The ability of two different pseudomonads, Pseudomonas oleovorans and Pseudomonas putida to metabolize and produce polymers from functional carbon substrates, in particular phenyl-containing substrates was investigated. A comparison of the two bacteria revealed P. putida to be the superior microorganism in that it produced higher cell yields and polymer yields regardless of the substrate on which it was grown. The bacterial polyester produced when P. oleovorans was grown on a mixture of 5-phenylvaleric acid and nonanoic acid, included both a homopolymer, poly-3-hydroxyphenylvalerate (PHPV) and a copolymer, poly-3-hydroxynonanoate (PHN). The intracellular location of each of these polymers was determined by selective staining of the inclusion body granules with ruthenium tetraoxide. Examination of the stained granules by transmission electron microscopy showed that both types of polyesters occurred sequentially in the same granule. Poly-3-hydroxynonanoate was synthesized first, and was present in the center of the granule, while poly-3-hydroxyphenylvalerate accumulated afterwards around the PHN inclusion body. The enzymes associated with the inclusion bodies were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In all cases, two polymerase enzymes of molecular weights of 59 and 55 KD were present, which suggests that the same polymerase enzymes may have been responsible for the production of both PHN and PHPV. In addition, attempts were made to produce a random copolymer containing both alkyl and phenylalkyl repeat unit by varying the growth conditions, but a mixture of two polymers was produced instead. PHPV is a homopolymer and 100% isotactic. However, it does not exhibit crystallinity. Various different annealing experiments carried out on PHPV did not result in the induction of crystallinity. However, a crystalline phenyl-containing polymer, poly-3-hydroxy-5-(4$\sp\prime$-tolyl)valerate was produced when either P. oleovorans or P. putida was grown on 5-(4$\sp\prime$-tolyl)valeric acid. To the best of our knowledge, this is the first report of a crystalline phenyl-containing microbially produced polyester. Intracellular degradation studies were carried out on P. oleovorans cells which contained either PHN, PHPV or a blend of PHN/PHPV. Fairly rapid intracellular degradation occurred when PHN was the sole storage polymer. The intracellular degradation rate of PHPV was much slower. However, when both polymers were present as intracellular storage polymer the rate of degradation of PHPV was greater than when PHPV was the sole intracellular storage polymer. It is possible that both polymers are degraded by the same intracellular depolymerase, which is more efficient in the presence of PHN.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-9051 |
| Date | 01 January 1995 |
| Creators | Curley, JoAnne Mary |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
Page generated in 0.002 seconds