Surface display and characterization of recombinant α-l-Rhamnosidase from Emiliania huxleyi on Pichia pastoris.

An α-l-Rhamnosidase gene with an open reading frame of 3192 bp encoding a 1036-amino acid protein (EhRha) was cloned from Emiliania huxleyi for flavonoid hydrolysis on the cell surface of Pichia pastoris (P. pastoris) strain GS115 by fusing with the anchor protein (AGα1) from Saccharomyces cerevisiae. Fluorescence microscopy and flow cytometry assays revealed that EhRha was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity assay and substrate specificity analysis showed that the enzyme activity of displayed EhRha was 78 U/g (cell wet weight). EhRha demonstrated a preference for the α-1,6 linkage l-rhamnose in hesperidin and rutin as its optimal substrates, while showing low activity towards the α-1,2 linkage l-rhamnose in naringin. Furthermore, EhRha demonstrated optimal activity at pH 7.0 and 30 °C, maintaining stability within a pH range of 4.5-9.0 at temperatures below 50 °C, and remained functional at temperatures ranging from 15 °C-30 °C. The enzyme activity was significantly enhanced by the presence of 10 mM Mn and Fe, whereas 10 mM Ca and 1 mM Fe had an inhibitory effect. These findings suggested that displayed EhRha holds promise for enhancing the bioavailability of health-beneficial polyphenols in low-temperature processing applications.