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Int. J. Mol. Sci. 2015, 16(2), 4028-4042; doi:10.3390/ijms16024028

Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli

1
Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan
2
Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Bing Yan
Received: 26 December 2014 / Accepted: 3 February 2015 / Published: 13 February 2015
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Mouse 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
Keywords: acidic mammalian chitinase; allergy; asthma; catalytic domain; chitin-binding activity; chitinolytic activity; colloidal and crystalline chitin; food processing; innate immunity; mouse acidic mammalian chitinase; allergy; asthma; catalytic domain; chitin-binding activity; chitinolytic activity; colloidal and crystalline chitin; food processing; innate immunity; mouse
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

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.

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Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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