Enantioselective esterification of ibuprofen with ethanol as reactant and solvent catalyzed by immobilized lipase: experimental and molecular modeling aspects

BACKGROUND: In recent years enantioselective esterification of racemic ibuprofen performed in organic co‐solvent media such as isooctane and cyclohexane and catalyzed by lipases, has been proposed as an effective way to increase the concentration of S‐ibuprofen in the racemic mixture. In this contribution, the enantioselective enzymatic esterification of (R,S)‐ibuprofen with ethanol catalyzed by commercial Novozym 435 without the addition of a co‐solvent is thoroughly investigated. Experimental data are further analyzed considering the results of extensive molecular modeling calculations. RESULTS: The conversion of ibuprofen towards the ethyl esters and the enantiomeric excess towards S‐ibuprofen are greatly affected by the ethanol and water contents of the reaction media. The optimum conditions for the esterification of racemic ibuprofen in a batch‐type reactor were as follows: molar ratio of ethanol to ibuprofen = 7, 4.8% v/v of water, 160 mg of Novozym 435, 45 °C and 200 rpm. Under these conditions an enantiomeric excess of 54% and 63% of ibuprofen conversion were reached. CONCLUSIONS: Results showed that the reaction in excess of the esterifying alcohol in a system free of additional organic solvents is possible if the proper conditions are set. Molecular modeling calculations demonstrated that the formation of dead‐end compounds between the enzyme and ethanol/water may account for lipase inhibition at high concentrations of those compounds. Copyright © 2009 Society of Chemical Industry     

高选择性脂肪酶的筛选及其拆分布洛芬

为了实现脂肪酶在有机相中对外消旋布洛芬的高效拆分,筛选得到了一株具有高立体选择性的脂肪酶产生菌-扩展青霉TS414(Penicillium expansum TS414). 实验探讨了水分、温度、有机溶剂、酶浓度、醇结构和醇浓度对酶促拆分反应的影响,确立了酯化反应的最佳反应体系：布洛芬6.46 mmol/L,脂肪酶53.3 mg/mL,正丙醇40 mmol/L,异辛烷15 mL,水0.5%(j),60℃条件下反应50 h. 在此条件下,拆分反应的转化率和对映体选择率分别为42%和429.63. 结果表明,Penicillium expansum TS414脂肪酶是一种较为理想的用于外消旋布洛芬拆分的工具酶,在布洛芬手性拆分方面具有广阔的应用前景.     

酶促酯化拆分-化学消旋串联法制备S-布洛芬

以消旋布洛芬为反应底物，探究了酶促酯化拆分布洛芬的工艺。利用南极假丝酵母脂肪酶B的对映体选择性催化布洛芬选择性酯化，并对醇的种类、反应介质、底物摩尔比、酶量、干燥剂种类和添加量、温度及时间等反应条件进行了优化，以56%的收率得到了光学纯的S-布洛芬，接下来对酯化产物进行化学法消旋。以二甲亚砜为反应介质，利用二异丙基氨基锂催化布洛芬发生外消旋反应，完全消旋后再进行酶促拆分仍能达到之前的效果。如此循环7次布洛芬的利用率可达到90%以上。     

Penicillium expansum脂肪酶选择性拆分外消旋萘普生

为了实现固定化扩展青霉TS414(Penicillium expansum TS414)脂肪酶在有机相中对外消旋萘普生的高效拆分,实验考察了水分、温度、有机溶剂、酶浓度、醇结构和醇浓度对酶促拆分反应的影响,确立了优化的酯化反应条件为：异辛烷为溶剂,外消旋萘普生2.15 mmol/L,正丙醇34.3 mmol/L,固定化酶量12 g/L,水0.05%(j), 40℃恒温摇床中200 r/min反应100 h. 在此条件下,酯化拆分反应的转化率为48.3%. 结果表明,固定化Penicillium expansum脂肪酶是一种较为理想的用于外消旋萘普生拆分的工具酶.     

Lipase-catalyzed esterification of oleic acid and methanol in hexane—A kinetic study

The kinetics of immobilized lipase-catalyzed esterification of oleic acid and methanol in hexane were investigated. The reaction follows Michaelis-Menton kinetics as observed from the relationship of initial rate of the reaction, both as a function of enzyme and of substrate concentration. Inhibition by excess of methanol has been identified. The kinetic constants have been measured for the reaction in the absence of any significant external diffusional limitations. The kinetics of the enzymatic reaction are suggested to agree with a Ping-Pong Bi Bi mechanism.     

