Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7

Abstract

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Fig 1

Chitin degradation and cell morphology of Paenibacillus sp. strain FPU-7. (A) Colonies of strain FPU-7 on an α-chitin powder plate. Cells of strain FPU-7 were smeared on a 1.0% (wt/vol) α-chitin powder plate, and the plate was incubated at 30°C for 1 week. (B) Strain FPU-7 was grown at 30°C for 5 days with shaking, in bonito extract medium containing 5.0% (wt/vol) crab shell chitin flakes. The solid matter (chitin flakes) in the left flask disappeared after 5 days of incubation (right flask). (C) Cell morphology of strain FPU-7 (stationary phase) observed by SEM. Bar, 1 μm.

Fig 2

Phylogenetic analysis of the 16S rRNA genes of Paenibacillus type strains and FPU-7 (shaded). The numbers at the nodes indicate the percentage bootstrap values for each node based on 1,000 bootstrap resamplings estimated by ClustalW software, using the neighbor-joining method. The tree was rooted with the 16S rRNA gene of Bacillus subtilis as an outgroup. The scale bar indicates 0.01 substitution per nucleotide position.

Fig 3

Zymogram analysis of the active chitinases in culture supernatants. Strain FPU-7 was grown at 30°C for 2 days with shaking in polypeptone medium supplemented with Glc, GlcN, GlcNAc, or (GlcNAc)₂ as described in Materials and Methods. Aliquots of culture supernatants were loaded onto an SDS-PAGE gel containing 0.1% (wt/vol) ethylene glycol chitin, and activity staining was performed. Medium alone served as a negative control; unsupplemented cultures (polypeptone medium alone) and cultures supplemented with Glc, GlcN, GlcNAc, and (GlcNAc)₂ were tested.

Fig 4

Domain structures of FPU-7 chitinases. The bars and numbers indicate positions of amino acid sequences. Protein sequence was analyzed by using the Pfam server, and the locations of the following domains are indicated: signal peptide (black boxes), GH-18 catalytic domains (GH-18; Pfam ID, PF00704), carbohydrate-binding module (CBM-5/12; Pfam ID, PF02839), chitinase A N-terminal domain (ChitinaseA_N; Pfam ID, PF08329), fibronectin type III domain (hatched boxes; Pfam ID, PF00041), and SLH domains (boxes with horizontal lines; Pfam ID, PF00395).

Fig 5

Overproduction and purification of the recombinant His-tagged ChiW protein and localization of native ChiW in FPU-7 cells. (A) Arrangement of domains and deleted regions of ChiW. The solubility of the truncated mutants is indicated on the left. The ChiW with the SLH domain deleted was the only soluble form obtained. (B) SDS-PAGE of purified SLH domain deletion-containing ChiW (right lane). The purified enzyme was subjected to SDS-PAGE (15% gel). Protein bands were stained with CBB R-250. Lane M, molecular weight standards. The N-terminal sequences of the bands were SVHMN and NHKVH, as determined by protein sequencing. The site-specific proteolysis occurred between Asn282 and Ser283 during the purification step. (C) Expression and localization of ChiW in FPU-7. FPU-7 was grown in medium containing 1.0% (wt/vol) polypeptone N and 0.5% NaCl with or without 24 mM (GlcNAc)₂ at pH 7.5. ChiW protein was induced by (GlcNAc)₂ and was detected in the cell particulate fraction by immunoblotting.