title: Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene.
abstract: A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.
authors.affiliation: Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA.
authors.forename: Pyoung Il
authors.initials: PI
authors.lastname: Kim
authors.forename: Tae Heung
authors.initials: TH
authors.lastname: Kang
authors.forename: Kyoung Jin
authors.initials: KJ
authors.lastname: Chung
authors.forename: In Seon
authors.initials: IS
authors.lastname: Kim
authors.forename: Ki-Chul
authors.initials: KC
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase