Heterodisaccharide 4-O-(N-acetyl-ß-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase.

AIMS: To investigate the attractant effect of 4-O-(N-acetyl-ß-D-glucosaminyl)-D-glucosamine (GlcNAc-GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc-GlcN from N,N'-diacetylchitobiose (GlcNAc)(2). METHODS AND RESULTS: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane-migration method. The results demonstrated that GlcNAc-GlcN functions as an effective chemoattractant in the CE family 4 COD-producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc-GlcN appears to stimulate polar flagellum rotation. CONCLUSIONS: GlcNAc-GlcN is a specific chemoattractant for the CE family 4 COD-producing vibrios, V. parahaemolyticus and V. alginolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: It was clarified for the first time that GlcNAc-GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc-GlcN from (GlcNAc)(2).

Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin.

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.

Hydrolytic activity of alpha-galactosidases against deoxy derivatives of p-nitrophenyl alpha-D-galactopyranoside.

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) alpha-D-galactopyranoside were synthesized, and hydrolytic activities of the alpha-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger alpha-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (K(m) and Vmax) of the enzymes for the hydrolysis of PNP alpha-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.