自微生物純化與分離聚羥基烷酸之研究

Purification and Separation of Polyhydroxyalkanoatesfrom Bacteria

呂忠賢(Chung-Hsien Lu)

指導教授 ： 吳和生

元智大學/工程學院/化學工程與材料科學學系/碩士(2006年)

摘要

本研究中主要尋找適合Cupriavidus taiwanesis184 、Buskholderia sp.PTU9、 Pseudomouas putida.KT244、Recombinant E. Coli四種菌株所回收取得聚羥基烷酸(polyhydroxyalkanoates, PHAs)，在前處理部份主要是分為冷熱法、均質機、超音波震盪、化學消化法進行破細胞，在萃取的方法使用了溶劑萃取法（氯仿）、鹼處理法（氫氧化鈉、氫氧化鉀）、次氯酸鈉法、次氯酸鈉-硫酸十二酯鈉法、酵素和酵素-次氯酸鈉等不同的溶劑進行萃取，將萃取後之PHA進行純度、回收率、分子量和熱性質探討。
Cupriavidus taiwanesis184利用前處理冷熱法、使用次氯酸鈉4cm3在50oC的125rpm震盪槽中反應5min。聚羥基烷酸(polyhydroxybutyrate)PHB其純度可達99%、分子量1350000、回收率達94%、熔點為178 oC
Recombinant E. coli菌株利用硫酸十二酯鈉前處理在5cm350oC的125rpm震盪槽中反應5min，而後使用氫氧化鈉溶劑5cm350oC的125rpm震盪槽中反應5min，純度可達99%、回收率96%、分子量310,000和熔點178.2oC。
Buskholderia sp.PTU9菌株利用前處理冷熱法使用酵素-次氯酸鈉，使用1% 1cm3木瓜酵素pH6.7磷酸緩衝溶液反應5min在40oC的震盪槽中125rpm後，再使用次氯酸鈉4cm3反應5min在50oC的震盪槽中125rpm，PHB其純度可達89%，回收率78%、分子量480,000、熔點為169 oC
P.putida.KT2442使用利用前處理冷熱法，在使用35 cm3次氯酸鈉50oC的125rpm震盪槽中反應5min，PHB純度可達99%、回收率78%、分子量為53,000、熔點為41 oC。
在Cupriavidus taiwanesis184菌株所生產的PHB進行分子量的控制，分為不同溶劑、化學溶劑(反應溫度、體積)和培養條件(培養基、碳氮比)進行探討。
Cupriavidus taiwanesis184使用次氯酸鈉在不同溫度30oC、50 oC、80 oC、100 oC，分子量分別為140萬、131萬、120萬、96萬，在反應體積5cm3、10 cm3、15 cm3，分子量分別為86萬、53萬、22萬，而分子量須達50萬以上才可以熱壓成膜。在不同碳源的培養基和碳氮比並不會對分子量有影響，分子量大約為1,500,000左右。

關鍵字

聚羥基丁酸、回收率、純化

並列摘要

Study the separation intracellular of PHA from Cupriavidus taiwanensis184, Buskholderia sp.PTU9, Pseudomonas.Putida.KT2442, Escherichia coli. Cell disruption was used heat、homogenizer、sonicator and chemical at pretreatment. Separation and purification of PHAs was included solvent extraction, alkaline(sodium hydroxide, potassium hydroxide), sodium hypochlorite, sodium hypochlorite- sodium dodecyl sulfate, enzyme-sodium hypochlorite. The purity, recovery, molecular weight and thermal property of PHAs was determined as well.
The optimal recovery condition were heat pretreatment and 50°C、125rpm、5min of reaction time with 4cm3 sodium hypochlorite. Under such condition, a purity of 99%, a recovery of 94% and Mw of 1,350,000 were obtained from Cupriavidus taiwanensis184. And melting point of PHB was 178oC.
Crude cell were pretreatment with 50°C, 125rpm, 5min of reaction time with 5cm3 SDS. The optimal recovery condition were 50°C, 125rpm, 5min of reaction time with 5cm3 1N sodium hydroxide from Recombinant E. coli strain. The purity and recovery were 99% and 96%. The Mw was 310,000. And melting point of PHB was 178.2oC
The PHB purity and recovery was 89% and 78% from Buskholderia sp.PTU9, using enzyme-sodium hypochlorite method. Crude cell were heat pretreatment. Digestion used 1cm3 1% papain digest with 40°C, 150rpm, time 1hr .After enzymatic, digesting with 4cm3 sodium hypochlorite at 50°C, 125rpm, 5min of reaction time. And melting point of PHB is 169oC. The Mw was 480,000
The melting point of PHB was 41oC, using 35cm3 sodium hypochlorite digestion at 50°C、125rpm、5min of reaction time form P.putida.KT2442 strain. The purity and recovery were 99% and 78%. The Mw was 53,000.
For molecular weight of PHB from Cupriavidus taiwanensis184. The operating condition, such as different solvents, temperature, amount of sodium hypochlorite and culture condition (media, carbon and nitrogen ratio) were investigated to obtain various molecular weight.

For molecular weight reacted different temperature were 30oC of 1,400,000、50oC of 1,310,000, 80oC of 1,200,000, 100oC of 960,000 from Cupriavidus taiwanensis184. The molecular weight reacted different sodium hypochlorite amount were 5cm3 of 860,000, 10cm3 of 560,000, 15cm3 of 220,000. The membrane used hot press for Mw over 500,000 from Cupriavidus taiwanensis184. Cupriavidus taiwanensis184 was cultured at different carbon source, carbon and nitrogen ratio, was not effect molecular weight. The Mw was about 1,500,000

並列關鍵字

Polyhydroxybutyrate, Recovery, Purification, Cupriavidus taiwanesis184, Buskholderia sp.PTU9, Pseudomonas.putida.KT2442,Escherichia coli

參考文獻

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