Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol

Abstract

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.

Keywords: biocatalysis; glucosylglycerol; glycoside phosphorylases; protein engineering; sucrose phosphorylase.

Scheme 1

Synthesis of glucosylglycerol (GGo) using the promiscuous transglycosylation of sucrose phosphorylases.

Figure 1

Docking of 2‐GGo in the active site of BaSP (PDB code 2GDV). Shown are the residues selected for mutagenesis based on a sequence comparison of BaSP to LmSP and GGoP.

Figure 2

Docking of 2‐GGo in the active site of (a) BaSP and (b) a model of variant P134Q.

Figure 3

Kinetic stability of BaSP P134Q and LmSP. The enzymes were incubated at 52 °C and samples were taken regularly, after which their residual activity was compared to that of untreated enzyme (CV<10 %).