Reference : Characterization and Cloning of Chitin Deacetylases from Rhizopus Circinans
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/76474
Characterization and Cloning of Chitin Deacetylases from Rhizopus Circinans
English
Gauthier, Carole mailto [Université de Liège - ULg > Département des sciences de la vie > Biologie moléculaire et biotechnologie végétales >]
Clerisse, Fabienne mailto [Université de Liège - ULg > Département des sciences de la vie > Département des sciences de la vie >]
Dommes, Jacques mailto [Université de Liège - ULg > Département des sciences de la vie > Biologie moléculaire et biotechnologie végétales >]
Versali, Marie-France mailto [Université de Liège - ULg > Département des sciences de la vie > Biologie moléculaire et biotechnologie végétales >]
26-Jan-2008
Protein Expression & Purification
59
127-137
Yes (verified by ORBi)
International
1046-5928
[en] Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of approximately 75kDa. At 37 degrees C it shows optimal activity at pH 5.5-6. Its pH stability and thermal stability are good. Mn(2+) and Mg(2+) slightly enhance the activity of the enzyme and Cu(2+) strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of approximately 75kDa and, like the native enzyme, showed optimal activity at pH 5.5-6 at 37 degrees C. It was strongly inhibited by Cu(2+). The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.
Researchers
http://hdl.handle.net/2268/76474
10.1016/j.pep.2008.01.013

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