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標題: 應用乳酸菌Lactobacillus pentosus從再生性資源生成 D型、L型乳酸與D型、L型乳酸脫氫脢基因之分析
Production of D-lactic acid and L-lactic acid from regenerated resources by Lactobacillus pentosus and characterization of D-LDH and L-LDH gene.
作者: 羅毓秀
Luo, Yu-Shiou
關鍵字: Lactic acid
出版社: 生命科學系
摘要: 乳酸(lactic acid)及其衍生物的應用範圍極廣,舉凡於食品、醫藥、化工、皮革及生物可降解性塑膠等方面都大量的使用乳酸,微生物發酵產生的乳酸產物可以用來聚合產生聚乳酸(polylactic acid, PLA),PLA是一種可以再生利用、且可被微生物降解的塑膠。 PLA的物理特性可藉由控制L-(+)-和D-(-)-lactic acid isomers的比例來改變以迎合特別的用途。國際市場年需求量正以10%-20%的速度增長,現今全球乳酸需求量约40萬噸,而生產量卻只有25萬噸。目前乳酸生產多利用乳酸菌將植物澱粉降解發酵製得,而將有機廢棄物資源化和乳酸的生產結合是值得考慮的一環。Lactobacillus pentosus是一種進行同型發酵(Homo fermentation )的乳酸菌,其代謝醣類產生的大量乳酸,包含D-form 及L-form兩種形式,佔其總代謝產物的90%以上。本研究嘗試以民生下水污泥與廚餘當作基質,以L.pentosus進行發酵培養產生乳酸後,利用HPLC測得D-lactic acid及L-lactic acid產量,初步探討有機廢棄物資源化之可行性。D-lactic acid及L-lactic acid是由兩個不同基因所轉錄轉譯出的D-lactate dehydrogenase及L-lactate dehydrogenase進行個別的催化反應而生成,為了要對這兩種酵素及其基因做進一步的探討,便依據相近菌株的基因資料設計degenerate primer,找出D-lactic dehydrogenase及L-lacyic dehydrogenase的基因,並分別選殖到Escherichia coli BL21表現菌株中表現,以期能產生單一形式之乳酸。另一方面,構築包含適當的D-ldh或L-ldh基因single crossover質體,及構築包含適當的D-ldh或L-ldh基因double crossover質體,再將這些重組質體以electroporation的方式送入L. pentosus菌體內,期望利用homologus recombination的發生使染色體DNA上的D-ldh或L-ldh基因被破壞,以達到產生單依形式lactic acid的目的,但是很可惜一直未能成功。
Lactic acid and its derivative are used for many applications suck like food, medicine, chemical industry, leather and biodegradable plastic. Lactic acid is mainly produced through fermentation processes. Polylactic acid (PLA) is being developed as a renewable alternative for conventional petroleum-based plastics. That can be lost all mechanical properties when composted for five weeks. PLA can be tailored to specific applications by altering the ratio of the L- lactic acid and D-lactic acid isomers. The annual requirement of international market increases at the speed of 10%-20%. Global lactic acid demand was around 400,000 tons, but the amount of production was 250,000 tons. Lactic acid is ferment from agricultural crops by lactic acid bacteria. Lactobacillus pentosus is a homofermenative lactic acid bacterial with the capacity to produce high level of D-lactic acid and L-lactic acid. In this study, production of D-lactic acid and L-lactic acid from regenerated resources, sewage sludge and waste cola, by L. pentosus. In order to achieve the objective of sustainable development, we should place emphasis on the economical resource, reduce the emission of waste and make use of discarded or trashed materials. D-lactate dehydrogenase and L-lactate dehydrogenase convert pyruvate into D-lactic acid and L-lactic acid, respectively. The study uses molecular technology to finish the whole d-ldh and l-ldh gene sequence and tries to express the whole d-ldh or l-ldh gene into E. coli BL21(DE3) to product D-lactic acid and L-lactic acid. The study also clones many vector and transpormation the vector into L. pentosus by electroporation. Hope the d-ldh or l-ldh gene on chromosome DNA is destructed by homologus recombination and by this way to production single form lactic acid but it hasn't succeed so far.
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