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J Eukaryot Microbiol. 2016 Jan-Feb;63(1):37-45. doi: 10.1111/jeu.12246. Epub 2015 Jul 15.

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb).

Author information

1
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China.
2
Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China.
3
College of Life Sciences, Chongqing Normal University, Chongqing, 400047, China.

Abstract

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β-(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)(2-5) detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)(3-5). On the basis of structure-based multiple-sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site-directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.

KEYWORDS:

3D-modeling; GH19; enzymology; microsporidia; site-directed mutagenesis

PMID:
26108336
DOI:
10.1111/jeu.12246
[Indexed for MEDLINE]

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