Fang Yakun, Liu Fan, Shi Yi, Yang Ting, Xin Yu, Gu Zhenghua, Shi Guiyang, Zhang Liang
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, Jiangsu, China.
Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, Jiangsu, China.
Front Microbiol. 2023 Jun 26;14:1193955. doi: 10.3389/fmicb.2023.1193955. eCollection 2023.
TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low and high value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest / value of 363.88 ± 15.83 L⋅min⋅mmol. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.
TrLipE是一种嗜热脂肪酶，由于其在极端条件下的催化能力而具有潜在的商业应用价值。与大多数脂肪酶一致，TrLipE的盖子位于催化口袋上方，控制底物进入活性中心的通道，并通过构象变化调节酶的底物特异性、活性和稳定性。来自[具体来源未提及]的TrLipE具有潜在的工业应用价值，但其酶活性较弱阻碍了这一应用。在此，通过在TrLipE和结构相似的酶之间进行N端盖子交换，构建了18个嵌合体（TrL1-TrL18）。结果表明，嵌合体的pH范围和最适pH与野生型TrLipE相似，但温度范围较窄，为40-80°C，TrL17和其他嵌合体的最适温度分别较低，为70°C和60°C。此外，在最适温度条件下，嵌合体的半衰期低于TrLipE。分子动力学模拟表明，嵌合体具有较高的均方根偏差（RMSD）、均方根波动（RMSF）和B因子值。当使用不同链长的对硝基苯酚酯作为底物时，与TrLipE相比，大多数嵌合体的[具体数值未明确]较低而[具体数值未明确]较高。嵌合体TrL2、TrL3、TrL17和TrL18可以特异性催化底物苯甲酸对硝基苯酯，其中TrL17的[具体比值未明确]最高，为363.88±15.83 L·min·mmol。然后通过研究TrL17与苯甲酸对硝基苯酯的结合自由能来设计突变体。结果表明，单取代、双取代和三取代变体（分别为M89W和I206N；E33W/I206M和M89W/I206M；以及M89W/I206M/L21I和M89W/I206N/L21I）催化苯甲酸对硝基苯酯的速度比野生型TrL17快约2至3倍。我们的观察结果将有助于TrLipE性质的开发及其工业应用。
