Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. strain O-7

Abstract

The chitinase B (ChiB) secreted by Alteromonas sp. strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced. The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30 degrees C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0 degrees C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The kca)/Km value for the ChiB reaction at 10 degrees C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.

FIG. 1.

(A) Deduced amino acid sequence encoded by the chiB gene. The signal peptide sequence is underlined. The type 3 ChtBD is boxed, and the Fn3-like domain is shaded. The regions that are homologous to the consensus sequence of the type 3 ChtBD are indicated by white letters on a black background. The N-terminal amino acid sequence of ChiB purified from the culture supernatant is shown by bold letters. Solid circles above amino acid residues represent the consensus sequences of family18 chitinases. (B) Diagram of the domain structure of ChiB: signal peptide,▪; ChtBD, ░⃞; Fn3-like domain, ▩; catalytic domain, □.

FIG. 2.

Western blot analysis of ChiB. Alteromonas sp. strain O-7 was cultured at 27°C for 24 h. Culture supernatant samples were taken at 3, 5, 7, 9, and 24 h. Each of the culture supernatant samples (180 μl) was added to 20 μl of 20% trichloroacetic acid and centrifuged. The pellets were dissolved with 20 μl of SDS-PAGE sample buffer and subjected to SDS-12% PAGE. Lane M, prestained molecular weight marker.

FIG.3.

SDS-PAGE of recombinant ChiB, ChiBΔN1, and ChiBΔN1ΔN2. Lanes: M, molecular size standards; 1, ChiB; 2, ChiBΔN1; 3, ChiBΔN1ΔN2.

FIG. 4.

(A) Temperature dependence of ChiA, ChiB, and ChiC. Reactions were carried out at various temperatures for 30 min with 50 mM citrate buffer, pH 6.0. The ChiA, ChiB, and ChiC protein concentrations were 1.7, 1.1, and 1.9 ng/μl, respectively. Symbols: □, ChiA; •, ChiB; ▵, ChiC. (B) Thermal stabilities of ChiA, ChiB, and ChiC. The enzymes were incubated at 40°C in 50 mM citrate buffer (pH 6.0), and residual activities of timed aliquots were measured by using pNP-(GlcNAc)₂ as the substrate. The ChiA, ChiB, and ChiC protein concentrations were 1.7, 1.1, and 1.9 ng/μl, respectively. Symbols: □, ChiA; •, ChiB; ▵, ChiC.

FIG. 5.

Real-time quantitative PCR analysis of the chiB transcript. Alteromonas sp. strain O-7 was cultured in Bacto Marine Broth 2216 (open bar) or the same medium containing 1.0% GlcNAc (solid bar) at 17, 27, or 37°C. The data shown are the means ± the standard deviations of three independent experiments.