Cloning and expression of a thermostable -amylase gene from Bacillus stearothermophilus TISTR 329 into Escherichia coli | |
| Author | II, Alfredo Jalbuna Anceno |
| Call Number | AIT Thesis no.BT-03-11 |
| Subject(s) | Cloning Gene expression |
| 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. BT-03-11 |
| Abstract | Extremophilic organisms have been a focus of many studies. Thermophilic enzymes, produced by thermophilic organisms, can be used in applications where they need to be stable at high temperatures. In the industrial hydrolysis of starch, thermostable amylases lead to economic processes. In this work, DNA recombination has been attempted. Thennostable tit-amylase gene has been amplified from Bacillus stearothermophflus TISTR 329 and cloned into Escherichia colt shuttle vectors pET9sn1 and pGEM-T. Sequence analysis of the cloned gene revealed up to 93% homology with B. stearothermoPhilus a ctr-amylase gene sequences in the GenBank. The predicted translation also showed alignment to the conserved regions of Cit-amylase catalytic domain and glycosyl hydrolases. The gene was cloned into the lacZ region of pET9sn1 and pGEM-T where it can be expressed under the control of T7 lac promoter. E. coli Rosetta and TOPIOF' were used as host for the expression of (it-amylase gene. E. coli transformants were selected on the basis of resistance to Kanamycin and Ampicillin in case of strain Rosetta and TOPIOF', respectively. The expression of cloned oi-arnylase gene in the transformants was studied by performing SDS-PAGE on culture supernatants and cell lysates to confirm extracellular .and intracellular expression, respectively. Transformants were also grown on starch agar to visualize (it—amylase activity. Overall recombination and expression of the gene has been achieved. However, further optimization for enhanced productivity needs to be done. Enhanced production of the thermostable cat-amylase under normal conditions with better control could then be possible. |
| Year | 2003 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. BT-03-11 |
| 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 | Biotechnology (BT) |
| Chairperson(s) | Rakshit, Sudip K.; |
| Examination Committee(s) | Mair, Graham;Valyasevi, Ruud;Athapol Noomhorm,; |
| Scholarship Donor(s) | The Royal Netherlands Government; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2003 |