Characterization and cloning of chitin deacetylases from Rhizopus circinans.

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.

Glycoproteins from the cuticle of the Atlantic shore crab Carcinus maenas: I. Electrophoresis and Western-blot analysis by use of lectins.

The protein and glycoprotein content of four different neutral or acidic solvent extracts (0.5 M KCl, 10% EDTA, 0.1 N HCl, or 2% acetic acid) from the mineralized exoskeleton of a decapod crustacean, the Atlantic shore crab Carcinus maenas, were characterized by quantitative analysis of proteins, SDS-PAGE analysis, and probing with lectins on blots. The lectins used were Conconavalin A, Jacalin, soybean agglutinin, Maackia amurensis agglutinin II, and Sambucus nigra agglutinin. The results show that many proteins can be obtained from the crab cuticle without strong denaturants in the extraction medium. Many of the extracted cuticle proteins appeared to be glycosylated, bearing O-linked oligosaccharides and N-linked mannose-rich glycans. N-acetyl-galactosamine and N-acetylneuraminic acids were revealed, for the first time, as terminal residues on N-linked mannose-rich structures of crab cuticle glycoproteins. Sialylated glycoproteins might thus be involved in organic-mineral interactions in the calcified crab exoskeleton. The amount and variety of glycoproteins extracted with the acidic solvents are obviously different from those extracted with neutral solvents. HCl proved to be the best of the tested extraction solvents and a valuable alternative to EDTA.

Purification and characterisation of a 31-kDa chitinase from the Myzus Persicae aphid: a target for hemiptera biocontrol.

Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS-PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

Glycol chitosan improves the efficacy of intranasally administrated replication defective human adenovirus type 5 expressing glycoprotein D of bovine herpesvirus 1.

The ability of two soluble formulations, namely chitosan and glycol chitosan, when used as an intranasal adjuvant, to improve the immunogenicity of an intranasal human adenovirus type 5 replication defective expressing bovine herpesvirus 1 (BoHV-1) glycoprotein D based vaccine, was investigated in cattle. Their adjuvant effects on immune response by increasing clinical and especially virological protection against an intranasal BoHV-1 challenge were then evaluated. The best virological protection was obtained in calves immunized with the vaccine vector adjuvanted with glycol chitosan which decreased the challenge BoHV-1 virus excretion titres by 0.5-1.5 log when compared to those obtained in calves immunized with the vaccine vector alone or adjuvanted with chitosan. A slight difference in clinical scores was observed in calves immunized with the adjuvanted vaccine vector compared to calves immunized with the vaccine vector alone. The obtained data suggest that the tested soluble formulation of glycol chitosan has promising potential use as an intranasal adjuvant for recombinant viral vector vaccines in cattle.