Elucidation of exo-β-d-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9
AbstractAbstract A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli . The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose ((GlcN) 2 ) and cellobiose (CG 2 ) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β- d -glucosaminide (GlcN-PNP) and 4-nitrophenyl-β- d -glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent 1 H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k cat / K m of (GlcN) 2 hydrolysis was 14 times greater than that of CG 2 hydrolysis. These results suggested that the protein is an exo-β- d -glucosaminidase (EC 188.8.131.52) rather than a glucan 1,4-β- d -glucosidase (EC 184.108.40.206). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN) 2 -phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.