料,在温和条件下合成高产率的2-(4-羟基苯基)-3,3,3-三氟丙酸乙酯的一条新途径.该途径包括无水碳酸钾催化的苯酚与三氟丙酮酸乙酯间的亲电取代反应、由此形成的产物的选择性侧连羟基的氯代反应和随后的硼氢化钠还原反应.     

2-vinyloxy ethyl phthalimide polymerization initiated by 1-(isobutoxy) ethyl acetate in the presence of ethyl aluminum dichloride and either ethyl acetate or ethyl benzoate

2-Vinyloxy ethyl phthalimide (ImVE) was polymerized using 1-(isobutoxy) ethyl acetate as the initiator in the presence of ethyl aluminum dichloride and either ethyl acetate or ethyl benzoate. The resulting polymers have a narrow molecular weight distribution, and their molecular weight can be controlled within a narrow range by varying the monomer and initiator concentrations. Diblock copolymers with n-butyl vinyl ether can also be formed. The behavior of the polymerization is consistent with a living cationic mechanism. A brief comparison of the title system with other initiating systems is also presented. © 1996 John Wiley & Sons, Inc.     

Thermal degradation of poly(ethyl methacrylate) and its copolymer with poly(ethyl acrylate)

The thermal degradation behaviour of poly(ethyl methacrylate) homopolymers and poly(ethyl methacrylate) and poly(ethyl acrylate) copolymers synthesized by using the benzoyl peroxide-di-methyl aniline redox pair at different temperatures (18–35C) was investigated. Contrary to some reports in the literature, the thermal degradation of PEMA was observed to take place in multi steps. These are assigned to be loss of labile end groups, side chain scission, anhydride formation and main chain degradation steps. Dominating chemical formations at the end of these steps were characterized by FTIR spectroscopy.The homopolymer samples synthesized at 18C showed a greater thermal stability against degradation. Copolymerization with small amounts of ethyl acrylate was observed to impart thermal stability to PEMA by stabilizing mainly the end groups against degradations.     

The Effect of Synergism Behavior in Binary Mixtures of Some Surfactants with Ethyl Xanthat Absorbed on Clay for Wastewater Treatment

Surface tension, sedimentation, and turbidity experiments were conducted in order to study the synergism behavior of some surfactants with ethyl xanthat mixtures on the reduction of surface tension of their aqueous solutions and on the hydrophilicity of clay surface for using it as absorbent to oil in wastewater treatment. The following systems were studied: 1) potassium ethyl xanthat (KEX) with sodium cetyl sulphate (SCS); 2) potassium ethyl xanthat with cetyl trimethyl ammonium bromide (CTAB); and 3) potassium ethyl xanthat with ethoxylated palmetic acid (EPA). A synergistic effect of the combined reagents was found to enhance the hydrophilicity of clay and also to reduce the surface tension depending mainly on the surface properties of the mixtures.     

Determination of ethyl glucuronide and fatty acid ethyl esters in hair samples

Hair testing for alcohol biomarkers is an important tool for monitoring alcohol consumption. We propose two methods for assessing alcohol exposure through combined analysis of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) species (ethyl myristate, palmitate, stearate and oleate) in hair (30 mg). EtG was analysed by liquid chromatography–tandem mass spectrometry, while FAEEs were analysed by gas chromatography–tandem mass spectrometry using electron impact ionization. Both methods were validated according to internationally accepted guidelines. Linearity was proven between 3 and 500 pg/mg for EtG and 30–5000 pg/mg for FAEEs, and the limits of quantification were 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Precision and accuracy were considered adequate, processed EtG samples were found to be stable for up to 96 h left in the injector and processed FAEEs samples for up to 24 h. Matrix effects were not significant. Both methods were applied to the analysis of 15 authentic samples, using the cut‐off values proposed by the Society of Hair Testing for interpretation. The results agreed well with the self‐reported alcohol consumption in most cases, and demonstrated the suitability of the methods to be applied in routine analysis of alcohol biomarkers, allowing monitoring consumption using low sample amounts.     

Toxicity Test n-Hexane: Ethyl Acetate (3:7) Fraction of Sudamala (Artemisia vulgaris L.)

