Microplate method for regioselective lipase screening with structured punicic acid triglycerides: finding Lip4 lipase as sn-2-regioselective from Candida rugosa isoforms.

In this study, we propose a continuous assay that provides a high-throughput, efficient method for screening the regioselectivity of lipases at the sn-1,3 and sn-2 positions on triacylglycerols (TAGs). This assay measures the specific hydrolysis rates at the primary and secondary positions of TAGs derivates containing oleic (O) and punicic (P) acids. The method is based on the absorbance ratio of released punicic acid from the hydrolysis of sn-POP (sn-1,3 regiospecific lipases) and sn-OPO (sn-2 regiospecific lipases). The method was validated using pure lipases with known and unknown regioselectivity. Unexpectedly, we found that recombinant Lipase 4 from Candida rugosa (rCRLip4) exhibited significant sn-2 regioselectivity, indicating greater regioselectivity than recombinant lipase A from Candida antarctica (rCALA). In silico analysis and molecular docking studies were conducted to elucidate the main structural differences between CRLip4 and the non-regioselective isoform CRLip1. This continuous regioselective lipase assay on TAGs is versatile and can be used to screen for sn-1,3, sn-2 or non-regioselective lipases. It holds significant potential applications in the biocatalytic production of structured lipids and other industrial processes where regioselectivity is crucial.