Extracellular expression of fungal chitin deacetylase gene in the yeast Pichia pastoris | |
| Author | Imastini Dinuriah |
| Call Number | AIT Thesis no. BP-01-03 |
| Subject(s) | Chitin Pichia pastoris |
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. BP-01-03 |
| Abstract | The use of chitin deacetylase enzyme (CDA) for converting chitin into chitosan catches the attention of many scientists. CDA genes from various sources of organisms have been cloned into fast growing bacteria or yeasts to increase CDA production. Following the success on cloning the CDA gene from Colletotrichum lindemuthianum UPS9 into the yeast Pichia pastoris strain GS115 (Mut) and KM71 (Muts) via plasmid shuttle vector pPIC9K done by Shrestha (2000), the CDA gene expression of his recombinant clones was investigated. Selection of the best CDA producer among the recombinant clones in the expression medium was carried out based on the CDA activity of each clone on a partially deacetylated chitin substrate (PDC, 65% DD 200 mesh). CDA activity was analyzed by using Gas-Liquid Chromatography based on the release of acetic acid during the enzyme assay. Clone Cl-6 was selected as the best CDA producer out of 63 clones tested. The effect of several factors on the production and characteristics of the CDA of the best clone was also studied. The CDA production was better in buffered minimal methanol medium than in non-buffered minimal methanol medium. Enzyme levels were inhibited rather than enhanced if chitin with different levels of deacetylation was included in the expression medium. The optimum condition for CDA assay was at 50°C and pH 5.8. It was found that the CDA of clone Cl-6 was not inhibited by acetate during the enzyme assay. The test on thermostability showed that the CDA activity remaining was more than 90% even after exposing the CDA to 50°C for one hour before enzyme assay but not for more than 3 hours |
| Year | 2001 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. BP-01-03 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Bioprocess Technology (BP) |
| Chairperson(s) | Stevens, Willem F.; |
| Examination Committee(s) | Suwalee Chandrkrachang;Sunee Nithisinprasert ;Macaranas, Julie; |
| Scholarship Donor(s) | DUE Project Batch II, LPIU - UNSOED, Indonesia ; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2001 |