Journal
CARBOHYDRATE POLYMERS
Volume 252, Issue -, Pages -Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.carbpol.2020.117121
Keywords
Chitinase; Glycosynthase; Polymerization; Chito-oligosaccharides; Chitin; Substrate-assisted catalysis; Oxazoline donor; Synthetic chitin polymers
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Funding
- European Union's Seventh Framework Programme for research, technological development and demonstration [613931]
- MINECO, Spain [BFU2016-77427-C2-1-R]
- AGAUR from the Generalitat de Catalunya [2017SGR-727]
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The study focuses on engineering chitinases to produce chitin oligomers with good yields in the range of bioactive DPs by exploiting transglycosylation activity.
Depolymerization of chitin results in chitooligosaccharides (COS) that induce immunostimulatory effects and disease protective responses and have many potential applications in agriculture and medicine. Isolation of bioactive COS with degree of polymerization (DP) larger than six from chitin hydrolyzates is hampered by their water insolubility. Enzymatic synthesis by exploiting the transglycosylation activity of GH18 chitinases offers a potential strategy to access oligomers in the range of bioactive DPs. We engineered SpChiD chitinase as a glycosynthase by mutation of the assisting residue of the catalytic triad in the substrate-assisted mechanism for polymerization of an oxazoline substrate (DP5ox). The insoluble polymer containing DP10 was partially hydrolyzed due to the significant residual hydrolase activity of the mutant enzyme. Combined mutations that strongly reduce the hydrolytic activity, in which the original catalytic triad only retains the essential acid/base residue, together with neighboring mutations in the-1/+1 subsites region, render glycosynthase-like chitinases able to produce chitin oligomers with DP10 as major product in good yields.
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