ENZYMATIC SYNTHESIS OF GLYCOLIPIDS FOR BIOSURFACTANT APPLICATIONS BY ENGINEERING OF A TYPE II ENDO-GLYCOCERAMIDASE

Surfactants display a wide variety of chemical structures, of which certain are more adapted to particular applications. Alkyl polyglycosides (APGs) are nonionic surfactants that have a wide variety of applications and present quite low allergenic properties, making them useful in cosmetics.

Acid-catalyzed Fischer synthesis does not easily afford APGs displaying multiple sugar moieties, thus it is pertinent to seek alternative synthetic methods. In this respect, some retaining glycoside hydrolases (r-GHs) catalyze alkylation reactions involving alcohols and glycosides. However, most r-GHs display dominant hydrolytic properties, which out-compete transglycosylation. Moreover, most r-GHs fail to transfer to any significant level sugars onto long chain (approx. >6 carbons) alcohols. Therefore, there is a need for new r-GHs that efficiently perform transglycosylation using longer chain alcohols.

In this work, we have focused on the endoglycoceramidase EGCII from Rhodococcus strain M-777. We believe that this enzyme could make a good catalyst for APG synthesis, because it naturally recognizes glycosphingolipids, which are long chain aliphatic amino alcohol-sugar conjugates. However, EGCII’s natural transglycosylation abilities have not been characterized, although previously EGCII has been converted into a glycosynthase via mutagenesis.

First, to explore EGCII’s natural transglycolytic ability, we have synthesized two donor substrates (2-chloro-4-nitrophenyl lactoside and cellobioside), studied their EGCII-mediated transfer onto various alcohols with chain lengths of C₅ and C₈. Next, site-saturation mutagenesis was used to probe the influence on EGCII acceptor binding of secondary hydroxyl and ketone groups on the alkyl acceptor. Finally, a screening method for the detection of improved transglycosylation properties among EGCII mutants was developed and used to screen an epPCR mutant library.

In this poster, a digest of our results will be presented and discussed, notably with regard to our progress towards converting EGCII into a transglycosylase.