title: Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9.
abstract: 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 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). 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.
authors.affiliation: Department of Food Science, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan. honda@ishikawa-pu.ac.jp
authors.forename: Yuji
authors.initials: Y
authors.lastname: Honda
authors.forename: Nozomi
authors.initials: N
authors.lastname: Shimaya
authors.forename: Kana
authors.initials: K
authors.lastname: Ishisaki
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ebihara
authors.forename: Hajime
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000378:metabolism; D057075:Enzyme Assays; D004795:Enzyme Stability; D006596:Hexosaminidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010776:Photobacterium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
chemical_list: D004187:Disaccharides; D011994:Recombinant Proteins; D006596:Hexosaminidases