Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

Thesis (Ph.D)--Physics, Georgia Institute of Technology, 2009. / Committee Chair: M. Raymond Flannery; Committee Member: Daniel Goldman; Committee Member: Dewey H. Hodges; Committee Member: Li You; Committee Member: Turgay Uzer. Part of the SMARTech Electronic Thesis and Dissertation Collection.

published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

No description available.

The tyrosine recombinases XerC and XerD function in the monomerisation of circular dimer replicons in many bacteria. The recombining complex contains two synapsed recombination sites and two molecules each of XerC and XerD. Recombination proceeds through two sequential steps of DNA strand exchanges separated in time and space. A specific pair of recombinases initiates the reaction forming a Holliday junction intermediate, which undergoes a conformational change to allow resolution to recombinant products by the other pair of enzymes. In an attempt to understand the molecular basis of recombination machine assembly and coordination of catalysis, chimeras of XerC and XerD were constructed and their properties studied in partial and complete recombination reactions. XerC and XerD are two-domain proteins, whose C-terminal regions contain all of the catalytic residues. It is demonstrated here that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild type partners. However, the normal pattern of catalysis is dramatically altered: strand exchange by the recombinase variant is stimulated, while that by the wild type partner is impaired. The primary determinants for the mutant phenotype are shown to reside in the region of a-helix B of XerCD. It is also demonstrated that the exchange of the extreme C-termini of XerCD has a profound effect on the direction of HJ resolution. These observations confirm the importance of the cyclic C-terminal "donor-acceptor" interactions between XerC and XerD. Finally, the recombination reaction catalysed by ResD, a tyrosine recombinase encoded by the F-plasmid of E. coli, which is believed to function in the monomerisation of F-plasmid dimers, was reconstituted in vitro. Recombination is intramolecular and shows topological selectivity. ResD lacks a region corresponding to the N-terminal domains of XerCD, and hence its characterisation might supply further insights about the roles of the N-terminal domains of tyrosine recombinases.

Thesis (M.Sc.). / Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Junghuei Chen, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis supervisor: Robert E. Malone. Includes bibliographic references (p. 258-279).