A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Technical Science, School of Environment, Resources and Development

Natural non-digestible polysaccharides such as cellulose and chitin are of great technological importance, as they are available in massive amounts and possess unique properties which are not found in synthetic polymers. Chitin is the most abundant natural biopolymer after cellulose. It exists in the exoskeleton of insects, crustaceans, in cell wall of fungi and recently detected in some vertebrates. The direct application of chitin is yet to be found whereas the deacetylated form of it, known as chitosan, is a high-value biopolymer. Chitosan is more tractable than chitin and finds its applications in biomedical products, including word dressings and artificial skin, in cosmetics, in food and in many sectors of industry. Chitosan is mainly obtained by thermochemical deacetylation of chitin, which produces large amounts of proteinoid alkaline waster and results heterogeneous product. The use of the enzyme chitin deacetylase (CDA, EC 3.5.1.41) for biocatalytic deacetylation offers an opportunity to overcome these problems. In an attempt to find out the best source of chitin deacetylase, 6 different strains of Colletotrichum lindemuthianum were investigated. C. lindemuthianum UPS9 was selected for the isolation of the cda open reading frame (ORF) of the cda gene coding for the chitin deacetylase to clone and express CDA in a production microorganism. The recombinant strain carrying the cda ORF extended with the nucleotides for six histidine residues at its C-terminal end was constructed. In order to select the best CDA producer, a microfluorimetric technique was developed for the CDA assay. The method is simple, easy and inexpensive to screen large numbers of CDA producing microorganisms. The microorganisms Escherichia coli, Corynebacterium glutamicum and Pichia pastoris were investigated for the expression of CDA. The yeast, P. pastoris was found to be the best producer. The recombinant enzyme was secreted in large quantity by growing P. pastoris in a bioreactor. Purification of the recombinant CDA from the culture medium, based on the binding of the 6xHis tag to chelating HiTrap column, was performed in a single step without prior concentration. A yield of 7.2 mg pure CDA was obtained per litre of culture medium. The molecular mass of the recombinant CDA was determined to be of culture medium. The molecular mass of the recombinant CDA was determined to be 25.19 kDa. The enzyme is active on various chitinous substrates. The optimum temperature and pH for the activity are 60°C and 8.0, respectively. The enzyme is thermostable and stable in a wide range of pH. The presence of Co ions enhances the activity significantly. The Kcat/Km ratio was found to be better for hexa N-acetyl chitohexaose compared to tetra N-acetyl chitotetraose and penta N-acetyl chitopentaose. The availability of a reproducible process for the large scale production and purification of CDA offers new perspectives to design an industrial process based on the biocatalytic conversion of chitin into chitosan and to elucidate the three dimensional structure and working mechanism of this enzyme.

Thesis (Ph.D.) - Asian Institute of Technology, 2004