Hydrophobic Residues Promote Interfacial Activation of Candida rugosa Lipase: A Study of Rotational Dynamics.

Microbial lipases constitute a class of biocatalysts with the ability to cleave ester linkages of long-chain triglycerides. This property makes them particularly attractive for industrial applications ranging from food processing to pharmaceutical preparation. Among such enzymes, Candida rugosa lipase (CRL) is one of the most frequently used in biotransformation. A notable feature of CRL, among many lipases, is its propensity for interfacial activation: these enzymes exhibit elevated catalytic rates when acting at the interface between aqueous and hydrophobic phases. Notably, this phenomenon can be attributed to the presence of a mobile lid domain, which in its closed state occludes the enzyme active site. To advance our understanding of interfacial activation, we explore the dynamics of CRL rotation at the octane-water interface in this work. To do so, we employ molecular dynamics and umbrella sampling to evaluate the free energy of rotation of the enzyme at the interface. We identify a global minimum in the rotational landscape that coincides with lid opening at the interface. Additionally, we investigate the role of surface residues outside the lid domain as they serve to instigate rotation of the lid toward the aqueous phase. In doing so, we identify a patch of leucine residues which when mutated to glycine impose a barrier to rotation that maintains the enzyme in the inactive (closed lid) state on the order of 1 μs. Importantly, this study presents a novel quantification of the rotational free energy corresponding to CRL lid opening at the octane-water interface. The accompanying mutagenesis study likewise clarifies the role of hydrophobic surface residues in the transition. As such, this work provides valuable insight into the phenomenon of interfacial activation that might open up new avenues for manipulating the microenvironment of industrially relevant lipases, affording enhanced control over the enzyme state.