title: Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.
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.
authors.affiliation: a Department of Bioscience , Fukui Prefectural University , Fukui , Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Itoh
authors.forename: Ikumi
authors.initials: I
authors.lastname: Sugimoto
authors.forename: Takao
authors.initials: T
authors.lastname: Hibi
authors.forename: Fumiko
authors.initials: F
authors.lastname: Suzuki
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuo
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Fujii
authors.forename: Akira
authors.initials: A
authors.lastname: Taketo
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kimoto
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D002686:Chitin; D002688:Chitinases
keywords: Paenibacillus sp. FPU-7; cell surface-expressed chitinase; chitin; glycoside hydrolase family 18; multi modular protein