Chitinoligosaccharides (COs), β-1,4-linked oligomers of N-acetylglucosamine, are an important class of signaling molecules involved in plant-biosphere cell-cell interactions. COs having a degree of polymerisation (DP) from 6 to 8 are potent inducers of immune responses in rice and wheat among others . Despite their biological interest and potential agronomical usefulness, COs with well-defined structure remain poorly accessible. While chemical or enzymatic degradation of chitin allows the production of COs of DP 2 to 6, higher oligomers are hardly accessible by depolymerisation methodologies. Therefore, enzymatic synthesis of COs has been a matter of research by exploiting the transglycosylation activity of retaining glycoside hydrolases (GH).
In the present work, we report the engineering and expression of Hen Egg-White Lysozyme (HEWL, GH-22) in the methylotrophic yeast Pichia pastoris. Site-directed mutagenesis on the essential aspartate 52  was carried out, three mutants devoid of hydrolytic activity were produced and one of them (D52S) displayed an efficient glycosynthase activity. Polycondensation reactions of α-chitintriosyl fluoride led to the formation of COs up to DP 15. Afterwards, we took advantage that a de-N-acetylated oligomer at the non-reducing end cannot behave as an acceptor for HEWL (+1 subsite does not accept a glucosaminyl residue). In a one-pot sequential procedure, the donor was first specifically de-N-acetylated at the non-reducing end by the action of Nod B chitin deacetylase, and then condensed on COs acceptors with mutant HEWL to give single addition products with size varying from hexa- to octamer.
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