碩士 / 國立中興大學 / 生命科學系所 / 102 / Green algae have a photosynthetic system similar to plants but can produce H2 by hydrogenase (HydA, encoded by hydA) using sun light under anaerobic condition, as O2 is a strong inhibitor of HydA. The catalytic H-cluster of the HydA is located in close proximity to O2 accessible area, termed the gas channel. The mutated amino acid residues around the gas channel could narrow the channel and in turn lower the access of O2. In this study, we attempted to mutate certain amino acids around Zone 1 and Zone 3 of gas channel so that the mutated HydA can have high O2 tolerance to enhance H2 production in Chlorella sp. DT (DT).
The specific amino acids along the O2 pathways were modified by site-directed mutagenesis with the goal of preventing the O2 entrance to the H-cluster. The residues A105 and V265 of Zone 1, and the residues G113 and V273 of Zone 3 proximal to the active site around the gas channel were replaced with amino acids I and W with bulky group by site-direct mutagenesis. The mutated hydA of pHyg3-hydA was inserted to DT mutants genomic DNA by electroporation. The hydA of the DT mutants could be transcribed but not that of DT-WT under aerobic and sulfur-supplied condition. We confirmed the key amino acids of A105I, V265W, G113I and V273I around the gas channel of hydA were successfully mutated. By western blotting analysis, using anti-HydA, the detectable signal of HydA protein band of 48 kDa was visualized in DT mutants but not in the DT-WT under aerobic and sulfur-supplied condition. The HydA protein expression levels of Zone 1 and Zone 3 DT mutants were 10 and 14 fold higher than that of the DT-WT. The H2 contents of Zone1 and Zone 3 DT mutants are 5.8 and 6.4 fold higher than that of DT-WT under 5% O2 condition. These results suggested that the Zone1 and Zone 3 DT mutants with higher O2 tolerance than DT-WT can produce more H2.
| Identifer | oai:union.ndltd.org:TW/102NCHU5105054 |
| Date | January 2014 |
| Creators | Da-Wei Yang, 楊大緯 |
| Contributors | Lee-Feng Chien, 簡麗鳳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 72 |
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