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

Yun Wang; Jin-Zhu Song; Qian Yang; Zhi-Hua Liu; Xiao-Mei Huang; Yan Chen

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, Km was 4.92 mg ml−1, and Kcat showed 6.25 s−1, thus the ratio of Kcat and Km was 1.27 ml s−1 mg−1. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.

CHITIN; ASPERGILLUS nidulans; ESCHERICHIA coli; GENE expression; CLONING

Applied Biochemistry & Biotechnology, 2010, Vol 162, Issue 3, p843

0273-2289

Academic Journal

10.1007/s12010-009-8772-z