Degradation of Chitosans with Chitinase G from Streptomyces coelicolor A3(2): Production of Chito-oligosaccharides and Insight into Subsite Specificities

We have studied the degradation of soluble heteropolymeric chitosans with a bacterial family 19 chitinase, ChiG from Streptomyces coelicolor A3(2), to obtain insight into the mode of action of ChiG, to determine subsite preferences for acetylated and deacetylated sugar units, and to evaluate the potential of ChiG for production of chito-oligosaccharides. Degradation of chitosans with varying degrees of acetylation was followed using NMR for the identity (acetylated/deacetylated) of new reducing and nonreducing ends as well as their nearest neighbors and using gel filtration to analyze the size distribution of the oligomeric products. Degradation of a 64% acetylated chitosan yielded a continuum of oligomers, showing that ChiG operates according to a nonprocessive, endo mode of action. The kinetics of the degradation showed an initial rapid phase dominated by cleavage of three consecutive acetylated units (A; occupying subsites -2, -1, and +1), and a slower kinetic phase reflecting the cleavage of the glycosidic linkage between a deacetylated unit (D, occupying subsite -1) and an A (occupying subsite +1). Characterization of isolated oligomer fractions obtained at the end of the initial rapid phase and at the end of the slower kinetic phase confirmed the preference for A binding in subsites -2, -1, and +1 and showed that oligomers with a deacetylated reducing end appeared only during the second kinetic phase. After maximum conversion of the chitosan, the dimers AD/AA and the trimer AAD were the dominating products. Degradation of chitosans with varying degrees of acetylation to maximum degree of scission produced a wide variety of oligomer mixtures, differing in chain length and composition of acetylated/deacetylated units. These results provide insight into the properties of bacterial family 19 chitinases and show how these enzymes may be used to convert chitosans to several types of chito-oligosaccharide mixtures.