Reaction pattern of Bacillus cereus D-11 chitosanase on chitooligosaccharide alcohols.

The purified endochitosanase (Mw 41 kDa) from bacterium Bacillus cereus D-11 hydrolyzed chitooligomers (GlcN) 5-7 into chitobiose, chitotriose, and chitotetraose as the final products. The minimal size of the oligosaccharides for enzymatic hydrolysis was a pentamer. To further investigate the cleavage pattern of this enzyme, chitooligosaccharide alcohols were prepared as substrates and the end products of hydrolysis were analyzed by TLC and HPLC. The chitosanase split (GlcN) 4 GlcNOH into (GlcN) 3 + (GlcN) 1 GlcNoH, and (GlcN) 5 GlcNOH into (GlcN) 4 + (GlcN) 1 GlcNOH and (GlcN)3+(GlcN)2GlcNOH. The heptamer (GlcN) 6 GlcNOH was split into (GlcN) 5 [thereafter hydrolyzed again into (GlcN)3+(GlcN) 2 ]+(GlcN) 1 GlcNOH, (GlcN)4+(GlcN) 2 GlcNOH, and (GlcN)3+(GlcN)3GlcNOH, whereas (GlcN) 1-3 GlcNOH was not hydrolyzed. The monomers GlcN and GlcNOH were never detected from the enzyme reaction. These results suggest that D-11 chitosanase recognizes three glucosamine residues in the minus position and simultaneously two residues in the plus position from the cleavage point.