title: Characterization of two Listeria innocua chitinases of different sizes that were expressed in Escherichia coli.
abstract: Two putative chitinase genes, lin0153 and lin1996, from the nonpathogenic bacterium Listeria innocua were expressed in Escherichia coli, and the gene products were characterized. The genes were close homologs of chitinases from the pathogenic bacterium Listeria monocytogenes, in which chitinases and chitin-binding proteins play important roles in pathogenesis in mice-infection models. The purified recombinant enzymes that are different in size, LinChi78 (lin0153 product) and LinChi35 (lin1996 product)-with molecular masses of 82 and 38 kDa, including vector-derived additional sequences, respectively-exhibited optimum catalytic activity under neutral and acidic conditions at 50 °C, respectively, and were stable over broad pH (4-11) and temperature (4-40 °C) ranges. LinChi35 displayed higher k cat and K M values for 4-nitrophenyl N,N-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2] than LinChi78. Both enzymes produced primarily dimers from colloidal chitin as a substrate. However, LinChi78 and LinChi35 could hydrolyze oligomeric substrates in a processive exo- and nonprocessive endo-manner, respectively, and showed different reactivity toward oligomeric substrates. Both enzymes could bind chitin beads but were different in their binding ability toward crystalline α-chitin and cellulose. The structure-function relationships of these chitinases are discussed in reference to other bacterial chitinases.
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Honda
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Wakita
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.|Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kawakita
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Oyama
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan. bt11532@ns.kogakuin.ac.jp.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D000595:Amino Acid Sequence; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008087:Listeria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases
keywords: Characterization; Chitin; Chitinase; Cloning; Expression; Listeria species