which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chi4 genes encoding various deletions of chitinase Al were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase Al specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase Al does not bind were not significantly afected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type HI modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.Various organisms, including bacteria, fungi, plants, and some vertebrates, produce chitinases, enzymes which hydrolyze the ,-1,4 linkage of chitin. Bacterial chitinases are thought to be important in the digestion of chitin for utilization as a carbon and energy source, and, from the ecological point of view, such chitinases serve an important role in recycling chitin in nature.Bacillus circulans WL-12 is one of the bacteria which excrete chitinases into culture media (28). The chitinase system of the bacterium includes at least six different chitinase molecules: chitinases Al, A2, Bi, B2, C, and D. Chitinase A2 is a derivative of Al and is generated by proteolytic modification; likewise, B2 is thought to be a derivative of Bi. Chitinase Al is assumed to be the key enzyme in the chitinase system, as it (and A2) is the most abundantly produced form as well as the enzyme with the highest colloidal chitin-hydrolyzing activity and very high affinity toward chitin. The gene encoding chitinase Al (chiA) and the one encoding chitinase D (chiD), which is located immediately upstream of the chiA gene, have been cloned and sequenced (27,29). Amino acid sequence analysis suggests that chitinase Al comprises at least three discrete functional domains; namely, a C-terminal domain, a region consisting of two type III modules (domains), and a large N-terminal domain. In previous reports, we showed that the type III modules of chitinase Al were related to those of fibronectin, a multifunctional extracellular and plasma protein of higher eukaryotes (29), and that glutamic acid 2...
