臺灣博碩士論文加值系統

聚三氫氧基丁酸 (poly-b-hydroxybutyrate, PHB)為細菌體內碳源及
能源的儲存物。此類可由生物分解之聚合物,在許多應用方面,均與傳統石
化原料合成之塑膠相似。我們選用Alcaligenes eutrophus H16為標準菌,
以建立生產PHB細菌及合成基因篩選系統。本研究首先選出高碳氮比例(C/
N=76)及糖蜜(5%)培養基用以生產PHB。結果顯示PHB產量可達到
Alcaligenes eutrophus菌體乾重的44%。使用長度780 bp之phbC基因高保
留片段為探針,利用菌落雜合選殖帶有Alcaligenes eutrophus PHB合成基
因之重組大腸桿菌。為篩選具PHB合成能力之本土菌株,利用PCR篩選並配
合南方雜合法與PHB萃取進行驗證。本研究結果顯示五株本土細菌菌株(
Corynebacterium sp. CB, Pseudomonas fluorescenceND, Alcaligenes
faecalis NE與未定名菌株T2,NF),經雜合反應,於780 bp處有訊號顯示。
同時,Corynebacterium sp. CB也有長條狀(smear)訊號出現。經由PHB萃
取與穿透式電子顯微鏡觀察,證明本土細菌菌株Corynebacterium sp. CB
與菌株T2的PHB產量分別為0.1 g/l及0.08 g/l。

Poly-b-hydroxybutyrate (PHB) is an intracellular carbon and
energy storagematerial accumulated in many bacteria. Its
biodegradable properties can be usedin various ways similsr to
plastics derived from conventional petrochemicals.To establish
PHB-producing and biosynthesis gene screening systems,
Alcaligenes eutrophus H16 was used as a reference bacteria.
First, high C/N ratio (C/N=76)and molasses (5%) media were
selected for PHB production. Under thesis conditions,PHB content
was accumulated to 44% of bacteria dry weight. PHB biosynthesis
genesfrom Alcaligenes eutrophus H16 were cloned by colony
hybridization probed with 780 bp conserved phbC fragment. In
order to screen the PHB-producing indigenousbacteria, PCR
followed by Southern blotting analysis was carried out. Result
showed that a 780 bp PCR amplified fragment was detected in
three (Alcaligenesfaecalis NE and unidentified strains T2, NF)
of five indigenous bacteria, including;Corynebacterium sp. CB
showed a smear from 780 bp to 300 bp by touchdown PCR.Production
of PHB in both Corynebacterium sp. CB and unidentified strain T2
wereconfirmed by extraction of PHB and transmission electro-
microscopy. The PHB contents in Corynebacterium sp. CB and
unidentified strain T2 were 0.1 g/l and 0.08 g/l respectively.