荧光分光光度法研究酶在非水相体系中的转酯化反应活性

以4-肼基-7-硝基-2,1,3-苯并氧杂二唑肼为荧光底物,乙烯基羧酸酯、伯醇为转酯化反应底物通过荧光分光光度法比较了几种能以固体聚合物为底物的商品化酶制剂在有机溶剂中催化转酯化反应的性质.该方法使用微量溶剂和底物进行反应,测定快捷,具有很高的直观性.实验所用4种酶制剂均可在异辛烷、甲苯和乙腈中催化转酯化反应,催化能力是脂肪酶LPL-3>蛋白酶Alcalase 3.0 T>蛋白酶subtilisin Carhberg>蛋白酶bacillolysin,但有机溶剂的疏水性对转酯化反应影响较大,疏水性高的有机溶剂比较适合做酶催化转酯化反应的溶剂.和月桂酸乙烯酯相比,小分子的丙酸乙烯酯更有利于酶催化转酯化反应的进行.     

一种新型极性荧光探针的合成与应用

合成了一种新型的基于查尔酮衍生物的极性荧光探针, 该探针是由4'-N,N-二甲氨基-4-氨基查尔酮与蒽醛通过希夫碱反应生成荧光物质R. 对比研究该荧光探针在不同溶剂中的荧光光谱, 发现荧光强度随着溶剂极性的增加显著降低, 可以用来检测有机溶剂中的水含量. 建立了一种新的乙醇中水含量荧光分析方法. 在水的体积分数为0～4.00%时, 荧光强度与水含量成良好的线性关系, 检测限为0.0115%.     

微波作用下高分子咪唑盐催化合成含氟芳香物

高分子咪唑盐1是一种新型高分子相转移催化剂,具有制备简单、稳定性好、催化活性高等特点。TG分析表明,其分解温度可达350℃左右。在微波作用下,采用此高分子咪唑盐相转移催化剂于中等极性的非质子溶剂中成功制备了系列含氟硝基苯类化合物,其收率为72.1%～94.4%,反应时间较常规加热最多可缩短60%以上,拓宽了卤素交换氟化反应溶剂的选择空间。选用中等极性溶剂,反应中还可突出地体现出微波对反应的“非热效应”。     

季铵盐表面活性剂引起非水混合溶剂的分相

当季铵盐表面活性剂在极性溶剂中具有合适溶解度时, 它可使该极性溶剂与另一种非极性溶剂形成的混合溶剂自发分相, 形成稳定的两相界面. 借鉴表面活性剂双水相现象的概念, 这种现象被称为表面活性剂非水双相(NSTP), 也可简称为双油相. 产生此种现象的原因被归结为季铵盐表面活性剂在极性溶剂中达到合适的溶解度时, 从而逐出与之不亲合的非极性溶剂.     

基于香豆素荧光团的新型极性检测荧光探针的开发及其成像应用

极性是生物微环境的重要参数之一, 在很大程度上, 生物体内许多生命活动都受到极性变化的影响, 本工作通过改变香豆素母体上的推-拉电子基团, 设计并合成了一种具有较大斯托克斯位移的新型极性荧光探针COM-PO, 该探针的荧光强度和波长会随着测试体系的极性变化而发生改变. 当极性增加时, COM-PO的激发态能量会通过偶极-偶极的相互作用散失在溶剂中, 荧光发射强度降低, 而在低极性溶剂中荧光发射强度增强, 利用这种特性实现了对极性的检测. 本工作通过荧光光谱、荧光成像实验表明COM-PO能够在样品中实现极性检测, 该探针有望实现与极性相关的疾病的早期诊断.     

L-异亮氨酸键合色谱固定相的制备及其性能

利用异氰酸丙基三乙氧基硅烷与L-异亮氨酸反应合成了一种新型的硅烷偶联剂,并进一步将其与硅胶反应制得键合有L-异亮氨酸的亲水色谱固定相。 通过核磁共振氢谱表明亮氨酸功能化硅烷偶联剂的成功合成、元素分析表征证明亮氨酸已成功键合到硅胶表面。 将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中,以一系列经典的极性小分子作为探针,考察了这些探针分子在固定相上的色谱行为。 极性化合物的保留时间随着流动相中有机溶剂含量提高而逐渐增大,表现出典型的亲水保留特征。 进一步研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对分析物在固定相上的保留的影响。 在优化了相关参数后,将固定相应用于碱性化合物、水溶性维生素以及核苷类极性物质的分离当中。 在等度洗脱下,5种碱性化合物、6种水溶性维生素和8种核苷类物质分别在8、18及25 min内被成功分离。 分离结果表明了合成的L-异亮氨酸键合亲水色谱固定相具有较好的色谱性能,在极性化合物的分离上具有良好的应用前景。     

