Cloning of a Heat-Stable Chitin Deacetylase Gene from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and its Functional Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Yun Wang Jin-Zhu Song Qian Yang Zhi-Hua Liu Xiao-Mei Huang Yan Chen |
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Institution: | (1) Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, 150001, China; |
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Abstract: | 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. |
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Keywords: | |
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