Lipase engineering, production and application : Expanding the lipids tailoring toolbox with applied bioinformatics

Enzymes, as efficient and environmentally friendly catalysts, have gainedsignificant scientific and industrial attention for their mild operating conditions,high substrate selectivity and reusability when immobilized. Among them, lipasesdominate the global industrial enzyme market, catalyzing various reactions with afocus on substrate specificity and selectivity. This thesis investigates theoptimization of Rhizopus oryzae lipase (ROL) for lipid modification, emphasizingits potential for industrial applications.The research is structured into four key phases aimed at optimizing ROL and itsapplication: 1) Screening the binding interactions between different fatty acidsubstrates and the ROL molecule, and investigating relevant structural features(Paper Ⅰ); 2) Designing ROL mutants based on structural insights from phase 1,with the objective of altering substrate selectivity (Paper Ⅲ); 3) Developing anefficient and convenient production system for ROL in the oleaginous yeastYarrowia lipolytica (GRAS) (Paper Ⅱ); and 4) Evaluating ROL immobilizationusing promising methods and carriers (Paper Ⅳ).Through the integration of molecular docking, molecular dynamics (MD)simulations and enzymatic experiments, this study explored the interactionsbetween various fatty acids and the ROL enzyme, correlating these interactions withsubstrate selectivity. Structural insights guided the creation of ROL mutants withtargeted mutations in the lid region, leading to the development of a lid-swappedchimera mutant that exhibited increased selectivity for long-chain fatty acids overmedium-chain fatty acids – a novel contribution to the field.Additionally, a high-yield production platform for ROL was developed inY. lipolytica using minimal media, simplifying lipase purification and enabling theevaluation of different expression factors.Lipase immobilization is crucial for ensuring efficient applications, especially innon-aqueous environments. This study investigated immobilization strategiesinvolving hydrophobic interactions and a combination of hydrophobic interactionand covalent coupling. The results indicate that polyacrylic supports functionalizedwith a short alkyl chain and a minimal number of epoxy groups are effective forROL immobilization. Furthermore, the study reveals that ROL immobilizationinfluences its fatty acid selectivity, regardless of whether the process involves pureadsorption or a combination of adsorption and covalent coupling.