title: Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2.
abstract: A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extracellular chitinase was purified to >90% homogeneity from the culture filtrate. The purification involved hydrophobic-interaction and gel-filtration chromatographic separations with a yield of 58%. The purified enzyme (ChiNCTU2) is a monomeric protein with an estimated molecular mass of 36.5 kDa and a pI of 6.3. It is thermally stable at 60 degrees C and pH 6-8 for more than 3 h. The optimal activity is in the range of 50-60 degrees C at pH 7.0. Chitobiose is the predominant product throughout the enzymic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. Chito-oligosaccharides [with degree of polymerization (DP) values of 4-6] are good substrates of the purified enzyme, whereas a DP3 oligomer was slowly hydrolysed to form DP1 and DP2 sugars. The first 15 N-terminal amino acids of the enzyme were determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. A PCR cloning technique was employed to obtain the corresponding gene from Bacillus NCTU2. The gene sequence was determined to be 1080 bp, encoding a polypeptide of 360 amino acids with the first 27 amino acids as the signal peptide.
authors.affiliation: Department of Applied Chemistry, The National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
authors.forename: Chih-Min
authors.initials: CM
authors.lastname: Wen
authors.forename: Chien-Sheng
authors.initials: CS
authors.lastname: Tseng
authors.forename: Chih-Yu
authors.initials: CY
authors.lastname: Cheng
authors.forename: Yaw-Kuen
authors.initials: YK
authors.lastname: Li
mesh_terms: D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D002688:Chitinases