Characterization and Cloning of Chitin Deacetylases from Rhizopus Circinans

in Protein Expression & Purification (2008), 59

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 ... [more ▼]

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. [less ▲]

Les chitosanes – nouveaux adjuvants pour la vaccination par voie muqueuse chez les animaux

in Annales de Médecine Vétérinaire (2003), 147(5, OCT-NOV), 343-350

The advantage of mucosal vaccination is the induction of an immune response at entry sites of pathogens. Because vaccines alone are poorly bioavailable after mucosal administration, they need to be co ... [more ▼]

The advantage of mucosal vaccination is the induction of an immune response at entry sites of pathogens. Because vaccines alone are poorly bioavailable after mucosal administration, they need to be co-administered with penetration enhancers, or adjuvants. Numerous studies have demonstrated that chitosans and their derivatives are safe and effective mucosal absorption enhancers of hydrophylic macromolecules such as peptides and proteins. Chitosan is a cationic polysaccharide derived from chitin present in the covering layer of arthropods and in the cell walls of many fungi. Association of vaccines to chitosans, their derivatives or some of their particulate systems, such as nano- and microparticles, has also shown to enhance antigen uptake by mucosal lymphoid tissues, thereby inducing mucosal and systemic immune responses against these antigens. Chitosan and its derivatives are promising adjuvants for mucosal vaccine delivery in animals. [less ▲]