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  • Title: Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.
    Author: Itoh T, Sugimoto I, Hibi T, Suzuki F, Matsuo K, Fujii Y, Taketo A, Kimoto H.
    Journal: Biosci Biotechnol Biochem; 2014; 78(4):624-34. PubMed ID: 25036959.
    Abstract:
    Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a β-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.
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