Biyobozunur Plastik Polihidroksialkanoatların Bacillus Megaterium’da Genetik Mühendisliği Yoluyla Üretiminin Artırılması

Abstract

Fossil fuel-derived plastic materials, have currently production and consumption values at increasing quantities, due to their lightness, easy malleability and economical reasons. Owing to their poor degradation rates and their carcinogenic components, fossil-resource based plastics pose serious health problems on humans and the environment. With the rapid depletion of limited fossil fuel resources, one of the alternative materials that can replace petroleum based plastics is polyhydroxyalkanoates (PHA) with their biodegradability and biocompatibility properties. PHAs are polyesters synthesized to be used during cellular stress, in a carbon-rich nutrient medium that is poor in terms of components such as oxygen, nitrogen and phosphorus. However, in order for these biopolymers to be preferred commercially, the production efficiency in bacteria should be increased. In this thesis study, genetic engineering methods have been applied to produce PHA at high efficiency, from recombinant (r)-Bacillus megaterium bacterium with various genetic modifications. PHA synthesis genes phaC and phaR found in the B. megaterium genome were cloned into an expression vector and a new (r)-B. megaterium strain was developed. The production efficiency and structure of PHA produced by the wild type Bacillus megaterium NRRL B-14308 strain, the (r)-Bacillus megaterium strain overexpressing phaC and phaR, and our previously developed (r)- Bacillus megaterium strain overexpressing phaC, were compared. After 72 hours of incubation in an orbitally shaken bioreactor, PHA was obtained from (r)-B. megaterium NRRL B-14308 strains containing pC-HIS1623hp-phaC-phaR, pC-HIS1623hp-phaC and as a control group pC-HIS1623hp plasmids with yields of 2.3 g L-1, 2.2 g L-1 and 2.1 g L-1, respectively. Following the confirmation of PhaC and PhaR proteins required for PHA synthesis via SDS-PAGE method, the structural and thermal properties of biopolymers obtained by chloroform purification method were characterized by Gas Chromatography (GC-FID), Fourier Transform Infrared Spectroscopy (FT-IR), Nuclear Magnetic Resonance Spectroscopy (1H-NMR), Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses. As a result of the characterization of PHAs obtained from the recombinant strains, it has been determined that the polymers are in copolymer (PHB-co-PHV) structure, and consist of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units.