A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.In the last several years, a growing number of industrial applications have required the use of lipases in the presence of partly oxidized triglycerides. Biodiesel (fatty acid monoalkyl esters) has attracted considerable attention during the past decade as a renewable, biodegradable, and nontoxic fuel (36). It is produced from various vegetable oils and has a viscosity close to that of diesel, but it is derived from triglycerides by transesterification with alcohol. Recently, enzymatic transesterification using lipases has become more attractive for biodiesel production, since the glycerol produced as a by-product can easily be recovered, and the purification of fatty methyl esters is simple to accomplish. Owing to the higher price of biodiesel relative to petroleum fuel, waste triglycerides (e.g., frying oils) are often used as cheaper feedstock for the biodiesel synthesis (6,8,26), thus helping also to reduce the cost of wastewater treatment in sewage systems and generally assisting in the recycling of resources (12,19,23,24).With enzymatic technology it is also possible to produce new fats according to specific requirements. Special triglycerides of the ABA-type containing medium-chain fatty acids (e.g., C 8 ) in the sn-1,3 positions and long-chain unsaturated fatty acids (e.g., C 16 to C 22 ) in the sn-2 position are effective energy sources for patients with malabsorption, e.g., pancreatic insufficiency. Polyunsaturated fatty acids like eicosapentaenoic acid