【作者】 任昌琼；

【导师】 郑穗平；

【作者基本信息】 华南理工大学 ， 生物制药， 2010， 硕士

【摘要】 糖酯是一类重要的非离子型表面活性剂,具有无毒、无刺激和可生物降解等优点,在食品、化妆品和医药等领域有着广泛的用途和良好的发展前景。与化学法合成糖酯相比,酶法合成糖酯具有反应条件温和、选择性强、副产物少以及产品易于纯化等优点。基于酵母表面展示技术得到的表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B, CALB)的毕赤酵母全细胞催化剂,具有固定化酶的特性及优点,且制备方法简单,易于回收与再生利用,用于在有机相中合成表面活性剂(糖酯),克服了游离酶在分离纯化、再生以及循环利用等方面的困难,且与目前商品化的固定化脂肪酶相比,其制备方法简单、成本较低、可重复利用,使酶法合成表面活性剂前景广阔。本文利用本实验室自制表面展示南极假丝酵母脂肪酶B的全细胞为催化剂,以葡萄糖和果糖为酰基受体,以月桂酸为酰基供体合成糖酯。首先建立了糖酯定性定量分析方法,用薄层色谱进行糖酯定性。薄层色谱最佳条件为:甲苯/乙酸乙酯/甲醇/水(10: 5: 4.5: 0.2,v/v/v/v)。将薄层色谱条件运用于硅胶柱层析,对产物进行初步分离,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱(HPLC-MS),核磁共振(NMR)技术鉴定纯品结构。此外,建立了利用高效液相色谱-蒸发光散射检测器(HPLC-ELSD)对糖酯进行定量分析的方法,色谱条件为:甲醇/乙酸乙酯/水(52: 35: 13, v/v/v),漂移管温度55℃,氮气流速30 psi。本研究对全细胞催化剂催化合成两种单糖酯的反应体系进行了优化。在以葡萄糖为酰基受体的反应体系中,考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、全细胞催化剂添加量、底物浓度、水活度和温度等几个影响酯化反应的因素。结果表明:在5 mL反应体系中,以叔戊醇/二甲基亚砜(30% DMSO, v/v)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5 g,葡萄糖0.5 g,月桂酸1.0 g,60℃下反应72 h后,葡萄糖月桂酸单酯的产率可达到48.7%。在以果糖为酰基受体的反应体系中,考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、全细胞催化剂添加量、底物浓度、底物摩尔比、温度、水活度和分子筛等因素对全细胞催化剂催化合成果糖月桂酸单酯和果糖月桂酸二酯的影响。在单因素优化的基础上,运用响应面分析方法对酯化反应过程进行了优化,考察了温度、底物摩尔比、全细胞催化剂添加量之间的交互作用对全细胞催化剂催化合成果糖酯的影响。结果表明:在5 mL应体系中,以叔戊醇/二甲基亚砜(20% DMSO, v/v)为反应介质,添加初始水活度为0.11的全细胞催化剂0.3 g,果糖0.36 g,月桂酸1.08 g,56℃下反应72 h后,果糖月桂酸单酯的产率达到82.5%。为了实现全细胞催化剂高效率的循环使用,考察了全细胞催化剂催化合成果糖月桂酸酯的操作稳定性。全细胞催化剂重复利用15批次(24 h/批次)后,果糖月桂酸单酯的相对产率仍可达最高反应批次的77%。此外,该全细胞还具有良好的有机溶剂稳定性,从而为糖酯的进一步开发应用提供了可能性。更多还原

【Abstract】 Sugar fatty acid esters are significant nonionic surfactant which produced from renewable and inexpensive substance. Because they are completely biodegradable under aerobic and anaerobic conditions, non-toxic, no-skin irritants, odourless and tasteless, so widely used as emulsifler in food cosmetic and pharmaceutical industries. Sugar esters can be porduced by enzymatic or chemical methods. However, chemical synthesis of sugar fatty acid esters is generally performed as a high temperature esterification in the persence of alkaline catalyst, which is accompanied by high energe consumption, browning of products and low selectivity toward the various hydroxyl groups in sugars. Comparing with the chemical method,enzymatic synthesis can be performed under milder reaction conditions with a higher regioselectivity.Candida antarctica Lipase B (CALB) has a good potential in industrial application, with a wide range of monosaccaride fatty acid esters often as single regiosomers. The free lipase has the drawbakcs of purification and recycling, while the high cost of commercial immobilized CALB limits its industrial application. Compared with immobilized enzymes, enzyme- displaying yeast whole-cell biocatalyst seems to be an alternative due to its simplicity, high enzymatic activity and cost-effective as well. Lipase-displaying yeast whole-cell biocatalyst has attracted more and more attentions since it can be used for the synthesis of useful product, such as bio-surfactant.In this paper, the recombinant CALB displayed on the surface of Pichia pastoris was constructed and used as a whole-cell biocatalyst to catalyze the esterification of sugers and fatty acids. Glucose and fructose as the acyl acceptors and lauric acid as the acyl donor. The thin layer chromatogaphy (TLC) to qualitatively analyze fatty acid ester was developed. The optimal conditions of TLC were: toluene-ethyl acetate-methanol-water (10:5:4.5:0.2, v/v/v/v) as the mobile phase, coloration by sprayingα-naphthol-sulfuric acid. Products was isolated by silica gel column chromatography at first, using the same condition as TLC, and then by preparative liquid chromatography, sugar fatty acids were identified by Liquid Chromatography-Mass Spectrometry and Nuclear Magnetic Resonance.The influential factors on whole-cell synthesis of glucose laurate monoester were studied, such as solvent composition, percentage of Dmethyl sulfoxide in 2-Methyl-2-butanol (v/v), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: 5 mL total reaction volume, 2-Methyl-2-butanol/Dmethyl sulfoxide (30% Dmethyl sulfoxide, v/v), 0.5 g glucose, 1.0 g lauric acid, 0.5 g whole-cell biocatalysts, the best initial water activity of whole-cell biocatalyst was 0.11, temperature 60°C, 200r/min. The maximum yield could be 48.7% after 72 h.In the synthesis of fructose laurate ester catalyzed by whole-cell catalyst, reaction conditions such as solvent composition, biocatalyst concentration, substrate concentration, water activity, amount of molecular sieves and temperature, were also studied. On the basis of single factor optimization, Response Surface Methodology was used to analyse the interaction of fators: catalyst dosage, substrate concentration and temperature. The highest conversion was obtained in 2-Methyl-2-butanol/Dmethyl sulfoxide, Dmethyl sulfoxide percentage 20% (v/v) in 5 mL total reaction volume, fructose 0.36 g, lauric acid 1.08 g, 0.3 g whole-cell biocatalysts, the best initial water activity of whole-cells biocatalyst was 0.11, temperature 56°C, 200 r/min, and 82.5% of the maximum yield achieved after 72 h.To further examine the potential of the whole-cell biocatalyst for sugar esters synthesis, the operational stability of the whole-cell biocatalyst in organic solvents was investigated. The results indicated that the whole-cell catalyst owned good operation stability, after being reused for 15 batches (24 h/batch), the residual activity remained above 77%, compared with the best batch.更多还原