Thesis (M.Sc.) - Asian Institute of Technology

A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science, School of Environment, Resources and Development

Thesis (M. Sc.) - Asian Institute of Technology, 2002

Chitin, which occurs in the exoskeleton of arthropods and in the cell wall of fungi, is an abundant renewable natural resource. Chitosan, the deacetylated form of chitin has a broad variety of industrial and biomedical applications. It is traditionally produced by a harsh thermochemical procedure that is environmentally unsafe and not easily controlled. The use of chitin deacetylases offers the possibility of an alternative procedure to the chemical process for the deacetylation of chitin. Molecular cloning of CDA gene into a suitable host is therefore explored to achieve efficient production of the enzyme. This work aimed at the cloning of CDA gene from Mucor rouxii and the constructing of CDA-overexpressing strains of Pichia pastoris. Chitin deacetylase (CDA) gene after amplification from Mucor rouxii ATCC 24905 was cloned into two of the E.coli-Pichia pastoris shuttle vectors, pPIC3.5K and pPIC9K. In case of pPIC9K the gene was cloned in phase with the a-secretion signal factor present in the parent vector for the secreted expression of CDA. The gene was put in both vectors under the transcriptional control of Pichia pastoris alcohol oxidase gene promoter. The gene was transformed into Pichia pastoris GS115 (methanol utilization fast, Mut) to obtain overexpressing strains. Pichia transformants were selected on histidine deficient medium and screened for their ability to use methanol as a sole source of carbon. The selected clones were screened for transformants resistant to G418 (Geneticin). Integration of the gene in Pichia genome was confirmed through PCR analysis of the transformants. The intracellular and secreted expression of the cloned CDA gene in Pichia transformants was analyzed through SDSp AGE analysis.