Hen egg-white lysozyme engineering for the synthesis of chitinoligosaccharides

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Hen egg-white lysozyme engineering for the synthesis of chitinoligosaccharides

Antoine Rousseau 1 , Sylvie Armand 1 , Sylvain Cottaz 1 , Sebastien Fort 1

1 Centre De Recherches sur les Macromolécules Végatales (CERMAV, UPR-CNRS 5301), Grenoble, France

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 [1]. 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 [2] 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.

Figure 1. Synthesis of long chitinoligosaccharides by HEWL glycosynthase.

Zipfel C.; Oldroyd G.E.D. Nature 2017, 543, 328-336.

Vocadlo D.J.; Davies G.J.; Laine R.; Withers S.G. Nature 2001, 412, 835-838.

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