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Green alga Chlorella sp. hydrogenase mutants have higher O2 tolerance and produce more H2
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|摘要:||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.|
綠藻有類似植物光合作用系統，可以將太陽能轉換成電子提供給產氫? (hydrogenase, HydA)，HydA可在缺氧 (O2) 條件下產生氫氣 (H2)，但是光合作用產生的O2是HydA的抑制劑。O2可經由HydA氣體通道 (gas channel) 進入其催化中心的H-cluster而抑制HydA。突變gas channel的特定胺基酸可以使gas channel縮小，進而減少O2進入H-cluster。本篇的研究中，我們嘗試突變HydA gas channel的Zone1和Zone3幾個特定胺基酸，期望綠藻Chlorella sp. DT (DT) 的HydA 具有較高O2耐受性及H2產量。 利用pHyg3-hydA載體以定點突變 (site-direct mutagenesis) 方法，使位於Zone1的A105和V265以及位於Zone3的G113和V273，突變為有較大支鏈的I和W。再將含有突變hydA的載體轉殖至DT，並篩選DT突變株。利用聚合連鎖反應，偵測到DT突變株含有轉殖hydA片段，得知突變hydA已成功被轉殖於DT。在有O2和S條件下，偵測到DT突變株hydA的轉錄，但DT野生株則沒有。又定序DT突變株之genomic DNA，確定gas channel的胺基酸A105I、V265W、G113I及V273I突變成功。在有O2和S條件下，利用西方墨點法分析DT突變株之蛋白表現，於48 kDa位置偵測到HydA蛋白，而DT野生株則沒有HydA蛋白表現。Zone1和Zone3 DT突變株之HydA蛋白表現量相較於DT野生株高出了10倍和14倍。在5% O2的條件下，Zone1和Zone3 DT突變株之H2產量相較於DT野生株則高出了5.8倍和6.4倍。綜合以上結果，Zone1和Zone3 DT突變株相較於DT野生株有較高的O2耐受性並且有較多H2產量。
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