Date of Graduation
1997
Document Type
Dissertation/Thesis
Abstract
Approximately 95-99% of soil phosphorous is unavailable to plants, with {dollar}\\sim{dollar}90% of phosphate fertilizers also being rendered unavailable upon application. 2-Ketogluconate (2KG) has been shown to solubilize insoluble phosphates in vitro. If plants themselves could solubilize the insoluble phosphate in the soil, this would greatly reduce the need for phosphate fertilizers. Two greenhouse experiments were conducted to examine in vitro and in situ solubilization of insoluble phosphates (calcium phosphate, RP, and iron phosphate, IP) with increasing concentrations of 2KG, as seen through plant (Lycopersicon esculentum) growth and Pi uptake. In the first experiment (in situ solubilization), water alone showed a significantly greater effect than 350ug/ml 2KG. In the second experiment, there was no significant difference between the water and 350ug/ml 2KG between the in vitro and in situ solubilizations of RP. But, there was a significantly greater uptake of Pi in the in vitro solubilization treatment groups for RP. Again all groups in both experiments showed a negative linear relationship to the increasing concentrations of 2KG. There was no significant difference found in the treatments using IP in either experiment. The main purpose of this project was to clone the gene(s) responsible for the production of Gluconate dehydrogenase (Gln DH), especially the 22kD subunit, and insert it into a plant to have the plant produce Gln DH. Gln DH was delipidated and deglycosylated and the 22kD subunit was found to consist of 4kD lipid and 6kD glycans. The activity of the holoenzyme was also found to be based on lipidation and glycosylation. Triple quadrapole ESMS sequencing at the University of Michigan resulted in three different ambiguous sequences, possibly sequencing partially delipidated and deglycosylated subspecies of the 22kD subunit or a contaminating protein. A three step sequential HPLC protocol, separating the holoenzyme, the 22kD subunit, then the delipidated and deglycosylated 22kD subunit, resulted in the delipidated and deglycosylated 22kD subunit being isolated to HPLC homogeneity. An attempt to sequence the protein again at the University of Michigan come up with negative results.
Recommended Citation
Gordon, John David, "Isolation and characterization of the 22 kD subunit of gluconate dehydrogenase derived from Pseudomonas aeruginosa PAO1." (1997). Graduate Theses, Dissertations, and Problem Reports. 8938.
https://researchrepository.wvu.edu/etd/8938