Enzymatic and genetic characterization of the DasD protein possessing N-acetyl-β-d-glucosaminidase activity in Streptomyces coelicolor A3(2)

Authors


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: a-saito@ms.sist.ac.jp

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.

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