Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn-1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N-glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris, the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.

中文翻译：

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通过引入 N-糖基化增强米根霉脂肪酶的热稳定性和催化活性


米根霉(Rhizopus oryzae)脂肪酶(ROL)具有显着的sn-1,3立体选择性和催化活性，但其较差的热稳定性限制了其在生产1,3-二油酰-2-棕榈酰甘油(OPO)（人类甘油的优质替代品）中的应用。乳脂）。在这项工作中，提出了一种半理性方法来设计 4M（我们之前研究中的 ROL 突变体）的热稳定性和催化活性。首先以4M为模板进行计算机辅助设计，然后在毕赤酵母中重组表达并筛选N-糖基化突变体，最优突变体N227在45℃下的半衰期为298.8 h，即7.23 -折叠长度超过4M。其催化活性也达到了1043.80±61.98 U/mg，比4M（808.02±47.02 U/mg）提高了29.2%。 N227的分子动力学模拟表明，聚糖的引入增强了蛋白质的刚性，而聚糖和蛋白质之间形成的强氢键稳定了脂肪酶的结构，从而提高了其热稳定性。利用固定化N227成功进行了油酸（OA）与三棕榈酸甘油酯（PPP）的酸解反应，PPP的摩尔转化率为90.2%。该工程策略指导脂肪酶的修饰，而本研究获得的糖突变体在OPO的生物合成中具有潜在的应用。