在非水介质中进行酶促反应的几个重要问题

介绍了在非水介质中酶促反应的最新进展。较深入地讨论了介质性质对酶的催化活性及立体选择性的影响，有机溶剂中必需水含量对酶催化反应的重要性，描述了非缓冲体系中的盐及其它极性添加剂对酶活性的影响，介绍了在非均相的低共熔体系中进行酶促合成肽的研究。     

强极性中药组分的亲水作用色谱保留行为

采用亲水作用色谱模式,以二醇基硅胶为固定相,水-有机溶剂为流动相,通过改变流动相中有机溶剂种类及浓度、缓冲盐、有机酸种类及其浓度、柱温等条件,研究了强极性中药组分在亲水作用色谱中的保留行为。结果表明:流动相中水的比例在0～100%(V/V)变化时,溶质保留呈U型曲线,属于亲水色谱和反相色谱的混合保留机理。水含量在0～50%(V/V)范围时,亲水作用控制溶质的保留。溶质保留随流动相中缓冲盐浓度的增大而增强。对于酸性溶质,其保留随有机酸三氟乙酸、甲酸、乙酸的酸性降低而增大。     

水溶性1，8-萘酰亚胺衍生物的合成及其对有机溶剂中少量水的识别

萘二甲酸酐与2-(2-氨乙基氨基)乙醇在乙醇中反应合成了一种新型水溶性1,8-萘酰亚胺衍生物--N-[2-(2-羟乙基氨基)-乙基]-1,8-萘酰亚胺(3),其结构经1H NMR,IR,MS和元素分析表征.荧光光谱分析发现3在不同溶剂中具有不同的荧光性质,在非(或弱)极性-非质子性溶剂、极性-非质子溶剂中几乎不产生或产生较弱的荧光,而在极性-质子性溶剂中产生较强的荧光,在水中的荧光最强.这种特性使3有望用于识别有机溶剂中的少量水.     

银纳米粒子表面油酸盐吸附的分子模拟

采用分子力学方法模拟了油酸盐分子在银纳米粒子(111)表面的两种主要吸附形态: 双键吸附和羧基吸附, 计算了不同溶剂环境中油酸盐分子的吸附能和振动频率. 结果表明: 作为银纳米粒子保护剂的油酸盐能够稳定地存在于极性和非极性溶剂环境中, 与银表面的作用方式随溶剂极性的改变而改变, 水相中以双键吸附为主, 而在有机相中主要是羧基吸附. 这与红外光谱实验的推论基本吻合. 从而在分子水平上为实验结果提供了有价值的理论支持和微观信息.     

Lipase catalysis in organic solvents

The conditions for esterification and transesterification catalyzed by porcine pancreatic lipase in organic media were studied. It was found that the enzyme reaction was dependent on the following factors: the pH at which the enzyme powder was prepared from its solution, the polarity of organic media, the reaction temperature, the water content in reaction system, and the substrate structures. Effects of the above factors on enzyme activity were discussed.     

Enhanced catalytic activity of lipase encapsulated in PCL nanofibers

Use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Enzyme immobilization as an effective strategy for improving the enzyme activity has emerged from developments especially in nanoscience and nanotechnology. Here, lipase from Burkholderia cepacia (LBC), as an example of the luxuriant enzymes, was successfully encapsulated in polycaprolactone (PCL) nanofibers, proven by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Evaluated in both organic and aqueous medium, the activation factor of the encapsulated enzymes in the hydrolysis reaction was generally higher than that in the transesterification reaction. Enhanced catalytic activities were found when 5-20 w/w % of LBC was loaded. The effect of different solvents pretreatment on the activity of immobilized LBC was also investigated. The highest activation factor was found up to 14 for the sample containing acetone-treated LBC/PCL (10 w/w %). The encapsulated lipase reserved 50% of its original activity after the 10th run in the transesterification reaction in hexane medium. The mechanism of activation of lipase catalytic ability based on active PCL nanofiberous matrix is proposed.     

