Complex oligosaccharides of human milk have received considerable attention due to their biological importance. Despite the advances made in chemical and enzymatic synthesis (1), large scale production of human milk oligosaccharides is still not possible and these compounds are not available in infant formulas.
Our group studies the Bifidobacterium bifidum lacto-N-biosidase responsible of hydrolysis of the lacto-N-tetraose, one of the main core structures of these oligosaccharides, characterized by lacto-N-biose (Galbeta1,3GlcNAc) and lactose. Few information of this enzyme is available (2,3): it belongs to the 20 glycosyl hydrolases family following a retaining mechanism via substrate-assisted catalysis and it consists of five different domains which catalytic domain presents high similarity to beta-N-acetylhexosaminidases (EC 3.2.1.52).
We have cloned and expressed the full-length protein in E. coli. With the aim of developing a new synthetic strategy for this tetrasaccharide, we have generated specific mutants of the putative catalytic domain to enhance synthase activity. Here we present the abilities of these mutants to transglycosylate p-nitrophenyl lactobioside and also, act as glycosynthase-like mutant using the lacto-N-biosyl oxazoline derivative as donor.
(1) Faijes and Planas, Carbohydr.Res. 342, 1581-1594 (2007)
(2) Wada, Ando, Kiyohara, Ashida, Kitaoka, Yamaguchi, Kumagai, Katayama, Yamamoto, Appl.Environ. Microbiol. 74 (13), 3996 (2008)
(3) Hattie, Debowski, Stubbs, ChemBioChem 13, 1128-1131 (2012)
Acknowledgement
Cristina Val acknowledges financial support from Fundación INSTITUTO DANONE.
