Yeast-derived high N-mannosylation modulates acyl chain length selectivity and biochemical properties of recombinant Cordyceps militaris lipase

Abstract

Recombinant Cordyceps militaris lipase (rCML) expressed in Komagataella phaffii is more highly N-mannosylated (Man8–11GlcNAc2) than the native lipase. The lipase activity of rCML was enhanced compared to its deglycosylated form (drCML), but the increases in the Vmax values of rCML for substrates with chain lengths of C4:0–C14:0 were 1.8 to 2.9-fold greater than those of drCML, whereas the difference was narrower for C16:0–C18:0. Changes in acyl chain length selectivity correlated with the conformational changes rather than hydrophobicity changes induced by N-mannosylation, which may have altered active site accessibility by shifting the favorable binding locations depending on acyl chain length. Nevertheless, high N-mannosylation did not significantly affect the melting temperature of rCML compared to that of drCML (55.5 °C and 57.0 °C, respectively) during thermal unfolding, but alleviated harsh deactivation in the thermal-transition state (60–70 °C). Furthermore, high N-mannosylation enhanced the long-term storage stability at –20 °C, and loss of activity was 5.2 ± 1.1% and 32.8 ± 1.2% for rCML and drCML, respectively. Overall, the introduction of yeast-derived high mannose moieties endowed short- to medium-chain selectivity and improved the biochemical properties of recombinant lipase.