Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
AbstractMouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases. View Full-Text
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Kashimura, A.; Kimura, M.; Okawa, K.; Suzuki, H.; Ukita, A.; Wakita, S.; Okazaki, K.; Ohno, M.; Bauer, P.O.; Sakaguchi, M.; Sugahara, Y.; Oyama, F. Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli. Int. J. Mol. Sci. 2015, 16, 4028-4042.
Kashimura A, Kimura M, Okawa K, Suzuki H, Ukita A, Wakita S, Okazaki K, Ohno M, Bauer PO, Sakaguchi M, Sugahara Y, Oyama F. Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli. International Journal of Molecular Sciences. 2015; 16(2):4028-4042.Chicago/Turabian Style
Kashimura, Akinori; Kimura, Masahiro; Okawa, Kazuaki; Suzuki, Hirotaka; Ukita, Atsushi; Wakita, Satoshi; Okazaki, Kana; Ohno, Misa; Bauer, Peter O.; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka. 2015. "Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli." Int. J. Mol. Sci. 16, no. 2: 4028-4042.