| Summary: | 碩士 === 元智大學 === 生物科技與工程研究所 === 98 === Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are accumulated in various microorganisms as intracellular carbon and energy storage material under nutrient-limited conditions. Polyhydroxybutyrate (PHB) is a kind of PHAs. In this study, we found that the Vibrio sp.M20 could be able to accumulate PHB under different fermentation strategies. We also found the PHB content decreased with the addition of K+ ion. In order to understand the relationship between PHB depolymerase and K+ addition. In this study, we construct a technical platform for expression of the intracellular PHB depolymerase from Cupriavidus taiwanensis 184 to identify the phenomena of PHB content decreased with the addition of K+ ion.
The intracellular PHB depolymerase (IPHBD) could be produced by the recombinant E. coli CYC04. The experimental data showed that the IPHBD could degrade artificial PHB but it is unable to degrade denatured PHB. The specific activity and the recycling production rate of the IPHBD will be 139.47 U/mg and 44.45 %, respectively, for the 3-hydroxybutyric acid content approximately contains 2.79 g/L at 50 ℃ after 90 minutes reaction. Moreover, optimal operations conditions (e.g., temperature, pH value) for the expression of PHB depolymerase were also identified. It was found that recombinant E. coli CYC04 was able to express 163 % relative activity of depolymerase when it was grown under the optimal operation conditions of 50℃ and pH 8. Furthermore, the expression of depolymerase was induced by the existence of Cu2+. The relative activity of depolymerase was significant increased to 176 % when the medium contained 10 mM of CuCl2. In contrary, the relative activity of depolymerase will be inhibited to 68 % in the medium contained 10 mM of NiCl2.
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