Glycosynthase-type GH18 mutant chitinases at the assisting catalytic residue for polymerization of chitooligosaccharides.

Chitooligosaccharides (COS), the depolymerization products of chitin, have many potential applications in agriculture and medicine since they induce immunostimulating effects and disease protective responses. Most of their biological activities require degrees of polymerization (DP) larger than the tetrasaccharide, but structurally well-defined COS with DP larger than six are difficult to produce due to their high insolubility and complex isolation from chitin hydrolysates. Enzymatic synthesis by exploiting the transglycosylation activity of chitinases offers a potential strategy for the assembly of oligomers in the range of bioactive DPs. We here explore the glycosynthase-like activity of six GH18 chitinases from bacterial and archaeal origin by mutating the catalytic assisting residue in the substrate-assisted mechanism of this enzyme family. The alanine mutants at the assisting residue have a significant, but not essential, effect on the hydrolase activity. We studied the ability of the alanine mutants at the assisting residue to catalyze the polymerization of an oxazoline derivative as donor substrate, selecting the oxazoline of pentaacetylchitopentaose (DP5ox) with the aim of obtaining larger oligomers/polymers that, being insoluble, might be resistant to further reactions by the hydrolytically compromised mutant enzymes. For all the enzymes, insoluble polymeric material was obtained, with DP10 as major component, but other COS with different DPs were also obtained, limiting the practical application to produce oligomers/polymers with a defined DP. The balance between the residual hydrolase activity of the mutant enzymes and the solubility/precipitation kinetics still lead to hydrolysis and/or transglycosylation reactions on the newly formed products. From the selected enzymes, the Thermococcus kodakaraensis ChiA D1022A mutant gave the best results, with the formation of insoluble polymers in 45% yield (w/w) and containing about 55% of the target DP10 product.

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