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Development of D-form and L-form lactic acid producer Lactobacillus pentosus and application in the production of poly-lactic acid
|摘要:||乳酸(lactic acid)及其衍生物的應用相當廣泛，舉凡食品、醫藥、皮革、化工等。乳酸有三種結構形式，即兩種旋光異構型-L型、D型和一種無光學活性結構-DL型，各種形式乳酸皆可在真空狀況下相互脫水縮合成為減水乳酸( Lactide )，並進一步聚合成為聚乳酸(poly-lactic acid, PLA)，PLA為生物可降解材質，能在特定條件下被微生物分解成水及二氧化碳，具有無毒、無刺激性、生物相容性好等特點，目前常被開發為手術縫線、骨釘、骨板等醫療用品。由於其物理特性可藉由L-(+)和D-(-)異構物混合比例來做調整，可開發在各種不同用途上，其性質和傳統塑膠相似，更被期待能夠取代廣泛使用的塑膠製品。然而乳酸的生產成本是PLA目前無法廣泛開發使用的主要原因之一，目前乳酸生產多利用乳酸菌發酵醣類或經前處理後的植物澱粉而製得，其前處理及後續光學異構物分離純化所需的成本是造成PLA價格高居不下的主要原因。本研究嘗試以這兩方面為目標對乳酸生產菌Lactobacillus pentosus作改造。L. pentosus為兼性異型發酵(fermentative hetero-fermentation)的乳酸菌株，在代謝六碳醣時能夠產生大量的(D型、L型)乳酸。本研究建立了L. pentosus的轉殖系統，並利用同源重組的方式破壞L型乳酸脫氫酶基因，獲得的突變株PeL能夠產生高純度(97%)的D型乳酸，乳酸產量也較野生株提高了40%。破壞D型乳酸脫氫酶基因的突變株PeD，L型乳酸的生產與純度只有微幅提高的情形，實驗結果顯示L. pentosus很可能帶有lactate racemase基因，將產生的L型乳酸轉換成D型乳酸，但是基一步嘗試破壞，卻一直未能成功。本研究也以B. licheniformis D2的endoglucanase基因構築重組質體pIN15E-L826-CMC，再以同樣策略建構能利用纖維素物質發酵產生單一型D型乳酸的轉形株PeL-CMC。以PCR檢驗此轉形株確認endoglucanase基因確實插入染色體DNA，卻未同時破壞L型乳酸脫氫酶基因，目前尚未了解其原因。以DNS法知PeL-CMC的endoglucanase酵素活性會隨時間增加而活性持續下降，研判和乳酸菌生長後期pH大幅降低有關，未來在乳酸菌利用纖維素物質生產乳酸的研究和實際應用上，須要能克服此問題。|
Lactic acid and its derivatives are used for many applications such as food, medicine, chemicals, leather processing and biodegradable plastic. Lactic acid is a simple chiral molecule that exists as two enantiomers, L- and D-lactic acid, these enantiomers can be used to synthesize lactide by condensation under vacuum and processing to polylactic acid (PLA) . PLA is a biodegradable material, it can be metabolized by microorganisms to H2O and CO2. It’s characteristics are nontoxic and highly biocompatible, and it has been used in medicine like metallic implants and sutures. PLA can be tailored to specific applications by altering the ratio of the L-lactic acid and D-lactic acid isomers. It has similar property of plastic and can be processed to many kinds of product, so it is expected to reduce the plastic usage. However the price of producing lactic acid are much higher than plastic, this is a baulk of developing PLA. Nowdays lactic acid is produced from the fermentation of glucose by lactic acid bacteria, and then followed by steps of separation and purification D-lactic acid and L-lactic acid. The cost of raw material and optical isomers isolation are the main reasons that cause the high price of PLA at present time. To resolve these problems, this study attempted to construct mutants of Lactobacillus pentosus, a facultative hetero-fermentative bacterium, so that they would only produce D-lactic acid or L-lactic acid, or they would produce lactic acid by utilizing cellulose. By means of targeted homologus recombination, L-lactate dehydrogenase gene-deficient Lactobacillus pentosus ( PeL ) and one D-lactate dehydrogenase gene-deficient L. pentosus ( PeD ), were obtained. Compared to wild type L. pentosus PeL produced 40% more D-lactic acid, and the optical purity of D-lactic acid increased to 97%. However PeD mutant strain did not increased the production and purity of L-lactic acid obviously, we deduce that lactate racemase converted L-lactic acid to D-lactic acid. A putative racemase gene was found, but attempt to disrupted it was fail so far. In this study we also tried to transfer and integrate an endoglucanase gene of B.licheniformis into the L-lactate dehydrogenase gene of L. pentosus with the same strategy of targeted homologus recombation, a transformant with endoglucanase activity was obtained, but experiment results displayed that its L-lactate dehydrogenase gene was not knocked out. When measured by DNS method, the highest activity can be found after growing for 12 hr, then decreased reached zero at 48 hr due to low-pH caused by lactic acid production.
|Appears in Collections:||生命科學系所|
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