Title page for ETD etd-05112006-154826


Type of Document Dissertation
Author Spencer, C. Thomas
URN etd-05112006-154826
Title Overproduction of xylose isomerase in recombinant Escherichia coli,
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Potts, Malcolm Committee Chair
Claus, G. W. Committee Member
Gregory, E. M. Committee Member
Larson, T. J. Committee Member
Rivard, Christopher J. Committee Member
Keywords
  • intramolecular oxidoreductases
  • recombinant enzyme
  • ´╗┐Escherichia coli
Date of Defense 1996-08-01
Availability unrestricted
Abstract
Aspects of recombinant enzyme production were characterized in genetically engineered Escherichia coli. Five strains transformed with the xylose isomerase overproduction system (pRK248/pTXI-1) were compared, based on parameters of cell metabolism and inducible enzyme activity in shake flask cultures. E. coli strain LE392 (pRK248/pTXI-l) performed the best with respect to nearly all of the parameters tested. The stability of the overproducing plasmids was tested in prolonged serial shake flask cultures. Segregational instability occurred in cultures lacking antibiotic selective pressure. The onset of this instability was influenced by host strain, plasmid construct and cultivation media. Despite its occurrence, the degree of plasmid instability, even in the worst case, would not be detrimental in the proposed application of this overproduction system. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-1) was characterized in four batch cultures. It was determined that both carbon source starvation and the accumulation of acetate as a byproduct of glucose metabolism diminished the E. coli's ability to produce xylose isomerase. The peak enzyme production was determined to be 1320 International Units (IU) of xylose isomerase activity per liter of culture. The cell mass and enzyme production of strain LE392 (pRK248/pTXI-l) was studied in fed-batch cultures. The amount of xylose isomerase the cells produced was dependent on the degree of glucose limitation they experienced during cultivation. The peak enzyme production was 3250 IU/L. The results of these experiments were interpreted in the context of this overproduction system and its proposed application for the production of ethanol from plant biomass.

Files
  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  LD5655.V856_1996.S734.pdf 4.21 Mb 00:19:29 00:10:01 00:08:46 00:04:23 00:00:22
[BTD] next to an author's name indicates that all files or directories associated with their ETD are accessible from the Virginia Tech campus network only.

Browse All Available ETDs by ( Author | Department )

dla home
etds imagebase journals news ereserve special collections
virgnia tech home contact dla university libraries

If you have questions or technical problems, please Contact DLA.