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標題: Aeromonas caviae NCHU1發酵生產幾丁酵素之純化及特性
Purification and Characterization of Chitinases Produced by Aeromonas caviae NCHU1
作者: 洪哲瑋
Hung, Che-Wei
關鍵字: Aeromonas caviae NCHU1;Aeromonas caviae NCHU1;chitin;chitinase;purification;fermentation-modified chitin;regeneration;幾丁質;幾丁酵素;純化;發酵修飾幾丁質;再生
出版社: 化學工程學系所
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本研究之菌種篩選自台南縣下營鄉田間土壤,經食品工業發展研究所鑑定為Aeromonas caviae,並命名為Aeromonas caviae NCHU1;以幾丁質粉末當作其主要生長源,為一株具有生產幾丁酵素之菌株。所得發酵上清液經硫酸銨沉澱、透析去除鹽類、陰離子交換層析(DEAE-Sepharose)及膠體過濾層析(Gel filitration Sephadex G-75)之純化步驟。獲得單一之酵素經SDS-PAGE測得分子量為26.2kDa,其最適反應溫度、最適反應pH值、熱穩定性及pH穩定性分別為60°C、pH6、10-40°C及 pH5-10。在酵素水解產物之幾丁寡醣分析中,由HPLC圖譜得知,反應後產物為N-乙醯幾丁單醣、二醣、五醣及六醣,因此可以推測Aeromonas caviae NCHU1具有內切型幾丁酵素。
第二階段之研究內容是以Aeromonas caviae NCHU1發酵修飾片狀幾丁質為載體,利用幾丁質對幾丁酵素之親和性來進行純化,即幾丁質親和性層析。以SEM圖觀察幾丁質經發酵修飾後之表面,發現幾丁質由平滑的表面變成多孔狀的結構,推測為幾丁質的直鏈結構,因此推知,經發酵修飾之幾丁質在吸附幾丁酵素時減少了立體障礙,進而提高對酵素的吸附量。文中探討幾丁酵素吸附之最適化條件,溫度、 pH值分別為10°C及pH7。於蛋白質脫附實驗中發現,以0.05M、pH10之Glycine緩衝液可脫附幾丁酵素,純化倍率達3.97倍;接下來探討修飾後幾丁質之再生與再利用性,以0.2M、pH10之Glycine緩衝液清洗載體3次,可脫附殘留於載體上之大部分蛋白質;而經過再生之載體也具備再次吸附酵素能力。

In this investigation, a bacterial strain with chitinase activity was isolated from soil in Tainan and identified as Aeromonas caviae NCHU1. This strain can product chitinases when it is grown in a medium containing chitin power of marine waste. A chitinase was purified from culture broth of Aeromonas caviae NCHU1 by a series of purification steps, i.e. ammonium sulfate precipitation, DEAE-Sepharose, and gel filitration with Sephadex G-75. The molecular weight of NCHU1 chitinase is 26.2kDa by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) analysis. The optimum temperature and pH, thermal and pH stability ranges of NCHU1 chitinase are 60°C, pH6, 10-40°C, and pH5-10, respectively. To determine the oligosaccharide contents of NCHU1 chitin hydrolysates, it was found that (GlcNAc), (GlcNAc)2, (GlcNAc)5, and (GlcNAc)6 existed. This implies that Aeromonas caviae NCHU1 may produce endo-chitinase.
Furthermore, the affinity chitin chromatography with fermentation- modified chitin was employed for purification of chitinase. By observing the structural changes of chitin and fermentation-modified chitin by SEM, it was found that chitin surface became porous and branching structure, which might reveal more active sites and less the steric hinderance to the adsorption of chitinase. For chitinase adsorption capacity, factors such as loading temperature, pH and elution solutions were studied. The optimal loading conditions were found at 10℃, pH7. The elution solution was 0.05M glycine buffer. Under these conditions, a purification fold of 3.97 was obtained. Then, the modified chitin can be regeneration and reused.
其他識別: U0005-1307200715061100
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