脂肪酶动力学拆分薄荷醇的研究进展

从脂肪酶拆分薄荷醇的转酯化、水解反应的工艺和影响因素等方面,分析了脂肪酶动力学拆分过程的转酯化和水解反应的催化路径与特点,指出了脂肪酶拆分薄荷醇的最佳路径和工艺选择. 综述了近10年来脂肪酶拆分制备工业重要的手性香料l-薄荷醇的研究进展,特别综述了具有工业化前景的脂肪酶催化拆分和单元操作工艺的研究进展. 同时展望了未来脂肪酶拆分薄荷醇的研究方向.     

基于BP神经网络和GA研究啤酒糟不溶性膳食纤维的酶法脱脂工艺

采用脂肪酶对酶碱法制备啤酒糟不溶性膳食纤维(IDF)的脱脂工艺进行研究,并对制备得到的IDF 成分和功能特性进行分析。在正交试验的基础上,基于BP 神经网络建立脂肪酶脱脂模型,利用遗传算法优化工艺条件。BP 神经网络建立的脱脂模型误差为0.0001,具有较强的逼近能力。优化得到的最佳工艺条件是加酶量0.7g、酶解温度39.6℃、酶解时间5.6h。在此条件下,脂肪去除率达74.1%。制备得到IDF 的溶胀性达6.05mL/g,持水力达318.2%,具有较好的生理活性。     

江米酒脂肪酶对米酒奶脂解风味的影响

利用传统江米酒制备凝乳剂来制作米酒奶、其中江米酒脂肪酶活力为1.0～1.5LU/mL。通过酸度值(ADV)分析发现,脂肪酸的积累主要发生在凝乳期间。采用气相色谱- 质谱联用仪(GC-MS)检测各游离脂肪酸的含量,棕榈酸、油酸、豆蔻酸和硬脂酸占有较高的比例。     

高效产脂肪酶菌株Burkholderia cepacia C1产酶条件优化

从油菜地土壤中分离到一株高效产脂肪酶菌株C1,经鉴定为Burkholderia cepacia。为了进一步提高C1 脂肪酶的产量,对其产酶的发酵条件进行优化。首先采用单因子试验筛选出最佳碳源为麸皮,最佳氮源为蛋白胨。通过Plackett-Burman 设计对发酵产酶的11 个相关因子进行试验,筛选出3 个主效因子,即蛋白胨质量浓度、橄榄油质量浓度及装液量。利用Box-Behnken 试验设计和响应曲面法分析确定主效因子的最优水平,得出产脂肪酶菌株C1 的最优产酶条件：1.50g/100mL 麸皮、1.05g/100mL 蛋白胨、1.63g/100mL 橄榄油、0.2g/100mL K2HPO4、0.05g/100mL MgSO4、初始pH 值为7.0、装液量为30mL。在优化条件下30℃,180r/min 培养72h,脂肪酶活力达到89.65U/mL,比未优化前的28.50U/mL 提高了2.15 倍。     

响应面法优化脂肪酶的固定化条件及其水解橄榄油的特性研究

采用Sepharose CL-4B和7种大孔树脂为载体固定Palatase 20000L脂肪酶,对载体进行筛选,以酶活力为指标,采用响应面法优化固定化条件,并考察所制得固定化酶的稳定性和水解橄榄油的酶学性质。结果表明：以大孔树脂HPD-600为载体制得的固定化酶具有较高的活性和良好的稳定性。其最优的固定化条件为：pH3.9,酶与载体比例为9.1mg/g,吸附时间1.8h。在最优条件下制得固定化酶在最适合条件下测得的酶活力达到1440U/g,酶活回收率大于50%。固定化酶最适作用温度为50℃,最适作用pH值为8.0。固定化酶的Km值为0.130g/mL,高于游离酶的Km(0.069g/mL)。固定化酶的热稳定性有一定程度的提高,其重复操作5次后相对酶活力仍保持在58%以上。     

Integrated synthesis and extraction of short‐chain fatty acid esters by supercritical carbon dioxide

