Studies of the use of enzymes to both produce and degrade polyhydroxyalkanotes (PHAs) under in vitro conditions are presented in this thesis. PHAs were produced using a lipase-catalyzed ring-opening polymerization of the corresponding lactones. Poly(3-hydroxybutyrate), PHB, was obtained in up to 89% yield with a degree of polymerization of 3--12. Polymerization of other lactones, including beta-propiolactone, gamma-butyrolactone, and e -caprolactone, also yielded the corresponding polyesters in good yields with degrees of polymerization up to 25. MALDI-TOF mass spectroscopy was used to characterize the polymer products. The growth and kinetics of PHB granules produced in an in vitro polymerization were studied using TEM and CRYO-TEM in conjunction with image analysis. The in vitro reaction was confirmed to be a pseudoemulsion polymerization. The average granule diameter and volume increased with reaction time while the number of granules fell throughout the reaction due to coalescence. Basic kinetic parameters including KM, Vmax, and the rate constants of polymerization were determined and compared to those obtained for the in vivo biosynthesis of poly(3-hydroxybutyrate). / With the aim of improved understanding of the mechanism of depolymerase action on water insoluble crystalline PHAs, folded chain lamellar single crystals of PHAs were partially degraded with PHA depolymerases and examined using TEM. Enzymatically degraded single crystals of bacterial PHB were found to be splintered by PHB-depolymerase A from Pseudomonas lemoignei parallel to their long axes into a needle-like morphology. These results support an "edge attack" model for the degradation of PHB single crystals and suggest that PHB-depolymerase A has both endo and exo activity. A further study was performed using single crystals of a number of PHAs which were partially degraded with depolymerases from Pseudomonas lemoignei and examined by TEM. In contrast to previous results with single crystals of bacterial PHB, the predominant effect observed with all crystals was a significant narrowing of the lamellae. This suggests an edge attack mechanism which because of lateral disorder of the crystals (caused by the introduction of valerate or repeat units of opposite stereochemistry) leads to a narrowing of the crystalline lamellae as opposed to the splintering effect previously observed.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37549 |
| Date | January 1997 |
| Creators | Nobes, Geoffrey A. R. |
| Contributors | Marchessault, R. H. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001615621, proquestno: NQ44533, Theses scanned by UMI/ProQuest. |
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