Stability analysis of a plant glycosyltransferase

Background: Glycosylation can significantly improve physicochemical or biological properties of small molecules such as vitamins, fragrances and antibiotics. Glycosyltransferases (GTs) are carbohydrate-active enzymes that can efficiently catalyse such reactions. We recently discovered a promising biocatalyst (GT-1) for the glycosylation of a wide variety of compounds, but its thermostability will be crucial for industrial applications. Materials and Methods: GT-1 was recombinantly expressed and purified via Ni-NTA chromatography. Thermal half-life time (t50(50°C)) at 50 °C was assessed by sampling at fixed intervals until the residual activity had dropped to 50%, while the melting temperature (Tm) was determined via differential scanning fluorimetry. The bio-informatic tools YASARA and 3DM were used to select target positions for engineering. Results: GT-1 was succesfully expressed and yields up to 40 mg purified protein per liter culture were obtained. Further, t50(50°C) and Tm values of respectively 90 min and 56 °C were found. Discussion: The kinetic stability of GT-1 was rather low. Indeed, after 1 min of incubation at 50 °C, residual activity already dropped to 70%. Clearly, enzyme engineering strategies need to be developed. Therefore, a homology model was constructed and structural analysis revealed a strikingly large amount of open spaces. Filling these cavities with larger amino acids should increase hydrophobic packing, yielding a more stable enzyme. Conclusion: The kinetic and thermal stability of GT-1 was assessed. Engineering the enzyme’s stability by a combination of smart (sequence-based) and rational (structure-based) mutagenesis will be key to biocatalyst development.