Sudamala (Artemisia vulgaris L.) is commonly used in the community as anti-tumor in digestive organ, including in oral cavity. However, there have been no scientific studies about oral anti-carcinogenic active substances of Sudamala. The purpose of this study was to analyse the effect of per oral administration of n-hexane: ethyl acetate (3:7) fraction of Artemisia vulgaris L. on healthy mice. This experimental study used male Swiss Webster (Balb C) strain mice (Mus musculus) 2.5 months old, 20-30 grams by weight. Group 1, the mice received 0.5% CMC- Na fraction solvent, as a control. Group 2, the mice received 200 mg/kgbw n-hexane: ethyl acetate (3:7) fraction of Sudamala (Artemisia vulgaris L). The fraction was given once daily for 8 weeks per oral according to the mice's weight. At the end of 8^th week, the mice were killed and oral mucosal tissue was taken for biopsy specimens. Tunnel assay for Apoptosis and immunohistochemistry were undertaken for PCNA. T test analysis showed there was no significant difference between control and treatment groups. The fractions of n-hexane: ethyl acetate (3:7) of Sudamala (Artemisia vulgaris L) was not toxic, it was not harmful to living tissue in healthy mice.     

超高效液相色谱-串联质谱法同时测定食品中4种常用香精

建立了超高效液相色谱-串联质谱法同时测定食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的方法。样品用水提取,固相萃取小柱净化,目标化合物采用UPLC^TMBEH C18色谱柱(50 mm×2.1 mm, 1.7 μm)分离,以甲醇和含0.002 mol/L乙酸铵及0.1%(v/v)甲酸的水溶液为流动相进行梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。4种香精在5~500 μg/L或10~1000 μg/L质量浓度范围内线性良好,相关系数均在0.9995~0.9998之间;回收率为75.8%~116%,相对标准偏差(RSD, n=6)为1.58%~4.01%。该方法灵敏、准确,检测范围广,分析速度快,适合食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的检测。     

Synthesis of biodegradable poly (methylparaben/glycine ethyl ester) phosphazene and further research of its degradation in vitro

Polydichlorophosphazene(PDCP)was synthesized from PCl5 and NH4Cl via a one-pot approach,and then further reacted with glycine ethyl ester and methylparaben via a two-step nucleophilic substitution approach to give poly(methylparaben/glycine ethyl ester)phosphazene(PMGP),which was a kind of mix-substituted biodegradable polymer.NMR and FT-IR were used to characterize its structure.A series of experiments were conducted to study the effects of the ratio of methylparaben to glycine ethyl ester and the experime...     

静电喷雾法制备聚膦腈微球

用静电喷雾法制备了粒径窄分布的甘氨酸乙酯-苯丙氨酸乙酯取代聚膦腈的微球.主要探讨了溶剂、溶液浓度、流速、喷射电压及接收距离对微球形貌和粒径的影响.微球的表面形貌与溶剂性质和聚合物溶液浓度密切相关,微球粒径受聚合物溶液流速的影响比较显著,随流速增大粒径呈单调上升趋势.结果显示对于[η]=0.1 dL/g的(甘氨酸乙酯)0.3(苯丙氨酸乙酯)0.7取代聚膦腈,以0.25 g/mL的四氢呋喃溶液进行静电喷雾,可获得粒径分布窄(1~2μm)且近球形的微粒,其工艺条件可进一步用于载药聚膦腈微球的制备和控制释放研究.     

由α-氰基肉桂酸乙酯/N-溴代苯甲酰胺合成2-噁唑啉衍生物

作为重要的杂环化合物,合成新的2-噁唑啉衍生物以及发展其新的合成方法具有重要意义,为此以α-氰基肉桂酸乙酯衍生物为底物,以N-溴苯甲酰胺为反应试剂,在无水碳酸钠(相对于底物3为110%摩尔分数)促进下,在丙酮溶剂中,室温下,建立了合成相应2-噁唑啉衍生物的新方法,共合成了11个新化合物,其结构由核磁共振波谱仪(~1H NMR,~(13)C NMR)和高分辨质谱(HRMS)确认。结果显示,各种α-氰基肉桂酸乙酯衍生物(3a~3k)可被顺利的转化成相应的2-噁唑啉衍生物(5a~5k)。在室温下,丙酮作溶剂,以Na_2CO_3为促进剂时,相应产物的最高收率可达90%。不仅α-氰基肉桂酸乙酯衍生物(3)可被用作该反应的底物,而且α-乙氧甲酰基肉桂酸乙酯(6)也适用于该反应。实验结果还证明,除了N-溴代苯甲酰胺外,N-溴代对硝基苯甲酰胺(8)及N-溴代乙酰胺(9)也适用该反应,证明该方法具有广泛的适应性。根据实验结果,提出了可能的反应机理,该机理支持了形成2-噁唑啉衍生物的区域选择性。