Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2

Chih Min Wen, Chien Sheng Tseng, Chih Yu Cheng, Yaw-Kuen Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

181 Scopus citations


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 pl of 6.3. It is thermally stable at 60°C and pH 6-8 for more than 3 h. The optimal activity is in the range of 50-60°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 ANNLGSKLLVGYVVHN, 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.

Original languageEnglish
Pages (from-to)213-219
Number of pages7
JournalBiotechnology and Applied Biochemistry
Issue number3
StatePublished - 1 Jan 2002


  • Bacillus cereus
  • Chitin
  • Chito-oligosaccharide

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