A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced a-anomer by hydrolyzing b-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc) 6 ] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc) 2 and (GlcNAc) 4 . The evidence from this work suggested that the subsite structure of the enzyme was ()2)()1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure ()3)()2)()1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.Keywords: Aeromonas; chitinase; hydrolytic mechanism; oligosaccharide; subsites.Next to cellulose, chitin, b-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc), is one of the most commonly occurring organic compounds on earth, and attracts special interest as a reusable material. Chitinases (EC 3.2.1.14) degrade chitin by hydrolyzing the glycosidic linkages, and are widely distributed in various organisms [1]; from the ecological viewpoint, chitinases play an important role in recycling chitin in nature. The enzymes sequenced thus far are divided into two families (family 18 and family 19) on the basis of their amino acid sequences [2]. The chitinases of the two families do not share sequence similarity, and possess completely different folds in their three-dimensional structures. From X-ray crystallographic studies of the chitinases [3][4][5][6][7][8], the family 18 enzymes were found to have an (a/b) 8 -barrel fold, while the family 19 enzymes are rich in a-helices and composed of two lobes. When the b-1,4-glycosidic linkage is hydrolyzed by the catalytic action of the family 18 chitinases, oligosaccharide fragments with b-anomer are produced (retaining mechanism) [9], while the a-anomer is produced by the family 19 enzymes (inverting mechanism) [10]. Family 18 chitinases are sensitive to allosamidin, but family 19 chitinases are insensitive [11]. The family 18 enzymes have been found widely in bacteria, fungi, viruses, animals, and plants (classes III and V). Most of the family 19 chitinases, however, have been isolated from plants, and are subdivided into different classes (classes I, II and IV) [12]. The class I and IV chitinases have a cysteine-rich domain at their N-terminus, which is involved in substrate binding. Class IV chitinases are smaller than class I chitinases due to deletions in both the cysteine-rich domain and the catalytic domain. Class II chitinases are homologous to those of class I...