Chakraborty Kajal, Paulraj R
Physiology Nutrition and Pathology Division, Central Marine Fisheries Research Institute, Kerala, India.
J Agric Food Chem. 2008 Feb 27;56(4):1428-33. doi: 10.1021/jf073176u. Epub 2008 Feb 1.
Lipase derived from Bacillus licheniformis MTCC 6824 was purified to homogeneity by anion exchange chromatography on Amberlite IRA 410 (Cl-) and gel filtration using Sephadex G-100 as judged by denaturing polyacrylamide gel electrophoresis. The purified lipase was used for hydrolysis of triacylglycerol in sardine oil to enrich Delta5-polyunsaturated fatty acids (Delta5-PUFAs) namely, arachidonic acid (5,8,11,14-eicosatetraenoic acid, ARA, 20:4n-6) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid, EPA, 20:5n-3). The individual fatty acids were determined as fatty acid methyl esters (FAMEs) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAMEs and N-acyl pyrrolidides. The enzyme exhibited hydrolytic resistance toward ester bonds of Delta5-PUFAs as compared to those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 degrees C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued down. Lipase-catalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided free fatty acids containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea complexation proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil.
通过在Amberlite IRA 410（Cl-）上进行阴离子交换色谱和使用Sephadex G-100进行凝胶过滤，将地衣芽孢杆菌MTCC 6824产生的脂肪酶纯化至同质，经变性聚丙烯酰胺凝胶电泳判断。纯化后的脂肪酶用于水解沙丁鱼油中的三酰甘油，以富集Δ5-多不饱和脂肪酸（Δ5-PUFA），即花生四烯酸（5,8,11,14-二十碳四烯酸，ARA，20:4n-6）和二十碳五烯酸（5,8,11,14,17-二十碳五烯酸，EPA，20:5n-3）。通过气液色谱和气相色谱-质谱法将各个脂肪酸测定为脂肪酸甲酯（FAME），作为FAME和N-酰基吡咯烷化物。与其他脂肪酸相比，该酶对Δ5-PUFA的酯键表现出水解抗性，并被证明对提高沙丁鱼油中EPA和ARA的浓度有效。利用这种脂肪酸特异性，通过脂肪酶介导的水解，然后在4℃下进行尿素分级分离，从沙丁鱼油中富集了EPA和ARA。纯化后的脂肪酶在9小时后对饱和脂肪酸和单不饱和脂肪酸产生了最高程度的水解（分别为沙丁鱼油中初始含量的81.5%和72.3%）。脂肪酶催化转化的曲线在6小时前呈稳定上升，此后趋于平稳。脂肪酶催化水解沙丁鱼油，然后与甲醇进行尿素加成，在饱和和不饱和程度较低的脂肪酸络合后，分别提供了含有55.4% EPA和5.8% ARA的游离脂肪酸。酶促水解和尿素络合相结合被证明是一种从沙丁鱼油中获得高浓度EPA和ARA的有前途的方法。
