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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/14764

Title: Aeromonas salmonicida MAEF108 菌株所產洋菜酶的純化與性質之探討
Studies on Purification and Properties of Agarases from Aeromonas salmonicida MAEF108
Authors: Tze-Yuan Chen
陳姿元
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 洋菜酶;純化;高效能液相層析法
agarase;purification;HPLC
Date: 2005
Issue Date: 2011-06-30T07:44:16Z
Abstract: Aeromonas salmonicida MAEF108 為一株具有分解洋菜能力的海洋細菌。將此菌於 MMB-MAEF108 培養液中振盪培養 (20oC、240 rpm) 24 hr,收集離心去除菌體後之培養液,進行洋菜酶的分離與純化。首先,培養液經濃縮透析後之比活性為 2225.10 AU/mg,純化倍數為 2.32倍,回收率為 24.66%;再以 DE-52 離子交換樹脂層析,可分離出三個具分解洋菜能力的蛋白質波峰劃分區域,個別收集後命名為 MAEF108-agarase Ι、ΙΙ、及 ΙΙΙ,其比活性分別為 855.35 AU/mg、174.47 AU/mg、及 1069.46 AU/mg,純化倍數分別為 0.89 倍、0.18 倍、及 1.11 倍,回收率分別為 4.97%、0.27%、及13.32%。MAEF108-agarase Ι 在經第二次 DE-52 離子交換樹脂層析及 Bio-Gel P-100 膠體過濾層析其純化後之比活性為 233.49 AU/mg、純化倍數為 0.24 倍、以及回收率為 0.40%。 MAEF108-agarase Ι 的最適作用 pH 值為 6.0 或 7.5-8.0,最適作用溫度為 45oC,但是於40oC 下反應 60 min 活性減少 50%。MAEF108-agarase ΙΙ 的最適作用 pH 值為 5.0 或 7.5,最適作用溫度為 45oC,於 50oC 下反應 20 min 後活性幾乎喪失,於其他溫度下反應 60 min 後活性減少 50% 以上。MAEF108-agarase ΙΙΙ 的最適作用 pH 值為 6.0 或 7.5,最適作用溫度為 45oC,於其他各溫度反應 60 min 活性均下降至 60% 以下。MAEF108-agarase Ι、ΙΙ、及 ΙΙΙ 以 high-melting-point agarose 為基質時所表現之基質特異性活性最佳 (分別可產相當於 3.76、2.17、9.46 mg D-galactose)。且此三種洋菜酶皆不能分解 □ι-carrageenan 和 □κ-carrageenan。利用 MAEF108-agarases、MAEF108-agarase Ι、ΙΙ、或 ΙΙΙ 水解中性洋菜,經 HPLC 分析水解產物發現水解產物可能是分子量小於半乳糖之單醣或介於半乳糖與乳糖間之醣類。利用 MAEF108-agarase Ι、ΙΙ、或 ΙΙΙ 水解新洋菜六糖 (neoagarohexaose),經 HPLC 分析產物發現水解產物可能是分子量小於半乳糖之單醣或介於半乳糖與乳糖間之醣類。
Aeromonas salmonicida MAEF108 is a marine bacterium, which has the ability to degrade agar. Strain MAEF 108 was grown at 20oC for 24 hr in MMB-MAEF108 broth with shaking speed of 240 rpm. Collected crude enzymes after removing the cells then to proceed separation and purification of the agarases. First, after concentrated and dialysed the specific activity, purification fold, and yield of the resulted solutions were 2225.10 AU/mg, 2.32 fold, and 24.66%, respectively. Chromatographed with DE-52 gel achieved the separation of three agarases in the concentrated and dialysised broth, they are named MAEF108-agarase Ι, ΙΙ, and ΙΙΙ, by their agarase activities in each group of eluted fractions. The following properties of these Ι, ΙΙ, and ΙΙΙ agarases were: specific activity, 855.35, 174.47, and 1069.46 AU/mg; purification fold, 0.89, 0.18, and 1.11 fold; and yield, 4.97%, 0.27%, and 13.32%, respectively. MAEF108-agarase Ι was purified further by second DE-52 and then Bio-Gel P-100 chromatographs. The final purified MAEF108-agarase Ι showed its specific activity, purification fold, and yield were 233.49 AU/mg, 0.24 fold, and 0.40%, respectively. The optimum pH for MAEF108-agarase Ι was found to be 6.0 or 7.5-8.0. The optimum temperature for MAEF108-agarase Ι was 45oC, but approximately 50% of the enzyme activity was inactivated in 60 minutes under 40oC. The optimum pH for MAEF108-agarase ΙΙ was found to be 5.0 or 7.5. The optimum temperature for MAEF108-agarase ΙΙ was 45oC, but almost inactivated in 20 minutes under 50oC and more than 50% of the enzyme activity was inactivated after exposure to the rest tested temperature. The optimum pH for MAEF108-agarase ΙΙΙ was found to be 6.0 or 7.5. The optimum temperature for MAEF108-agarase ΙΙΙ was 45oC, but more than 60% of the enzyme activity was inactivated while stand 60 minutes under the rest tested temperature. The best substrate specificity activities of MAEF108-agarase Ι, ΙΙ, and ΙΙΙ toward high-melting-point agarose were produced equivalent 3.76, 2.17, and 9.46 mg D-galactose, respectively. MAEF108-agarase Ι, ΙΙ, and ΙΙΙ did not have the capability to degrade □ι-carrageenan and □κ-carrageenan. Hydrolytic products of agarose digested by MAEF108-agarases, MAEF108-agarase Ι, ΙΙ, or ΙΙΙ were analyzed by HPLC, and the results showed that they could be monosaccharides, which molecular weight were smaller than galactose, and disaccharides, which molecular weight were between galactose and lactose. Hydrolytic products of neoagarohexaose digested by MAEF108-agarase Ι, ΙΙ, or ΙΙΙ were analyzed by HPLC. The products examined could be monosaccharides, which molecular weight were smaller than galactose, and disaccharides, which molecular weight were between galactose and lactose.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M93320019
http://ntour.ntou.edu.tw/ir/handle/987654321/14764
Appears in Collections:[食品科學系] 博碩士論文

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