The site-directed immobilization of enzymes has demonstrated significant potential in industrial applications due to its ability to minimize enzyme heterogeneity and maximize retained activity. However, existing approaches often require the introduction of unnatural amino acids or excessive specific ligase to achieve this goal. In this study, a self-catalyzed protein capture system (i.e., the SnoopCatcher/SnoopTag pair) was utilized for the directed immobilization of lipase on magnetic carriers. By tagging the Pseudomonas fluorescens lipase (PFL) with a SnoopTag at the C-terminal, the fused lipase PFL-SnoopTag (PSNT) readily conjugated with the SnoopCatcher partner via a spontaneously formed isopeptide bond between them. Novel magnetic particles functionalized by SnoopCatcher proteins were prepared using a co-precipitation method, achieving a loading capacity of around 0.8 mg/g carrier for the SnoopCatcher. This functionalized magnetic carrier enabled the site-directed immobilization of lipase PSNT at 81.4% efficiency, while the enzyme loading capacity reached 3.04 mg/g carriers. To further assess the practical performance of site-directed immobilized lipases, they were applied in biodiesel production and achieved a yield of 88.5%. Our results demonstrate a universal platform for the site-directed immobilization of enzymes with high performance, which offers significant advantages, e.g., single-step purification and catalyst-free immobilization of engineered enzymes, as well as easy recovery, highlighting its potential for industrial applications.
Keywords: SnoopCatcher/SnoopTag system; directed immobilization; lipase; magnetic carrier.