Characterization of chitinase genes from an alkaliphilic actinomycete, Nocardiopsis prasina OPC-131

Abstract

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.

FIG. 1.

SDS-PAGE and zymogram analyses of proteins in the culture supernatant of N. prasina OPC-131. (A) SDS-PAGE stained with Coomassie brilliant blue R-250. (B) Zymogram of chitinase activity. N. prasina OPC-131 was grown in medium supplemented with 0.5% chitin, 0.5% chitin plus 1% glucose, or 1% glucose. Samples were taken at 80 h for these experiments. The same volume of each culture supernatant was concentrated to 1/30 of its original volume by ultrafiltration with NanoSpin Plus. The concentrated samples were applied to SDS-PAGE. Lanes: M, marker proteins; 1, 0.5% chitin; 2, 0.5% chitin plus 1% glucose; 3, 1% glucose.

FIG. 2.

Restriction maps of pCHIA629 and pCHIB377 and domain structures of ChiA and ChiB. (A and C) Restriction maps of PCHIA629 (A) and pCHIB377 (B). The arrows indicate the ORFs and the direction of transcription. (B and D). Domain structures of ChiA (B) and ChiB (D). Solid, signal peptide; hatched, ChtBD; open, catalytic domain.

FIG. 3.

Comparison of amino acid sequences of catalytic domains of ChiA and ChiB with those of other proteins. (A) Amino acid sequence of the active-site region of ChiA compared with those of family 18 chitinases. The SXGG and DXXDXDXE motifs are boxed. A Glu residue identified as a proton donor is marked with an asterisk. The residue number of the first amino acid in each line is shown on the left. Residues that are identical are indicated by boldface letters. ChiA, N. prasina chitinase A; StChi30, S. thermoviolaceus chitinase 30; SlChiA, S. lividans chitinase A; ScChiA, S. coelicolor chitinase A; BcChiD, B. circulans chitinase D. (B) Amino acid sequence of the catalytic domain of ChiB compared with those of family 19 chitinases. ChiB, N. prasina chitinase B; SgChiC, S. griseus chitinase C; ScChiG, S. coelicolor chitinase G; ScChiF, S. coelicolor chitinase F; StChi35, S. thermoviolaceus chitinase 35; BnChi25, B. napus class I chitinase 25; HvChi26, H. vulgare class II chitinase 26. The residue number of the first amino acid in each line is shown on the left. Residues that are identical are indicated by boldface letters.

FIG. 4.

SDS-PAGE of recombinant chitinases. Lanes: M, marker proteins; 1, ChiA; 2, ChiB; 3, ChiBΔ.

FIG. 5.

Effects of pH and temperature on chitinase activities. (A) The following buffer systems were used: McIlvaine buffer (pH 3.0 to 6.0), 50 mM Tris-HCl buffer (pH 7.0 to 8.0), and 50 mM glycine-NaOH buffer (pH 9.0 to 11.0). The reaction mixtures were incubated at the optimum temperature for each enzyme (ChiA, 60°C; ChiB, 60°C; ChiBΔ, 50°C) for 10 min. The amount of each enzyme was 20 mU. (B) The reaction was carried out at various temperatures for 10 min at the optimum pH of each enzyme. The buffers used were 50 mM Tris-HCl buffer (pH 7.0) for ChiA and McIlvaine buffer (pH 6.0) for ChiB and ChiBΔ. The amount of each enzyme was 20 mU. •, ChiA; ▴, ChiB; ▵, ChiBΔ.

FIG. 6.

Binding assays and hydrolytic activities of ChiB and ChiBΔ. (A) Binding assay mixtures contained 1.5 μg of ChiB (open bars) or ChiBΔ (solid bars) and 5 mg each of insoluble polysaccharides in 20 mM HEPES-KOH (pH 7.0). The mixtures were permitted to stand for 20 min on ice. The chitinase in the supernatant was measured, and the activity lost from the supernatant was assumed to be the activity bound. (B) Hydrolytic activities of ChiB (open bars) were measured at 60°C for 10 min using McIlvaine buffer (pH 6.0), and those of ChiBΔ (solid bars) were measured at 50°C for 10 min using the same buffer. Colloidal chitin or glycol chitin was used as a substrate. The error bars represent standard deviations.

FIG. 7.

Antifungal activities of ChiA, ChiB, and ChiBΔ. The mycelium of T. reesei was directly inoculated onto the center of a potato dextrose agar plate. After 24 h at 27°C, paper disks were placed around the T. reesei colony, and samples were put onto the disks. 1, blank disk; 2, 80 μg of ChiA; 3, 80 μg of ChiB; 4, 80 μg of ChiBΔ.