Wilmar Innovation Centre, Wilmar International Limited, 28 Biopolis Road, Singapore 138568, Singapore.
Int J Mol Sci. 2022 Aug 23;23(17):9515. doi: 10.3390/ijms23179515.
1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.
1,3-区域特异性脂肪酶是在食品工业中大量用于生产结构三酰基甘油（TAG）的重要酶。脂肪酶（ROL）最近引起了人们的兴趣，因为这种酶具有高选择性和催化效率。然而，其低热稳定性限制了其在较低温度下进行反应的应用。最重要的是，由于反应中使用的底物熔点较高，该酶不能用于生产 1,3-二油酰基-2-棕榈酰甘油（OPO）和 1,3-硬脂酰基-2-油酰基甘油（SOS）。尽管已经进行了各种工程努力来提高 ROL 的热稳定性，但该酶无法在 60°C 以上的温度下发挥作用。在这里，我们描述了 ROL 的合理设计，以确定能够在高于 60°C 的温度下保留其活性的变体。经过两轮诱变和筛选，我们成功地鉴定出一种可以在 70°C 下保持大部分活性的突变体 ROL_10x。我们进一步证明，该突变体可用于 SOS 的合成，而 ROL_WT 则很少观察到产物形成。我们设计的酶为在高温下进行结构脂质的工业合成提供了一个有前途的解决方案。