Enhanced activity of α-chymotrypsin in organic media using designed molecular staples

We report the enhancement of α-chymotrypsin activity in organic solvents using modified peptides bearing two crown ethers. The transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol was used as model reaction. Co-lyophilization of crown ether modified peptides with α-chymotrypsin prior to use resulted in an increase of enzyme catalytic activity in non-aqueous media. The efficiency of enzyme activation is dependent on the amino acid sequence of peptidic additives and on the positions of the amino acids bearing the crown ligand.     

Specific anion effects on enzymatic activity in nonaqueous media

The present work shows that salt anions affect the activity of Pseudomonas cepacia lipase both in aqueous and in nonaqueous media (NAM) according to a Hofmeister series. The biocatalytic assay in water was the hydrolysis of p-nitrophenyl acetate, whereas the esterification between 1-hexyl-beta-D-galactopyranoside and palmitic acid was followed in an organic solvent. The solid lipase preparations to be used in NAM were obtained through lyophilization in the presence of concentrated solutions of Hofmeister salts (Na2SO4, NaH2PO4/Na2HPO4, NaCl, NaBr, NaI, NaSCN). Salts affect enzyme activity in organic media through two mechanisms: (1) enzyme protection during lyophilization; (2) enzyme activation during the reaction. At least in our case, the latter seems to be more important than the former. The decrease of the activation energy caused by the stabilization of the transition state due to "kosmotropic" anions might be the driving force of enzyme activation. According to the most recent findings, dispersion forces may be responsible of specific anion enzyme activation/deactivation in NAM.     

Improved catalytic performance of lipase accommodated in the mesoporous silicas with polymer-modified microenvironment

The highly ordered mesoporous silicas with elaborately controlled microenvironment were synthesized via covalent incorporation of long-chain polymers (M(w) = 2000 g mol(-1)) bearing specific hydrophilic/hydrophobic balance. The microenvironment (hydrophilicity/hydrophobicity) of the mesoporous silicas was quantitatively determined by gas adsorption experiments and investigated by lysozyme (LYZ) adsorption. The relative activity of lipase from Pseudomonas cepacia (PCL) encapsulated in the mesoporous silica with moderate hydrophobic microenvironment (hereafter denoted as MHM) reaches up to 281% compared with the free PCL, notably higher than that of PCL accommodated in the mesoporous silicas with hydrophilic or strong hydrophobic microenvironment (20.7-26.2% relative to the free PCL). Moreover, PCL entrapped in the nanochannels with MHM affords the highest initial rate in the kinetic resolution of (R,S)-1-phenylethanol relative to other immobilized PCL. The above results suggest that the MHM could render the active center of PCL entirely exposed to the substrates without interrupting its native conformation in the "interfacial activation". In addition, the nanochannels with MHM could markedly improve the thermal stability of PCL (preserving nearly 60% of the initial activity after the incubation at 70 °C for 2 h) and facilitate the recycling of the immobilized PCL in both aqueous and organic media. Our work demonstrates that the subtle modulation of the microenvironment of mesoporous silicas for enzyme immobilization designates a very promising strategy to fabricate the highly active and stable heterogeneous biocatalysts for industrial application.     

聚β-羟基丁酸酯和聚ε-己内酯的酯交换反应

以辛酸亚锡为催化剂 ,研究了聚 β 羟基丁酸酯 (PHB)与聚ε 己内酯 (PCL)在液相条件下的酯交换反应 .讨论了反应时间 ,反应温度和催化剂浓度对酯交换反应的影响 .采用1 3C NMR ,FTIR ,DSC ,WAXD和TGA等方法对PHB和PCL共聚酯 (PHB co PCL)的结构进行了表征 ,并对其结晶行为、晶体结构和热稳定性进行了研究 .结果表明 ,通过酯交换反应 ,所得到的共聚酯为嵌段共聚物 .提高反应温度和延长反应时间有利于酯交换反应的发生 .随着酯交换量的增加 ,PHB co PCL的结晶行为发生很大的变化 .但是 ,PHB co PCL晶体结构并没有因为PCL链段的引入而发生变化 ,而且它的热稳定性在空气气氛中略有提高     

