Cloning of a Heat-Stable Chitin Deacetylase Gene from Aspergillus nidulans and its Functional Expression in Escherichia coli

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans . Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum . The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml −1 for the glycol chitin substrates, and its specific activity was 4.17 U mg −1 for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K m was 4.92 mg ml −1 , and K cat showed 6.25 s −1 , thus the ratio of K cat and K m was 1.27 ml s −1 mg −1 . The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.