Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.18.104.22.168) and chitin deacetylase (E.C.22.214.171.124) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains.
The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the “NodB homology domain”(Caufrier et al., 2003) or “polysaccharide deacetylase domain”. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006).
Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996).
The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics.
The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified.
In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5’ extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3’ extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase.
The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.