We developed an efficient, integrated reaction‐extraction process for the production of short‐chain fatty acid ethyl esters (FAEE) from milk fat, using carbon dioxide as the only processing solvent. FAEE were synthesized using a short‐chain fatty acid selective lipase. The expansion of the liquid mixture of reactants by dense carbon dioxide enhanced the apparent lipase selectivity. In situ extraction of FAEE by a continuous flow of supercritical carbon dioxide proved to increase the lipase production rate. When the integrated process was operated with alternated periods of synthesis and product removal, the overall selectivity for short‐chain FAEE increased as well, as a result of the combination of the selectivities of lipase and extraction solvent. A two‐fold increase of the lipase productivity was achieved at these conditions, compared to a single batch reaction. The developed process enables the synthesis and isolation of high‐value fatty acid derivatives from a natural source such as milk fat. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Investigation of enzymatic biodiesel production using ionic liquid as a co‐solvent

In this study, the lipase catalysed esterification reaction for biodiesel production was investigated in the presence of the ionic liquid [BMIM][PF₆]. Unlike regular organic solvents, many ionic liquids have no vapour pressure, and are therefore considered non‐volatile. When used in systems with enzyme catalysts, ionic liquids may enhance their activity, selectivity, and stability. The use of an enzyme (lipase) as a catalyst, and the ionic liquid as a solvent/immobilization agent also represents an environmentally friendly, “green” technology. Methyl acetate was used as the acyl acceptor as opposed to the more commonly used methanol due to the negative effects methanol and the glycerol by‐product has on lipase enzyme activity. The results of this research indicate that methyl oleate (i.e., biodiesel) was successfully produced, with an 80% overall biodiesel yield in the presence of ionic liquid, at a 1:1 ratio (v/v) to the amount of oil. This verified that the presence of an ionic liquid, at a specified amount, improved the activity of the lipase and the overall biodiesel yield. Results also indicate the addition of ionic liquid facilitated the separation of the methyl esters from the triacetylglycerol by‐product. The best conditions investigated was found to be: 14:1 molar ratio between oil and acyl acceptor; 20% (w immobilised lipase/w of oil; and a temperature in the range of 48–55°C. However, additional purification is required in order for the produced biodiesel to meet ASTM standards.     

理性设计提高华根霉脂肪酶对不饱和长链脂肪酸的底物特异性及其在大豆油水解中的应用

为了提高华根霉脂肪酶在大豆油水解制备脂肪酸中的应用效果,通过理性设计的方法改造脂肪酶的脂肪酸特异性。对突变酶的酶学性质进行了研究,并通过单因素实验优化脂肪酶催化水解大豆油的反应条件。结果表明:利用定点突变技术获得了一系列脂肪酸特异性改变的脂肪酶,其中突变酶HQL增强了对不饱和长链脂肪酸的特异性,并且对p-NPP(C16)的水解活力相比野生型提高了2.72倍;所有突变酶的最适温度和最适p H均为40℃和8.0,与野生型脂肪酶一致;得到脂肪酶催化水解大豆油的最适反应条件为反应时间24 h、水油质量比1∶1、加酶量500 U/g(以油质量计)、p H 8.0、温度40℃,在最适反应条件下,野生型酶催化大豆油水解率仅为80%,而突变酶HQL由于增强了对长链不饱和脂肪酸的底物特异性,对大豆油水解率提高到98%。     

皱褶假丝酵母脂肪酶CRL1在毕赤酵母中的高效表达及其催化合成维生素E醋酸酯

本研究将经密码子优化的皱褶假丝酵母脂肪酶(Candida rugosa lipase) CRL1基因克隆到毕赤酵母分泌型表达载体pHKA,构建得到重组菌株GS115/pHKA-CRL1;经摇瓶发酵并结合甲醇诱导120 h处理,该菌株对底物对硝基苯酚丁酯(p NPB)的水解活力达到39.73 U/mL。通过对AOX1启动子和α分泌信号肽同时进行改造,进一步获得重组菌株GS115/pHKA-AOX1m/αm-CRL1,其脂肪酶活力提高达到63.63 U/mL。发酵液上清液经超滤浓缩、硫酸铵沉淀后,再经脱盐和阴离子交换层析处理,获得纯化的重组CRL1。该纯化工艺的纯化倍数为5.41倍,回收率为33.81%。而纯化重组CRL1的比酶活为984.5U/mg。重组CRL1的最适温度为40℃,最适pH为7.5;经40℃保温6 h,仍保留52.99%的相对活力;在偏酸性环境中较为稳定。以摇瓶发酵、真空冷冻干燥后的重组CRL1为催化剂,在无溶剂体系中催化合成维生素E醋酸酯,在最适反应条件下(200 mg D-α-生育酚、1 mL乙酸酐、100 mg CRL1、反应温度60℃、转速200 r/min、反应时间9 h),D-α-生育酚的转化率在97.00%以上。