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Engineering CGTase to improve synthesis of alkyl glycosides.

Authors
  • Ara, Kazi Zubaida Gulshan1
  • Linares-Pastén, Javier A1
  • Jönsson, Jonas1
  • Viloria-Cols, Maria1, 2
  • Ulvenlund, Stefan2
  • Adlercreutz, Patrick1
  • Karlsson, Eva Nordberg1
  • 1 Biotechnology, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden. , (Sweden)
  • 2 Enza Biotech AB, Scheelevägen 22, 22363 Lund, Sweden. , (Sweden)
Type
Published Article
Journal
Glycobiology
Publisher
Oxford University Press
Publication Date
Jun 03, 2021
Volume
31
Issue
5
Pages
603–612
Identifiers
DOI: 10.1093/glycob/cwaa109
PMID: 33270133
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties. © The Author(s) 2020. Published by Oxford University Press.

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