Purification and characterization of chitinase from Bacillus circulans No.4.1.
Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35 degrees C for 5 days. Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially. The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa. This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)(2)]. The optimal conditions for this chitinase were pH 8.0 and 40 degrees C. The isoelectric point of the chitinase was 5.1. The amino acid composition of the purified chitinase was determined. The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V). Knowledge of these properties of chitinase from B. circulans No. 4.1 should be useful in the development of genetically engineered Bacillus sp. as biopesticides.
Wiwat
C
C
Department of Microbiology, Faculty of Pharmacy, Mahidol University, Sri-Ayudhya Rd., Bangkok 10400, Thailand.
Siwayaprahm
P
P
Bhumiratana
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013696:Temperature
D001426:Bacterial Proteins; D002686:Chitin; C044872:O-(carboxymethyl)chitin; C031610:glycolchitin; D002688:Chitinases