title: Enzymatic and genetic characterization of the DasD protein possessing N-acetyl-β-d-glucosaminidase activity in Streptomyces coelicolor A3(2).
abstract: The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2)  temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin.
authors.affiliation: Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka, Japan. a-saito@ms.sist.ac.jp
authors.forename: Akihiro
authors.initials: A
authors.lastname: Saito
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ebise
authors.forename: Yukari
authors.initials: Y
authors.lastname: Orihara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Murakami
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sano
authors.forename: Akane
authors.initials: A
authors.lastname: Kimura
authors.forename: Yuuta
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujii
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Miyashita
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics* / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002686:Chitin; D000118:Acetylglucosaminidase