Plant Chitinase Mutants as the Catalysts for Chitooligosaccharide Synthesis Using the Sugar Oxazoline Derivatives

Sugar oxazolines, (GlcNAc)n-oxa (n = 2, 3, 4, and 5), were synthesized from a mixture of chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, and 5), and utilized for synthesis of (GlcNAc)7 with higher elicitor activity using plant chitinase mutants as the catalysts. From isothermal titration calorimetry, the binding affinity of (GlcNAc)2-oxa toward an inactive mutant obtained from Arabidopsis thaliana GH18 chitinase was found to be higher than those of the other (GlcNAc)n-oxa (n = 3, 4, and 5). To synthesize (GlcNAc)7, the donor/acceptor substrates with different size combinations, (GlcNAc)2-oxa/(GlcNAc)5 (1), (GlcNAc)3-oxa/(GlcNAc)4 (2), (GlcNAc)4-oxa/(GlcNAc)3 (3), and (GlcNAc)5-oxa/(GlcNAc)2 (4), were incubated with hypertransglycosylating mutants of GH18 chitinases from A. thaliana and Cycas revoluta. The synthetic activities of these plant chitinase mutants were lower than that of a mutant of Bacillus circulans chitinase A1. Nevertheless, in the plant chitinase mutants, the synthetic efficiency of combination (1) was higher than those of the other combinations (2), (3), and (4), suggesting that the synthetic reaction is mostly dominated by the binding affinities of (GlcNAc)n-oxa. In contrast, the Bacillus enzyme mutant with a different subsite arrangement synthesized (GlcNAc)7 from combination (1) in the lowest efficiency. Donor/acceptor-size dependency of the enzymatic synthesis appeared to be strongly related to the subsite arrangement of the enzyme used as the catalyst. The A. thaliana chitinase mutant was found to be useful when combination (1) is employed for the substrates.