An exo-chitosanase was purified from the culture filtrate of Gongronella butleri NBRC105989 to homogeneity by ammonium sulfate precipitation, followed by column chromatography using CM-Sephadex C-50 and Sephadex G-100. The enzyme comprised a monomeric protein with a molecular weight of approximately 47,000 according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 4.0, and was stable between pH 5.0 and 11.0. It was most active at 45 degrees C, but was stable at temperatures below 30 degrees C. The enzyme hydrolyzed soluble chitosan and glucosamine (GIcN) oligomers larger than tetramers, but did not hydrolyze N-acetylglucosamine (GIcNAc) oligomers. To clarify the mode of action of the enzyme, we used thin -layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to investigate the products resulting from the enzyme -catalyzed hydrolysis of chitosan and NI-acetylchitohexaose [(GIcN)5-GIcNAcl with a GIcNAc residue at the reducing end. The results indicated that the enzyme is a novel exo-type chitosanase, exo-chitobiohydrolase, that releases (G1cN)2 from the non reducing ends of chitosan molecules. Analyses of the hydrolysis products of partially N-acetylated chitooligosaccharides revealed that the enzyme cleaves both GIcN-GIcNAc and GIcNAc-GIcN bonds in addition to GIcN-GIcN bonds in the substrate. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.