Substitution of L133 with Methionine in GXSXG Domain Significantly Changed the Activity of Penicillium expansum Lipase

It is not clear how the amino acid composition of the X sites in GXSXG domain affects the properties of the lipase. To address this, saturation mutation at these sites of Penicillium expansum lipase (PEL) was carried out. After screening, a mutant L133M with significantly different activity was obtained. The activity of L133M was lower than that of wild-type PEL (WT) when the reaction time was 3–10 min. However, it was 1.61 times that of WT under long-term (48 h) catalysis. WT exhibited interfacial activation when the concentration of p -nitrophenylbutyrate was higher than 3 mM, whereas L133M could only show interfacial activation at higher micelle concentration ( p -nitrophenylbutyrate concentration > 5 mM), which seemed to be slower. Further analysis showed that a new hydrophobic interaction was generated between the methionine introduced at position 133 and the lid, which made the interface activation process more difficult. However, the binding energy between the activated mutant and the substrate was smaller, showing stronger activity. This work provides some reliable clues for further understanding the complex role of GXSXG domain in enzyme catalysis. Graphic Abstract