表面活性剂包衣Candida rugosa脂肪酶在异辛烷中的稳定性

The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction.At 30℃,the half-life of the activity of the coated lipase was ca 10h,the enzyme activity became less changed after 12h and the residual activity was 39% of the initial value ,The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9 J.mol^-1.     

一种选择性拆分布洛芬对映体的固定化脂肪酶

为了提高扩展青霉TS414脂肪酶(PEL)对布洛芬的拆分效率,建立了适于非水相中选择性拆分(R,S)-布洛芬的固定化方法. 结果表明,固定化介质的类型、冻干pH和外加水量等因素对固定化PEL酶促拆分(R,S)-布洛芬有较大影响. 在冻干pH为9.0、外加水量为0、以大孔吸附树脂AB-8为固定化载体的体系中,40℃反应30 h后,拆分反应的转化率可达47%,对映体过量值eeP可达98.75%. AB-8固定化后,PEL在有机相反应体系中的分散性得到了明显改善,大幅度提高了酶促拆分反应的效率;大孔吸附树脂AB-8固定化PEL具有较高的操作稳定性,连续10批拆分反应的平均转化率在47%以上,eeP值均稳定在98%以上.     

Enzymatic synthesis of wax esters from rapeseed fatty acid methyl esters and a fatty alcohol

Wax esters were transesterified from fatty acid methyl esters of rapeseed and a fatty alcohol (1-hexadecanol, 16:0). The amounts of both the substrates were fixed to 0.1 mmol and an immobilized enzyme, Lipozyme, was used as catalyst. The experiment was performed following a statistic central composite design with five variables. The enzyme/lipid ratio was varied between 0.3–0.9 of the substrate weight and the enzyme was equilibrated to different water activities varying from 0.11 to 0.44. A temperature range of 50–80°C was investigated and the reaction time lasted up to 40 min. A solvent, isooctane, constituted 0–30% of the substrate weight. The first experimental series was performed in small closed test tubes. In the second series the caps of the test tubes were off to evaporate the methanol produced during the reaction. The highest initial reaction rate was 9.6 g_{wax esters}/g_enzyme · h. It appeared when: the enzyme/lipid ratio was low, 0.3, the temperature was high, 80°C; no isooctane was present; and the water activity was below 0.11. The initial reaction rate was independent of the caps on the test tubes. With the large amount of enzyme the yield of wax esters was above 70% after 10 min in both experimental series. In the reaction with caps, the reaction reached equilibrium at 83% after 20 min at 80°C. However, without caps the continuous evaporation of methanol increased the equilibrium constantly, and after 40 min at 80°C a yield of 90% was reached.     

Enhancement of (S)-naproxen ester productivity from racemic naproxen by lipase in organic solvents

The kinetics of enantioselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase (triacylglycerol ester hydrolases, EC 3.1.1.3) were examined in various organic mixtures. The effects of solvent hydrophobicity on the activity, selectivity and stability of the enzyme and Naproxen solubility were investigated. Parabolic correlation for the dependence of the kinetic constants and Naproxen solubility on solvent hydrophobicity was found. A mixture of 60% isooctane and 40% toluene (v/v) was selected as the best reaction medium in which improvement of (S)-Naproxen ester productivity was obtained.     

Effect of light irradiation on esterification of oleic acid with ethanol catalyzed by immobilized Pseudomonas cepacia lipase

The present study demonstrates the effect of light irradiation on the esterification of oleic acid catalyzed by immobilized Pseudomonas cepacia lipase. The reaction rates of all the experiments under light irradiation were found to be higher than dark conditions. The kinetics of reactions supported the Ping‐Pong Bi‐Bi mechanism with dead end inhibition by both the alcohol and acid substrates. Moreover, circular dichroism (CD) spectroscopy was used to analyze the effect of light on lipase enzyme. The CD spectroscopic studies confirmed that the conformational changes in the secondary structure of the lipase enzyme increased the reaction rate of light‐illuminated experiments, which might have opened up the active sites of enzymes and thus, resulted in higher reaction rates compared to dark reactions. These results have successfully demonstrated that the light illumination positively influenced the rate of P. cepacia enzyme‐catalyzed esterification reactions.     

