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Title: Chitinoclastic enzymes from fungal and viral sources
Author: Ferguson, M. J. L.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1999
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The opportunistic pathogen, Candida albicans, was screened for the presence of a chitin deacetylase homologue of two Saccharomyces cerevisiae chitin deacetylase genes. A candidate chitin deacetylase gene was identified, that showed high sequence homology to both S. cerevisiae chitin deacetylase genes, and termed CaCDA1. The gene CaCDA1 was over-expressed in S. cerevisiae haploid yeast cells which were screened for evidence of chitin deacetylase activity. No chitin deacetylase enzyme product was identified in either S. cerevisiae cells containing over-expressed CaCDA1 or in C. albicans yeast, hyphas and chlamydospore cells. Northern analysis of CaCDA1 showed that the gene was not transcribed during yeast cell stationary phase growth or in early growth of hyphal cells. The chlamydospore of C. albicans was separated from other cell types and studied in detail using biochemical assays, scanning electron microscopy and fluorescent stains which revealed that the cell wall of the chlamydospore contains chitin and possibly dityrosine. Chitin deaceytlation is the first step in the alternative pathway of chitin degradation which is followed by chitosanase then glucosaminidase. A chitosanase enzyme was studied using an over-expressed algal virus, Paramecium bursaria Chlorella virus-1 (PBCV-1), gene in Escherichia coli. A putative chitosanase protein was purified and characterised along with three other PBCV-1 putative chitinase proteins. Kinetic studies of the PBCV-1 proteins showed that the chitosanase protein was active along with two of the chitinases although one of the chitinases was not active or performed some unknown function. In silico analysis of the amino acid sequences of the chitinases showed that the PBCV-1 sequences were more similar to bacterial and fungal sequences than to other viral chitinases. A model is presented to explain the possible roles that chitinase and chitosanase plays in the infection cycle of PBCV-1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available