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.

Continuous process technology for glucoside production from sucrose using a whole cell-derived solid catalyst of sucrose phosphorylase

“…The 2-GG selectivity of the enzymes was between 80 and 85%. Overall, therefore, when assessed as whole cell catalyst for 2-GG production, the P134Q variant of BaSucP served the purpose of a selective and stable catalyst, thus strongly supporting the antecedent molecular engineering of the enzyme (Franceus et al 2021).…”

“…The BaSucP is more stable but less regioselective (~65%) than LmSucP. The BaSucP_P134Q variant was developed (Franceus et al 2021) to confer regioselectivity (83±3%) to the stable enzyme. Measured in cell-free extract (N = 2) for 30 min using~0.3 M sucrose and~1.8 M glycerol, the specific activity at 30°C of LmSucP and BaSucP_P134Q was 792±86 and 794±57 U g −1 cell wet weight, respectively.…”

“…E. coli BL21 (DE3)-agp harboring pQE30 plasmid was used to produce SucP from L. mesenteroides (LmSucP, N-terminal His-tag) (Goedl et al 2007) and a SucP variant from Bifidobacterium adolescentis (BaSucP_P134Q, N-terminal His-tag) (Franceus et al 2021). The feature of deleted agp gene (α-glucose 1-phosphate phosphatase) in the E. coli strain used can be useful when working with glycoside phosphorylases, but it was not essential here.…”

Biocatalytic Production of 2-α-d-Glucosyl-glycerol for Functional Ingredient Use: Integrated Process Design and Techno-Economic Assessment

“…13 The used enzymes are stereospecific and show high regioselectivity on glycerol (>80% 2-GG). 14 The glycerol substrate is applied in excess (up to six-fold) for two reasons. First, sucrose conversion is driven to completion.…”

Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase

“…Prolonging the catalysis time could reduce impurity I, but impurity III increased simultaneously, with more time cost generally disfavored in industrialization 35. With the intention of controlling impurities, a semi-rational design strategy of the enzyme was further applied 36. The structures of impurities I and III indicated that their formation was attributed to the regioselectivity and promiscuity of SPase 29,33.…”