Lipases naturally function at the interface formed between amphiphilic molecules and the aqueous environment. Thermomyces lanuginosus lipase (TLL) is a well-characterised lipase, known to exhibit interfacial activation during which a lid region covering the active site becomes displaced upon interaction with an interface. In this study, we investigate the effect the amino acid sequence of the lid region on interfacial binding and lid dynamics of TLL. Three TLL variants were investigated, a wild-type variant, a variant containing an esterase lid region (Esterase), and a Hybrid variant, containing both wild-type lid residues and esterase lid residues. Multiple coarse-grained molecular dynamics simulations revealed that the interfacial binding orientation of TLL was significantly affected by the nature of amino acids in the lid region, and atomistic simulations indicated effects on the structural dynamics of the lid itself. The atomistic simulations, as well as steered molecular dynamics simulations, also indicated that the Esterase lid region was less flexible than the wild-type lid region, whereas the Hybrid variant displayed superior lid flexibility and stability in the open conformation both at the interface, and in aqueous solution. Additional experiments performed to investigate the activity and binding behaviour of the lipase variants indicated a slightly higher specific activity for the Hybrid variant compared to the wild-type variant, correlating the observations of increased lid flexibility. Together, these results are in line with previous experimental studies, highlighting the importance of the nature of the amino acid residues within the functional lid region of lipases, particularly regarding interfacial binding orientation, activation, and structural stability.

脂肪酶天然地在两亲性分子与水性环境之间形成的界面处起作用。嗜热单胞菌脂肪酶（TLL）是一种特性良好的脂肪酶，已知具有界面活化作用，在此期间，覆盖活性位点的盖区会在与界面相互作用时发生位移。在这项研究中，我们调查了盖区域的氨基酸序列对TLL的界面结合和盖动力学的影响。研究了三种TLL变体，一种野生型变体，一种包含酯酶有盖区（Esterase）的变体和一种杂种变体，其中既包含野生型有盖残基又包含酯酶有盖残基。多个粗粒度的分子动力学模拟表明，TLL的界面结合方向受盖子区域氨基酸的性质影响很大，而原子模拟表明对盖子本身的结构动力学有影响。原子模拟 以及转向分子动力学模拟也表明，酯酶盖区的柔韧性低于野生型盖区，而Hybrid变体在界面和水溶液中的开放构象中均表现出优异的盖柔度和稳定性。为研究脂肪酶变异体的活性和结合行为而进行的其他实验表明，与野生型变异体相比，杂合变异体的比活性略高，这与观察到的盖弹性增加有关。总之，这些结果与先前的实验研究一致，突出了脂肪酶功能性盖区中氨基酸残基的性质的重要性，特别是关于界面结合方向，活化和结构稳定性的重要性。