摘要
1-苯乙醇是一种重要的有机合成原料,有两个对映体(R,S-1-苯乙醇)。本文的主要研究内容是利用固定化脂肪酶拆分1-苯乙醇及其动力学研究。
本论文对交联法制备固定化酶的方法和条件进行了初步探索。利用氧化多壁碳纳米管(MWNT)为固定化载体,以N-羟基琥珀酰亚胺(NHS)和1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDAC)为交联剂,在pH 6.2的吗啉乙磺酸(MES)缓冲液中,将Candida.sp99-125脂肪酶(CSL)成功交联在多壁碳纳米管上,通过元素分析测定固定化酶的最大负载量为1.8mg CSL/mg MWNT,根据橄榄油乳化液水解滴定法测定固定化酶相对酶活收率达63%,由圆二色性光谱和傅里叶红外光谱测定固定化酶相对二级结构保持率为61.3%。
实验以正庚烷为溶剂,乙酸乙烯酯为酰基供体,在有机相中α-苯乙醇与乙酸乙烯酯在固定化脂肪酶(MWNT-CSL)的催化下发生转酯化反应,利用固定化脂肪酶的高度对映体选择性,R-1-苯乙醇发生转酯化生成R-1-乙酸苯乙酯,剩余未反应的底物为S-1-苯乙醇,反应混合物可以比较容易的进行分离,从而实现拆分1-苯乙醇的目的。
实验中利用带手性柱的液相色谱仪Shimadzu 10AVP来定量分析反应过程底物及产物的浓度变化,并计算各自的对映体过剩量e.e.s和底物转化率C。
本论文确立了以正庚烷为溶剂,乙酸乙烯酯为酰基载体的酶催化转酯化反应体系。考察了不同的反应温度、酰基载体与底物摩尔比对反应的影响,并对该酶的反应条件进行优化,最终确定了反应时间。结果表明该酶在底物浓度50mmol/L、酰基载体与底物摩尔比为6:1、反应温度为50℃、摇床转速为100rpm的条件下,反应时间为56h,转化率达到49.8%,e.e.s达到97.4%。
同时,我们还考察了该酶的重复使用效率。当固定化脂肪酶反复使用五次,反应56h时,底物的转化率均大于44%,只是底物的e.e.值有所降低,但产物的e.e.值没有下降,这就说明该酶的对映体选择性并没有受到不利影响。
1-Phenyl ethanol was one of the important chiral building blocks for organic synthesis.The objective of this work is to study the resolution of(R, S)-1-Phenyl ethanol by lipase immobilized on multi-walled carbon nanotubes.
Lipase was covalently attached onto multi-walled nano tubes(MWNTs) by using N-hydroxysuccinimide(NHS) and N-ethyl-N-(3-(dimethylamino) propyl) carbodiimide hydrochloride(EDC) as crosslinking agent in MES [2-(N-morpholino) ethanesulfonic acid]buffer.The high support surface area of MWNTs facilitated high enzyme loadings(up to 1.8 mg of CSL/mg of MWNT) as determined by elemental analysis.63%of hydrolytic activity was retained by the conjugate of MWNT with lipase.And the secondary structure of the conjugate was anlysed through FTIR Spectroscopy and Circular dichroism(CD) spectra.
In the resolution,n-heptane was used as solvent and vinyl acetate as acyl donor.And lipase and lipase-MWNT were used as catalyst.The productα-phenylethyl acetate was approximately 100%enantiomeric pureα-Phenyl ethanol was transesterified with vinyl acetate.
The concentrations of different enantiomer of substrate and product were analyzed by high performance liquid chromatography(HPLC) with chiral column,from which the enantiomeric excess of substrate and product was calculated.
Optimum conditions of transesterification of immobilized lipase were investigated,including the influence of temperature,molar ratio of vinyl acetate to(R,S) - 1-Phenyl ethanol and reaction time.It was found that the optimal temperature and molar ratio were 50℃and 6:1,respectively.At the optimized reaction conditions,when the reaction time was about 56h, conversion was 49.8%and enantiomeric excess was 97.4%.
The reuse of lipase-MWNT was studied.The results show that when reaction time reached 56h,conversion of substrate was higher than 44%,but the enantiomeric excess of R-phenylethyl acetate remained unvaried.
引文
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