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 degrees 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.
Wang
Yun
Y
Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
Song
Jin-Zhu
JZ
Yang
Qian
Q
Liu
Zhi-Hua
ZH
Huang
Xiao-Mei
XM
Chen
Yan
Y
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D000581:Amidohydrolases; C019866:chitin deacetylase