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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Acknowledgments
This research was supported by the projects of the Nature and Technology Heilongjiang province of China (C200609) and the Natural Scientific Research Innovation Foundation in Harbin Institute of Technology (HIT. NSRIF. 2008-18).
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Wang, Y., Song, JZ., Yang, Q. et al. Cloning of a Heat-Stable Chitin Deacetylase Gene from Aspergillus nidulans and its Functional Expression in Escherichia coli . Appl Biochem Biotechnol 162, 843–854 (2010). https://doi.org/10.1007/s12010-009-8772-z
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DOI: https://doi.org/10.1007/s12010-009-8772-z