Enzyme-catalyzed transesterification of vinyl esters on cellulose solids

Enzyme-catalyzed transesterification of several cellulose solids in organic media have been investigated. Several protease enzymes were made soluble in organic media through ion-paired enzyme–surfactant complexes. Subtilisin Carsberg was found to be catalytically active in the transesterification of cellulose with vinyl propionate and vinyl acrylate in anhydrous pyridine. The ester carbonyl groups in acylated cellulose derivatives were confirmed by Fourier transform infrared spectroscopy. The surfaces of these cellulose derivatives became hydrophobic as demonstrated by increased water-contact angles. The enzyme-catalyzed transesterification was confirmed to regioselectively target the primary hydroxyl group of cellulose by reactions on specifically substituted cellulose. The cellulose esters from enzyme-catalyzed transesterification could be hydrolyzed partially by the same enzyme in aqueous media, and were thus biodegradable. Surface grafting of cellulose acrylate was demonstrated using azobisisobutyronitrile-initiated polymerization of acrylonitrile in dimethylformamide. Polyacrylonitrile (PAN)-g-cellulose shows a different thermal behavior from cellulose, homopolymer PAN, and PAN/cellulose blends. The grafted PAN on PAN-g-cellulose at a 16% grafting add-on is incapable of cyclization. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1931–1939, 2001     

高浓度盐系统中指肪酶的固定化及其催化活力

选择了四十多个可溶性的盐，实现了脂肪酶在高浓度盐系统中的固定化，并以 固定化脂肪酶的盐为催化剂，研究了正已烷中脂肪酶催化丁醇和乙酸乙烯酯间的转 酯化制备乙酸丁酯的反应和水溶液中橄榄油的水解反应，考察了高浓度盐系统中脂 肪酶的催化活力。     

Synthesis and characterization of poly(β‐hydroxybutyrate) and poly(ϵ‐caprolactone) copolyester by transesterification

To synthesize the copolyester of poly(β‐hydroxybutyrate) (PHB) and poly(?‐caprolactone) (PCL), the transesterification of PHB and PCL was carried out in the liquid phase with stannous octoate as the catalyzer. The effects of reaction conditions on the transesterification, including catalyzer concentration, reaction temperature, and reaction time, were investigated. The results showed that both rising reaction temperature and increasing reaction time were advantageous to the transesterification. The sequence distribution, thermal behavior, and thermal stability of the copolyesters were investigated by ¹³C NMR, Fourier transform infrared spectroscopy, differential scanning calorimetry, wide‐angle X‐ray diffraction, optical microscopy, and thermogravimetric analysis. The transesterification of PHB and PCL was confirmed to produce the block copolymers. With an increasing PCL content in the copolyesters, the thermal behavior of the copolyesters changed evidently. However, the introduction of PCL segments into PHB chains did not affect its crystalline structure. Moreover, thermal stability of the copolyesters was little improved in air as compared with that of pure PHB. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1893–1903, 2002     

Hydrophobic surface induced activation of Pseudomonas cepacia lipase immobilized into mesoporous silica

Lipase from Pseudomonas cepacia (PCL) was successfully immobilized into siliceous mesocellular foams (MCFs) with various hydrophobic/hydrophilic surfaces. The catalytic performances of immobilized PCL were investigated using the transesterification reaction and hydrolytic reaction as model reactions. The specific activity of immobilized PCL greatly increased with enhanced surface hydrophobicity of MCFs, mainly because of lipase activation via hydrophobic interaction between alkyl groups in MCFs and the surface loop (so-called "lid") of PCL. Conformational changes of immobilized PCL were further investigated using time-resolved fluorescence spectroscopy with Trp as an intrinsic probe. When the immobilized PCL was suspended in phosphate buffer, short-lived τ(1) shortened and the fractional contribution of τ(1) significantly increased with the increasing level of surface hydrophobicity of MCFs. These results revealed that Trp(s) of the immobilized PCL were surrounded by a hydrophilic microenvironment because of the fact that the opened "lid" permitted the diffusion of water to the active site cleft. However, for the immobilized PCL suspended in n-hexane, long-lived τ(3) increased with the increase of surface hydrophobicity of MCFs. The reduced interaction between Trp(s) and the surrounding protein matrix was due to intercalation of n-hexane into the active site cleft when the lipase was in open conformation. The above results demonstrated that PCL immobilized into MCF with hydrophobic surfaces were in an activated open conformation.