新型温控离子液体介质中脂肪酶催化合成乙酸苯乙酯

A Novel Temperature-Controlled Ionic Liquid as the Medium for Phenylethyl Acetate Synthesis Catalyzed by Lipase

设计合成了三种同分异构离子液体1,3-二正戊基咪唑六氟磷酸盐([D(n-C5)Im]PF6)、1,3-二异戊基咪唑六氟磷酸盐([D(i-C5)Im]PF6)和1,3-二(2-甲基丁基)咪唑六氟磷酸盐([D(2-mb)Im]PF6).以假单胞菌脂肪酶Pseudomonascepacial催化合成乙酸苯乙酯为模型反应,分别考察了反应介质对酶行为的影响.结果发现,酶在离子液体[D(2-mb)Im]PF6中的活性及反应性能明显高于有机溶剂正己烷.基于[D(2-mb)Im]PF6离子液体的温控特点,提出了一种高温反应与低温分离相结合的乙酸苯乙酯合成路线.通过优化实验,得到合成乙酸苯乙酯的最佳反应条件为:30mg酶,1.0g离子液体,2%含水量,反应温度35oC,反应时间48h.此时,乙酸苯乙酯产率达92.3%.脂肪酶在[D(2-mb)Im]PF6中的稳定性是在正己烷中的7.4倍,且重复使用10次后催化活性没有明显降低.此外,采用圆二色谱和内源荧光光谱法研究了不同介质中脂肪酶结构的变化.结果表明,脂肪酶在[D(2-mb)Im]PF6中有较大的氨基酸残基裸露程度和良好的二级结构稳定性.

Three 1,3-dipentylimidazolium hexafluorophosphate ionic liquid isomers, 1,3-di-n-pentylimidazolium hexafluorophosphate （[D（n-C5）Im]PF6）, 1,3-di-isopentylimidazolium hexafluorophosphate （[D（i-C5）Im]PF6）, and 1,3-di（2-methylbutyl）imidazolium hexafluoro- phosphate （[D（2-mb）Im]PF6）, were designed and synthesized. They were used as media for the synthesis of phenylethyl acetate catalyzed by Pseudomonas cepacial lipase. The activity and reactivity of the lipase in [D（2-mb）Im]PF6 is obviously higher than that in organic solvent hexane. Based on the temperature-controlled characteristics of [D（2-mb）Im]PF6, we designed a new route to synthesize phenylethyl acetate catalyzed by lipase, in which the reaction and the separation of the product were carried out under a relatively high and low temperature, respectively. By investigating the influence of various factors on the yield of product, optimal conditions for the synthesis of phenylethyl acetate were obtained, i.e. the amount of lipase 30 mg, the ionic liquid 1.0 g, water content 2%, reaction temperature 35 oC and reaction time 48 h. Under the optimal conditions, the yield of phenylethyl acetate was up to 92.3%. Also, the stability of the lipase in the [D（2-mb）Im]PF6 medium was 7.4-fold that in hexane. The lipase in [D（2-mb）Im]PF6 was recycled 10 times without substantial diminution in activity. Moreover, circular dichroism and fluorescence spectroscopy were used to follow the changes of enzyme structure in different solvents. The results showed that the lipase in the ionic liquid has excellent secondary structure stability and slightly big exposure level of amino-acid residues.