Enzymatic and genetic characterization of the DasD protein possessing N-acetyl-β-d-glucosaminidase activity in Streptomyces coelicolor A3(2)
Corresponding Author
Akihiro Saito
Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka, Japan
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Correspondence: Akihiro Saito, Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555, Japan. Tel.: +81538 45 0188; fax: +81538 45 0110; e-mail: [email protected]Search for more papers by this authorHiroki Ebise
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYukari Orihara
Department of Applied Biochemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorSatoshi Murakami
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYukari Sano
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorAkane Kimura
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYuuta Sugiyama
Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka, Japan
Search for more papers by this authorAkikazu Ando
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorTakeshi Fujii
National Institute of Agro-Environmental Sciences, Tukuba, Ibaraki, Japan
Search for more papers by this authorKiyotaka Miyashita
National Institute of Agro-Environmental Sciences, Tukuba, Ibaraki, Japan
Search for more papers by this authorCorresponding Author
Akihiro Saito
Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka, Japan
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Correspondence: Akihiro Saito, Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555, Japan. Tel.: +81538 45 0188; fax: +81538 45 0110; e-mail: [email protected]Search for more papers by this authorHiroki Ebise
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYukari Orihara
Department of Applied Biochemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorSatoshi Murakami
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYukari Sano
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorAkane Kimura
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorYuuta Sugiyama
Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka, Japan
Search for more papers by this authorAkikazu Ando
Department of Nanobiology, Graduate School of Advanced and Integration Science, Chiba University, Matsudo, Chiba, Japan
Search for more papers by this authorTakeshi Fujii
National Institute of Agro-Environmental Sciences, Tukuba, Ibaraki, Japan
Search for more papers by this authorKiyotaka Miyashita
National Institute of Agro-Environmental Sciences, Tukuba, Ibaraki, Japan
Search for more papers by this authorAbstract
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.
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