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Engineering of a Thermostable Biocatalyst for the Synthesis of 2‐ O ‐Glucosylglycerol
Author(s) -
Franceus Jorick,
Ubiparip Zorica,
Beerens Koen,
Desmet Tom
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202100192
Subject(s) - glycerol , leuconostoc mesenteroides , biocatalysis , directed evolution , chemistry , regioselectivity , metabolic engineering , trehalose , protein engineering , biochemistry , enzyme , combinatorial chemistry , bacteria , catalysis , mutant , biology , lactic acid , reaction mechanism , gene , genetics
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.