sn-2位DHA结构甘油三酯的酶法不可逆合成及sn-2单甘酯酰基转移规律研究

Structured triacylglycerols with docosahexaenoic acid (DHA) at the sn-2 position are widely applied in food industry. In the previous studies, chemical and chemoenzymatic methods have been established for the synthesis of symmetrically structured triacylglycerols. Enzymatic synthesis is difficult due to acyl migration of 2-monoacylglycerols (2-MAGs) during the reaction. In this study, an algal oil rich in DHA will be used as starting material to obtain DHA 2-MAG by enzymatic ethanolysis at room temperature. Subsequently, enzymatic transesterification between DHA 2-MAG and capric acid vinyl ester is conducted to synthesize highly pure 1,3-dicaproyl-2-docosahexaenoylglycerol (CDC). A general method for the enzymatic synthesis of symmetrically structured triacylglycerols is studied using CDC synthesis as a model. To inhibit the acyl migration of 2-MAGs during the reaction, acyl migration kinetics of 2-MAG are studied under the different reaction conditions. Mechanism of 2-MAG acyl migration will be revealed by establishing the kinetic differential equations. The understanding on the kinetics of 2-MAG acyl migration will provide a theoretical support for the enzymatic synthesis of structured triacylglycerols with high purity in food industry. Compared to the previous methods for the synthesis of highly pure triacylglycerols, the reaction using vinyl ester as acyl donor has a lower reaction temperature, is irreversible and more effective. The establishment of enzymatic method for the synthesis of symmetrical triacylglycerols is very important for oil industry producing value-added structured glycerides.

二十二碳六烯酸（DHA）在sn-2位的对称型甘油三酯在食品工业中有广泛的应用，已有工作解决了高纯度对称型甘油三酯的化学和化学酶法合成，酶法合成因无法实现2-单甘酯酰基转移的控制而少有报道。本项目以DHA藻油为原料，通过酶法醇解制备sn-2 DHA单甘酯，制备的2-单甘酯与癸酸乙烯酯进行酶法转酯化合成DHA在sn-2位，癸酸在sn-1,3位的结构甘油三酯CDC，以CDC合成为模型，建立对称型甘油三酯酶促合成一般性方法。为实现2-单甘酯酰基转移的控制，随后研究各反应条件影响下的2-单甘酯酰基转移动力学，以揭示2-单甘酯酰基转移发生规律。2-单甘酯酰基转移规律的揭示为实现高纯度甘油三酯的酶法合成提供理论支撑。与已有的结构甘油三酯合成相比，以脂肪酸乙烯酯为酰基供体的转酯化反应具有反应温度更低、反应不可逆、更绿色的特点。对称型甘油三酯酶促合成方法的突破对油脂工业生产高功能性结构酯产品具有重要意义。

DHA或者ARA在sn-2位的对称型结构脂具有一些列重要的生理活性。目前富含DHA的对称型结构脂一般采用化学法合成，酶法合成存在sn-2 DHA单甘酯（2-MAG）酰基转移的问题，导致结构脂纯度和得率低等问题。本项目以富含DHA或者ARA的天然油脂为底物，酶法醇解制备2-单甘酯，随后以2-单甘酯和脂肪酸乙烯酯为底物，酶法不可逆制备对称型结构脂，在酶法酯交换过程中，研究反应温度和溶剂对2-单甘酯酰基转移的影响并探究其酰基转移动力学，其具体内容如下：. 采用酶法醇解天然油脂制备富含DHA的2-MAG，探究反应条件的影响。结果表明，在油脂与乙醇摩尔比为1:60、2h、30℃、Lipozyme 435添加量为8%的条件下，金枪鱼油中2-MAG含量在22.1%-27.4%、藻油中2-MAG含量在26.50%-30.95%。经正己烷和85%乙醇-水萃取后，金枪鱼油和藻油2-MAG的含量提高至95%以上，回收率在70%左右，而DHA含量分别达到53.22%和74.76%。然后通过2-MAG与乙腈之比1:10（w/v）、-40℃下结晶10h，使金枪鱼油2-MAG中DHA含量提高至67.63%，2-MAG含量保持在90%以上。. 其次，确立了以棕榈酸乙烯酯作为酰基供体酶法不可逆合成sn-2位富含ARA的对称性结构脂方法。探究了反应条件的影响。结果显示，不可逆酶促反应的最优条件为：棕榈酸乙烯酯和2-MAGs的底物摩尔比为2.5:1，脂肪酶Lipozyme RM IM的添加量为10%，在30℃下、0.5mL正己烷体系中反应2.5h。在该反应条件下，对称性结构脂在粗产物中约占72%，反应效率明显优于以棕榈酸作为酰基供体的可逆酶促反应。采用低温结晶法纯化结构脂，所得结构脂的纯度为98%，纯化回收率为85%。. 最后，研究了2-MAG在不同温度和不同溶剂中的酰基转移规律，结果表明，2-MAG酰基转移符合一级可逆反应模型。在无溶剂体系中，温度提高25℃，酰基转移程度增加5.6倍；不同溶剂对酰基转移影响不同，酰基转移速率常数的大小为正己烷>无溶剂体系>二氯甲烷>乙醇≈丙酮≈乙腈>叔丁醇，与所选溶剂的log P值呈正相关（40℃ R=0.932、50℃ R=0.939，p<0.01）。.