Investigation of regioselectivity and thermostability of free and immobilized Pleurotus citrinopileatus lipase.

A fungal lipase from the Basidiomycete Pleurotus citrinopileatus (PCI_Lip) has been investigated for its regioselectivity. Therefore, the conversion products of a triglyceride were analyzed using 1H-NMR to determine the position of hydrolysis within the substrate. PCI_Lip is a 1,3-selective lipase, as it preferentially hydrolyzes the fatty acids at the outer positions of the triglyceride. Furthermore, this study aimed to gain deeper insights into the stability of the lipase. The melting point of the enzyme was determined to be at 63 °C in ultrapure water, which could be increased to 71 °C by dissolving PCI_Lip in a 400 mM citrate buffer pH 6.0 with the addition of glucose and trehalose. To optimize the stability of the enzyme, PCI_Lip was immobilized via its affinity tag. Twenty-one supports were tested, with IB-HIS-19 being chosen as the most appropriate for PCI_Lip immobilization due to its high binding capacity and low leakage. This immobilized lipase displayed only minor loss of activity over 13 cycles of reuse. Immobilization of the wild-type preserved activity and selectivity, whereas it induced notable changes in the case of the double mutants. The increased stability of the immobilized PCI_Lip was further proven in a thermal inactivation experiment. Double mutants of PCI_Lip displayed even higher inactivation temperatures, while the immobilization raised this temperature by 5 °C. Overall, in this study, the 1,3-regioselective PCI_Lip identified from our group has been further characterized, focusing on the improvement of its thermal and kinetic stability. A major boost in stability can be achieved via immobilization of the enzyme to an appropriate support. KEY POINTS: IB-HIS-19 had the highest binding capacity and kept up enzyme activity over 13 cycles of the screened immobilization materials Immobilization increased the stability of PCI_Lip by 5 °C, protein engineering increased the stability of two double mutants up to 4 °C, and thermostability could be increased by 9 °C by using high molarity buffers PCI_Lip was found to be a 1,3-regioselective lipase by analyzing its products by 1H-NMR.