Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure - PubMed (original) (raw)

Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure

H Tsujibo et al. Appl Environ Microbiol. 1998 Feb.

Free PMC article

Abstract

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

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Figures

FIG. 1

FIG. 1

Restriction maps of the cloned gene and the domain structure of ChiC. The transformants carrying the plasmids with appropriate deletions were transferred to an LB agar plate containing 0.05% glycol chitin, 0.01% trypan blue, and 100 μg of ampicillin per ml. Production of chitinase was judged by the formation of clear halos around the colonies. +, visible halo; −, no halo. The box indicates the coding sequence and the domain structure of ChiC. ▪, signal peptide; ░⃞, chitin-binding domain; ▧, fibronectin type III-like domain; □, catalytic domain.

FIG. 2

FIG. 2

Nucleotide sequence of the chiC gene. The putative ribosome-binding site (GGAA) is boxed. The −10 and −35 regions of a possible promoter sequence are double-underlined. The deduced amino acid sequence of ChiC is given below the nucleotide sequence. The N-terminal sequences of the native and cloned ChiC are underlined. The signal peptide cleavage site is shown by an arrow. The amino acid residues which seem to be essential for chitinase activity are circled. The stop codon is indicated by asterisks. The inverted repeat sequence is indicated by facing arrows with solid lines.

FIG. 3

FIG. 3

SDS-PAGE of the cloned and native chitinases. Lane M contains molecular size standards: phosphorylase b (94 kDa), bovine serum albumin (66 kDa), ovalbumin (42 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20.1 kDa). Lanes 1 and 2 correspond to the native ChiC from Alteromonas sp. strain O-7 and the cloned ChiC, respectively.

FIG. 4

FIG. 4

Alignment of the N-terminal and middle regions of ChiC with those of other enzymes. The sequences of the N-terminal and middle regions of ChiC (ALTCHIC) are aligned with those of_Alteromonas_ chitinase 85 (ALTCHI85), _Aeromonas_chitinase II (AERCHIII), Aeromonas chitinase A (AERCHIA),Bacillus cellulase 1 (BCICEL1), _Bacillus_cellulase 2 (BCICEL2), Vibrio chitodextrinase (VIBCDE),Pseudomonas carboxyesterase (PSECARB),Pseudomonas esterase (PSEEST), Bacillus chitinase A (BCICHIA), Bacillus chitinase D (BCICHID),Streptomyces chitinase 63 (STOCHI63),Streptomyces exochitinase (STOCHI01),Streptomyces chitinase C (STOCHIC), _Alcaligenes_poly-3-hydroxybutyrate depolymerase (ALCPHB), _Cellulomonas_endoglucanase B (CELENGL), and _Clostridium_α-amylase–pullulanase (CLOAP). Identical amino acids are indicated by white on black.

FIG. 5

FIG. 5

Binding assays of ChiC and GST fusion protein. (a) Binding of ChiC to chitin, chitosan, and Avicel. (b) Binding of GST fusion protein to chitin, chitosan, and Avicel. Data are from five independent experiments; standard errors are indicated by vertical lines.

FIG. 6

FIG. 6

SDS-PAGE of GST, GST fusion protein, and the N-terminal region of ChiC. Lane M contains molecular size standards: ovalbumin (42 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), and lysozyme (14.4 kDa). Lanes 1 to 3 correspond to GST, GST fusion protein, and the N-terminal region of ChiC.

FIG. 7

FIG. 7

Adsorption of the isolated domain to chitin. The figure shows the equilibrium adsorption isotherms ([B] versus [F]). Each datum point is from six independent experiments; standard errors are indicated by vertical bars.

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