Aggeliki Martinou

Chitin deacetylases: new, versatile tools in biotechnology

Chitin deacetylases have been identified in several fungi and insects. They catalyse the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. Chitosans, which are produced by a harsh thermochemical procedure, have several applications in areas such as biomedicine, food ingredients, cosmetics and pharmaceuticals. The use of chitin deacetylases for the conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled, non-degradable process, resulting in the production of novel, well-defined chitosan oligomers and polymers.

Carbohydrate esterase family 4 enzymes: substrate specificity

The substrate specificity of selected enzymes classified under Carbohydrate Esterase family 4 (CE4) has been examined. Chitin deacetylase from Mucor rouxii and both a native and a truncated form of acetyl xylan esterase from Streptomyces lividans were found to be active on both xylan and several soluble chitinous substrates. Furthermore, the activities of all enzymes examined were significantly increased in the presence of Co(2+) when chitinous substrates were employed. However, the presence of this metal ion did not result in enhancing the activities of the enzymes when xylan was used as substrate. An acetyl xylan esterase from Bacillus pumilus, classified under Carbohydrate Esterase family 7, was found to be inactive towards all chitinous substrates tested. Finally, all enzymes examined were inactive towards cell wall peptidoglycan.

CHITIN DEACETYLATION BY ENZYMATIC MEANS : MONITORING OF DEACETYLATION PROCESSES

Abstract A method for monitoring enzymatic deacetylation processes of natural or artificial chitin substrates as well as N-acetylchitooligosaccharides by the direct determination of the acetate released is described. Furthermore, a new assay is presented for the determination of chitin deacetylase activity employing hexa-N-acetylchitohexaose [(GlcNAc)6] as substrate and measuring the acetate released enzymatically. The Km value for (GlcNAc)6 has been determined as well as the pH and temperature dependence of activity and the thermostability of the enzyme. Finally, initial studies on the effectiveness of the enzyme on various chitin and chitosan substrates are presented.

Isolation of chitin deacetylase from Mucor rouxii by immunoaffinity chromatography

Abstract The purification of chitin deacetylase from Mucor rouxii to homogeneity employing conventional methods has already been described. However, a lengthy protocol is required resulting in a low yield and specific activity for the enzyme. A 169-fold one-step purification of chitin deacetylase by immunoaffinity chromatography is reported, resulting in a homogeneous enzyme preparation. The enzyme purified using this procedure was judged to be electrophoretically homogeneous as tested by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate PAGE. Using antibodies of lower affinity, less severe chemical conditions were required for the desorption of immunoadsorbents. Chitin deacetylase purified by immunoaffinity chromatography exhibited a specific activity of 13 U mg −1 while a 30% yield was obtained, both much higher than the respective values obtained using conventional methodology.