非水相体系中枯草杆菌蛋白酶-表面活性剂离子对的形成条件

枯草杆菌蛋白酶在水相pH值小于其等电点条件下带正电,能与异辛烷中带负电的阴离子表面活性剂二辛基丁二酸磺酸钠（AOT）通过静电引力结合形成离子对进入有机相。详细研究了两相接触方式、水相初始pH值、水相离子强度等因素对酶转移率的影响。采用120 r/min磁力搅拌使两相混合能得到较好的酶转移率,作用时间为6 h。水相初始pH值5.0有利于离子对的形成及酶蛋白的转移。水相中CaCl2的加入有利于消除两相混合过程中的乳化;但是过高的CaCl2浓度会减弱酶分子与AOT间的静电引力,不利于酶分子与AOT的结合。水相中酶浓度不变,随着有机相中AOT初始浓度的增加,酶的转移率呈现增长趋势。固定异辛烷中AOT浓度,随着水相中初始酶浓度的增加,转移至有机相中酶量先增加后减少。进入异辛烷的酶分子仍旧保持活性,能催化转酯化反应的进行,但随着反应时间的延长,酶的催化效率出现下降。     

Esterification kinetics of long-chain fatty acids and fatty alcohols with a surfactant-coated lipase in n-hexane

The esterification reaction kinetics of long-chain fatty acids and fatty alcohols catalyzed with a surfactant-coated lipase in a microaqueous n-hexane system were studied. The biocatalytic complex, surfactant-lipase adduct, showed 40 times the activity after a reaction time of 5 h compared to the unmodified lipase in the same reaction system. Various factors that may affect the activity of the modified lipase were studied, such as the influence of substrate fatty acid chainlength, water content, and temperature. By varying the concentration of each of the two substrates while keeping that of the other substrate constant, it was found that the esterification reaction follows Michaelis-Menten kinetics. The surfactant-enzyme complex kinetic parameters were determined with respect to both substrates. It was suggested that the kinetics of the lipase-catalyzed esterification reaction model follow a Ping-Pong Bi Bi mechanism with no substrate or product inhibition.     

Enzymatic synthesis of phosphatidylcholine with fatty acids,isooctane, carbon dioxide,and propane as solvents

Phosphatidylcholine (PC) was synthesized from lyso-PC and long polyunsaturated fatty acids (PUFA) with phospholipase A₂. In previous investigations, performed in small glass tubes, the enzymatic synthesis reaction was optimized. This paper presents results from experiments performed in a high-pressure reactor filled with an immobilized enzyme (Im.E.). Fatty acids were used as the main solvent while isooctane, CO₂, or propane was used as an additional solvent. The water content was carefully controlled over wide ranges. The temperature was kept constant at 45°C for up to 50 h. The highest initial reaction rate was attained with pure fatty acids under relatively humid conditions (water=35% of dry Im.E.). The reaction rates were more than three times as high in the high-pressure reactor than in previous experiments in glass tubes. In all solvent systems, the best yield was attained after long times under dry conditions (water <15% of dry Im.E.). Addition of CO₂ to the PUFA reduced the yield, while addition of isooctane or propane increased the yield. After 20 h at 45°C, the best yield (25%) was attained at a solvent composition of 91% PUFA and 9% propane.     

表面活性剂包衣脂肪酶在有机溶剂中催化酯合成

用合成的谷氨酸二烷基酯核糖醇对来源于Candidarugosa的脂肪酶进行了包衣 ,以月桂酸与月桂醇的酯化为模型反应 ,研究了各种操作条件对包衣酶活性的影响。结果表明 ,包衣酶制备过程中的缓冲溶液的最适 pH为 6.8,谷氨酸双十二烷基酯核糖醇的包衣效果最好 ,最适反应温度为 3 0℃ ,最佳溶剂为异辛烷。在 10h内底物转化率达 94%。     

溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

Lipase-catalyzed synthesis of lysophosphatidylcholine using organic cosolvent for in situ water activity control

Lysophosphatidylcholines (LPC) were synthesized from l-α-glycerophosphatidylcholine (GPC) by lipase-catalyzed esterification in a solvent-free system. Adding small amounts of a water-mimicking solvent such as dimethylformamide (DMF) to the reaction media significantly enhanced the reaction rate and the product yield. The role of solvent was studied with regard to changes in substrate solubility, the water activity of the reaction system, and the water content of the enzyme. Whereas the solubility of GPC was virtually unaffected by the addition of DMF at controlled water activity, it was considerably affected by water activity. DMF itself lowered the water activity of the system and deprived Lipozyme IM of water. The LPC production was also controlled by varying the initial water content of the enzyme. When two kinds of controls were employed together, a synergistic effect was observed and a 90% conversion was achieved. As a result, an operating window was suggested for LPC production, including water activity of Lipozyme IM and concentration of DMF as two parameters.     

Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion‐paired complex with AOT

Candida rugosa lipase was extracted from aqueous solutions into organic solvents by forming an ion‐paired complex with sodium bis(2‐ethylhexyl)sulfosuccinate (AOT). The optimal aqueous pH for lipase recovery was 4.5 and the optimal CaCl₂ concentration was 10 mmol dm^?3. The lipase recovery decreased with increasing aqueous enzyme concentration but increased with increasing AOT concentration in the organic phase. The presence of polar co‐solvents in the aqueous phase did not obviously improve the lipase recovery, which was also little influenced by the type of hydrophobic organic solvent used for solubilising AOT. Surprisingly, no detectable activity of the ion‐paired C. rugosa lipase was observed for both the esterification of lauric acid with 1‐propanol in isooctane and the hydrolysis of olive oil in isooctane containing an appropriate amount of water. The ion‐paired C. rugosa lipase mediated the enantioselective crystallisation of racemic ketoprofen in isooctane, indicating the feasibility of using it as a chiral mediator for the enantioseparation of hydrophobic racemic compounds in organic systems. Copyright © 2006 Society of Chemical Industry     

Lipase ofGeotrichum candidum immobilized on silica gel

Silica gel is a useful support for the lipases ofGeotrichum candidum. Esterification of selected fatty acids and alcohols proceeded to 85–92% conversion in hydrocarbon solvents, and the degree of esterification was increased to 96–98% by adding 4Å molecular sieves at later stages of reaction. The equilibrium ratio of ester to fatty acid (butyl oleate to oleic) was determined for the supported lipase in a number of solvents and ranged from 92∶8 in hexane and isooctane to 16∶84 int-butanol. The essential character of the enzyme seemed unimpaired by deposition onto silica gel as judged by fatty acid selectivity and stereoselectivity.     

Fast synthesis of 1,3‐DAG by Lecitase® Ultra‐catalyzed esterification in solvent‐free system

Lecitase® Ultra, a phospholipase, was explored as an effective biocatalyst for direct esterification of glycerol with oleic acid to produce 1,3‐DAG. Experiments were carried out in batch mode, and optimal reaction conditions were evaluated. In comparison with several organic solvent mediums, the solvent‐free system was found to be more beneficial for this esterification reaction, which was further studied to investigate the reaction conditions including oleic acid/glycerol mole ratio, temperature, initial water content, enzyme load, and operating time. The results showed that Lecitase® Ultra catalyzed a fast synthesis of 1,3‐DAG by direct esterification in a solvent‐free medium. Under the optimal reaction conditions, a short reaction time 1.5 h was found to achieve the fatty acid esterification efficiency of 80.3 ± 1.2% and 1,3‐DAG content of 54.8 ± 1.6 wt% (lipid layer of reaction mixture mass). The reusability of Lecitase® Ultra was evaluated via recycling the excess glycerol layer in the reaction system. DAG in the upper lipid layer of reaction mixture was purified by molecular distillation and the 1,3‐DAG‐enriched oil with a purity of about 75 wt% was obtained. Practical applications: The new Lecitase® Ultra catalyzed process for production of 1,3‐DAG from glycerol and oleic acid described in this study provides several advantages over conventional methods including short reaction time, the absence of a solvents and a high product yield.     

硅铁合金催化臭氧氧化水中布洛芬

近几年,在各地的饮用水中不断检测到微量布洛芬,其环境毒性引起了广泛关注。选用工业硅铁作为催化剂,催化臭氧氧化去除水中的布洛芬,并通过单因素试验,确定了反应体系的最佳条件。结果表明,在硅铁的作用下,催化臭氧氧化可以明显提高布洛芬的去除率。在硅铁投加量为1 g/L、水溶液初始pH值为8、臭氧浓度9.0 mg/L、布洛芬初始浓度为10 mg/L时,经过80 min,水样总有机碳(TOC)的去除率可达75.5%,较单独臭氧氧化提高了38.0%。将碳酸氢钠作为自由基抑制剂加入反应体系,可明显降低TOC的去除率,间接证明了催化臭氧氧化布洛芬的反应遵循自由基机理。