Author/Editor | Jeraj, Nataša; Kunič, Barbara; Lenasi, Helena; Breskvar, Katja | |
Title | Purification and molecular characterization of chitin deacetylase from Rhizopus nigricans | |
Type | članek | |
Source | Enzyme Microb Technol | |
Vol. and No. | Letnik 39 | |
Publication year | 2006 | |
Volume | str. 1294-9 | |
Language | eng | |
Abstract | Chitin deacetylase is an enzyme, which catalyses the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. In the present report we purified the chitin deacetylase from mycelial extracts of a zygomycete Rhizopus nigricans by sequential ammonium sulfate precipitation, CM Sepharose chromatography and DEAE-cellulose chromatography. The progress of enzyme purification was followed by measurement of enzyme activity using partially O-hydroxyethylated chitin (glycol chitin) radiolabelled in N-acetyl groups as a substrate. The apparent molecular mass of chitin deacetylase obtained by SDS-PAGE was approximately 100 kDa. A cDNA library containing a chitin deacetylase gene from R. nigricans was constructed and the complete gene was sequenced. The complete gene contains an open reading frame of 1341 nucleotides, which encodes a sequence of 447 amino acid residues. The estimated molecular mass is 47 kDa, suggesting that carbohydrate content is 53% by weight of the protein. The gene sequence consists of nucleotides encoding a conserved polysaccharide deacetylase domain located in the middle, covering 34% of the entire sequence. Overall, there were eight possible N-linked glycosylation sites. The deduced amino acid sequence shows the highest identity with chitin deacetylases from other zygomycetes Phycomyces blakesleeanus, Gongronella butleri, Rhizopus oryzae and Mucor rouxii (55%, 48%, 43% and 41% identity, respectively). | |
Descriptors | RHIZOPUS CELL WALL CHITIN HYDROLYSIS CHROMATOGRAPHY, DEAE-CELLULOSE CHROMATOGRAPHY, AGAROSE BASE SEQUENCE |