Successes in engineering glucansucrases to enhance glycodiversification - Carbohydrate Chemistry: Chemical and Biological Approaches - page 624

Successes in engineering glucansucrases
to enhance glycodiversification
David Daud ´ , a Isabelle Andr ´ , a Pierre Monsan a and
Magali Remaud-Sim ´ on* b,c
DOI: 10.1039/9781849739986-00624
Carbohydrates are biomolecules that have an essential role in every form of life. The
reservoir of naturally occurring glyco-structures is incredibly large and involves a tre-
mendous number of carbohydrate-active enzymes (more than 280,000 released modules
in the Carbohydrate Active enZymes database) for their synthesis and degradation.
Nevertheless, natural enzymes do not necessarily present all the requested properties in
terms of e ciency, specificity or stability when considering their usage for carbohydrate
or glyco-derivative manufacturing. In addition, if existing, the identification of an enzyme
perfectly adapted to a specific function from the natural diversity may be critical due to the
lack of available biochemical data and may necessitate intensive screening efforts. To
circumvent such limitations and provide optimized solutions, protein engineering has
been considered. Leloir-type glycosyltransferases, for example, are mainly involved in the
biosynthesis of glycoconjugates in Nature and they have been widely studied and en-
gineered for this purpose. However, these enzymes are often found as membrane-bound
proteins, what renders di cult their isolation and purification. In addition, their need of
low-abundant activated sugars as glycosyl donors also impairs their usage. Alternatively,
enzymes that use more abundant glycosyl donor directly issued from agro-ressources
have been considered to access to new glyco-derivatives. This has promoted the use of
glucansucrases (GS) that catalyze transglycosylation reactions from sucrose substrate.
These enzymes are of particular interest for synthetic purpose and have found industrial
interest for pharmaceutical and fine chemical applications. To diversify their applications,
various approaches of engineering have been exploited to improve expression level,
stability, or change substrate or product specificity of these enzymes. In particular, the
range of molecules recognized and the osidic linkages formed by GS is broad but yet
limited. Therefore, protein engineering methods have been applied to further increase the
diversity of glycosylation reactions catalyzed by these enzymes. Sequence analysis and
mutagenesis experiments have enabled the identification of key amino acid residues of
glucansucrases either involved in catalysis or substrate specificity. Moreover, the de-
termination of three-dimensional structures of glucansucrases from both families 13 and
70 of Glycoside-Hydrolases (GH) have provided powerful information for understanding
the sequence-structure-function relationships and guiding structure-based rational and
semi-rational engineering of these proteins. To assist these efforts, high-throughput
screening and biomolecular methods have been developed for the directed evolution of
these enzymes. Here are reported some of the successes in the bioengineering of glu-
cansucrases from precursor work to latest results, as well as the methods developed for
screening and developing e cient variant libraries. The major progresses and break-
throughs in the field will be highlighted and further prospects will be considered and
discussed.