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Published on: December 30, 2016
Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two
Takafumi Itoh1, Ikumi Sugimoto, Takao Hibi
1a Department of Bioscience , Fukui Prefectural University , Fukui , Japan.
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.
Insights
This study overproduced and purified a novel chitinase, ChiW, from Paenibacillus sp. strain FPU-7. The enzyme effectively hydrolyzes chitin into N,N'-diacetyl-D-chitobiose, identifying key catalytic residues.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Paenibacillus sp. strain FPU-7 produces chitinases that effectively hydrolyze chitin.
- ChiW is a novel, monomeric, cell surface chitinase from P. FPU-7 with a molecular mass of 150 kDa.
Purpose of the Study:
- To overproduce and purify an active ChiW enzyme lacking N-terminal anchoring domains.
- To characterize the biochemical properties and catalytic mechanisms of ChiW.
Main Methods:
- Overproduction of truncated ChiW in Escherichia coli and purification.
- Biochemical characterization including substrate hydrolysis.
- Vacuum-ultraviolet circular dichroism spectroscopy.
- Site-directed mutagenesis to identify catalytic residues.
Main Results:
- Active ChiW, lacking N-terminal domains, was successfully purified.
- The enzyme hydrolyzed various insoluble chitins to N,N '-diacetyl-D-chitobiose.
- The N-terminal region exhibited a β-stranded-rich structure.
- Glu691 and Glu1177 were identified as the catalytic residues within the two GH-18 domains.
Conclusions:
- The purified ChiW enzyme is catalytically active and produces N,N '-diacetyl-D-chitobiose.
- The unique N-terminus may play a role in enzyme structure or function.
- Key catalytic residues Glu691 and Glu1177 are crucial for chitin hydrolysis.
