A chitin-degrading bacterial strain, KN1699, isolated from Yatsu dry beach (Narashino, Chiba Prefecture, Japan), was identified as Vibrio parahaemolyticus. Treatment of powdered chitin with crude enzyme solution prepared from the supernatant of KN1699 cultures yielded a disaccharide, beta-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN), as the primary chitin degradation product. The extracellular enzymes involved in the production of this heterodisaccharide, chitinase (Pa-Chi; molecular mass, 92 kDa) and chitin oligosaccharide deacetylase (Pa-COD; molecular mass, 46 kDa), were isolated from the crude enzyme solution, and their hydrolysis specificities were elucidated. These studies confirmed that (1) Pa-Chi hydrolyzes chitin to produce (GlcNAc)(2) and (2) Pa-COD hydrolyzes the acetamide group of reducing end GlcNAc residue of (GlcNAc)(2). These findings indicate that GlcNAc-GlcN is produced from chitin by the cooperative hydrolytic reactions of both Pa-Chi and Pa-COD.

Oligosaccharides can exhibit physiologically useful functions. Many of these oligosaccharides are prepared by enzymatic degradation of biomass polysaccharides, or by enzymatic conversion of oligosaccharides produced by higher plants. New oligosaccharides with potential therapeutic activities are currently being developed; the physiological properties of oligosaccharides obtained by the hydrolysis of chitin, a β (1,4) polymer of N acetyl D glucos amine (GlcNAc), are of particular interest. Studies have reported that hexa N acetylchitohexaose, (GlcNAc)6, exhibits antitumor 1 3) and antimicrobial activity 4,5) in mice by enhancing the immunological defense system. These findings have focused attention on GlcNAc and (GlcNAc)6 as potential agents for arthritis therapy and immunotherapy, respectively, and have raised the possibility that chitin oligosaccharides could have physiologically useful functions. Generally, GlcNAc and chitin oligosaccharides are obtained by hydrolysis of chitin using strong acid. Chitin, one of the most abundant of all biomass polysaccharides, is the major component of the shells of crustaceans such as crab and shrimp, the exoskeletons of insects, and the cell walls of fungi. The hydrolysis of this biomass polysaccharide by enzymes possessing high product specificity would be more efficient than acid hydrolysis for the production of specific oligosaccharides. Various enzymes involved in chitin hydrolysis [e.g., chitinase (EC 3.2.1.14), β N acetylhexosaminidase (EC 3.2.1.52), chitin deacetylase (EC 3.5.1.41), and chitin oligosaccharide deacetylase (EC 3.5.1. )] have been identified. Chitinase catalyzes the degradation of water insoluble chitin into water sol uble chitin oligosaccharides through hydrolysis. A number of chitinases have been isolated from bacteria and their properties investigated. 6) In addition, genes encoding a variety of chitinases have been cloned. Based on their amino acid sequences, these chitinases are classified as belonging to either glycoside hydrolase (GH) family 18 or 19 (http: afmb.cnrs mrs.fr CAZY ). β N Acetylhexosa minidase catalyzes hydrolysis of chitin oligosaccharides to release the monosaccharide GlcNAc. Enzymes from various sources have been classified in three GH families: 3, 20 (the main family), and 84. Microbial β N acetylhexo saminidases have been reviewed. 7) Chitin deacetylase and chitin oligosaccharide deacetylase are involved in hydrolysis of the acetamide group of the GlcNAc residue of chitin and chitin oligosaccharides. 8) Both enzymes, isolated from various sources, are classified as belonging to carbohydrate esterase (CE) family 4 (http: afmb.cnrs mrs.fr CAZY ). Several microbial chitin deacetylases and chitin oligosaccharide deacetylases have been isolated and characterized.9 13) Previously, high yield production of di N acetylchitobiose, (GlcNAc)2, had been accomplished by cultivating the exo type chitinase producing marine bacterium, Vibrio anguillarum E 383a, in a medium containing colloidal chitin. 14)To obtain the enzymes that produce spec...