Cai Haiying, Li Yang, Zhao Minjie, Fu Guanwen, Lai Jia, Feng Fengqin
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, China; Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou, China.
Hangzhou Kangyuan Food Technology Co., Ltd, Hangzhou, China.
PLoS One. 2015 Jul 28;10(7):e0133857. doi: 10.1371/journal.pone.0133857. eCollection 2015.
The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system.
1,3-二油酰基-2-棕榈酰甘油（OPO）是母乳脂肪的主要成分之一，其酶促合成一直受到sn-1,3特异性脂肪酶成本较高以及生物催化剂稳定性不足的阻碍。来自米曲霉的sn-1,3特异性脂肪酶（AOL）通过基于聚苯乙烯的疏水树脂D3520进行了高效固定化，与AOL粉末相比，在催化棕榈硬脂和油酸（OA）通过酸解合成OPO时，脂肪酶比活性显著提高了49.54倍。研究了最佳固定化条件，包括时间进程、初始蛋白质浓度和溶液pH值。评估了不同固定化条件下脂肪酶的sn-1,3特异性，并确定其与脂肪酶活性呈正相关，固定化溶液的pH值影响这些脂肪酶的区域特异性和合成活性。固定化的AOL D3520作为生物催化剂，用于通过棕榈硬脂和OA之间的酸解酶促合成结构化脂质OPO。对结构化脂质OPO的合成进行了以下条件优化：温度65°C；棕榈硬脂与OA的底物摩尔比为1:8；酶负载量为8%（w/w）；固定化脂肪酶的含水量为3.5%；反应时间为1小时。在这些条件下，实现了高效的C52产量（45.65%），体系中三棕榈酸甘油酯含量为2.75%，sn-2棕榈酸（PA）比例为55.08%。
