College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen, Fujian Province 361021, China; Xiamen Key Laboratory of Marine Functional Food, Xiamen 361021, China.
College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China.
J Biotechnol. 2022 Aug 20;355:1-9. doi: 10.1016/j.jbiotec.2022.06.009. Epub 2022 Jun 28.
As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times k/K as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.
作为磷脂降解的必需酶，磷脂酶 C（PLC）已被用于植物油的酶法脱胶和有价值的磷脂衍生物的生产。在这项研究中，我们基于 B 因子分析和分子动力学模拟分析进行了合理的工程设计，合理地确定了突变候选者，并从蜡状芽孢杆菌 HSL3 设计了一种 PLC 双突变体 F96R/Q153P。与野生型 PLC 相比，F96R/Q153P 表现出显著改善的热性能，包括更高的温度最佳值和更好的热稳定性。它显示了迄今为止报道的最高最佳反应温度（90°C）。F96R/Q153P 的 kcat 和 k/K 分别是野生型的 4.94 倍和 2.37 倍，并且底物适应性也得到了提高。结构见解表明，突变导致无规卷曲的比例降低，并限制了某些环的波动。这些结果表明，基于知识的合理设计在酶法脱胶过程中提高工业酶的催化特性方面具有巨大的潜力。
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