title: Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase.
abstract: Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.
authors.affiliation: Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
authors.forename: Lanxiang
authors.initials: L
authors.lastname: Huang
authors.forename: Ewelina
authors.initials: E
authors.lastname: Garbulewska
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Sato
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kato
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nogawa
authors.forename: Goro
authors.initials: G
authors.lastname: Taguchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Shimosaka
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007564:Japan; D008969:Molecular Sequence Data; D009346:Neisseriaceae / Q000201:enzymology* / Q000235:genetics*; D009876:Operon / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D014242:Trichoderma / Q000187:drug effects
chemical_list: D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine