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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15042

Title: Aeromonas salmonicida MAEF108 所產 Agarase AS-II 之純化與特性研究暨經 Agarase AS-II 水解所得藻類寡醣之生理活性探討
Studies on Purification and Characterization of Aeromonas salmonicida MAEF108 Agarase AS-II and Bioactivities of Agarase AS-II Digested Algal Oligosaccharides
Authors: Sheen-Kye Kang
康新楷
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 洋菜□;藻類;生理活性
agarase;algae;bioactivity
Date: 2006
Issue Date: 2011-06-30T07:57:40Z
Abstract: 從海洋細菌 Aeromonas salmonicida MAEF108 所產具有洋菜□活性劃分中純化單一酵素,命名為Agarase AS-II 並且利用此一純化酵素降解龍鬚菜或海菜萃取多醣評估其生理活性。A. salmonicida MAEF108 培養液經離心 (30,700 x g)、超過濾 (30 kDa MWCO)、以及離子交換層析得到 Agarase AS-II,其純化倍率為 44.03,經電泳分析後得到分子量估計為 95.4 kDa 的單一蛋白質。Agarase AS-II 的最適作用溫度以及 pH 分別為 45oC 及 7.0, 且存放在低於 40oC 2 小時之後仍具有 60% 以上的殘存活性;存放於 pH 6.0-8.0 的磷酸緩衝溶液中有較佳的穩定性。與不添加金屬離子的控制組相比,5 mM CaCl2 可以增加 Agarase AS-II 30% 的酵素活性。Agarase AS-II 的活化能與 Km 分別為 46.24 J mol-1 K-1 及 4.39% 的 agarose 基質。 此純化酵素對紅藻綱藻類及其精製萃取物具有基質特異性,並可在 1 分鐘時即已水解部分 agarose,且生成以新洋菜六醣 (neoagarohexaose, N6) 以及新洋菜四醣 (neoagarotetraose, N4) 組成的最終產物,因此被認為是一個可將 agarose 直接水解產生 N6 及 N4 的 beta-agarase。除此之外,Agarase AS-II 具有將 N2 生合成 N4 以及將半乳糖生合成未知寡聚合物的能力。 以 2 mg/mL 的 (1) 龍鬚菜萃取多醣 (Gra) 或海菜萃取多醣 (Mon)、(2) Gra 或 Mon 經 A. salmonicida MAEF108 粗酵素液降解所得降解物 (Gra-C108、Mon-C108)、以及 (3) Gra 或 Mon 經 Agarase AS-II 降解所得降解物 (Gra-AS-II、Mon-AS-II) 等做為三株雙歧桿菌 (bifidobacteria) 的生長碳源,以評估做為益生物質的能力。當 Bifidobacterium pseudolongum subsp. pseudolongum BCRC 14673 利用 Gra-C108 時,24 小時之內活菌數可以增加到超過 3 log cfu/mL。海菜萃取多醣及其水解物亦皆能夠做為雙歧桿菌生長利用的碳源。B. adolescentis BCRC 14608 以及 B. pseudolongum subsp. pseudolongum BCRC 14673 皆可利用上述 6 種藻類樣品且產酸降低 pH 值。B. longum BCRC 11847 較能利用龍鬚菜樣品產酸降低 pH 值。不同濃度的 6 種海藻樣品 (Gra、Mon、Gra-C108、Mon-C108、G-AS-II、或 Mon-AS-II) 分別與不同濃度的BHK-21 細胞進行培養,發現皆不具細胞毒性,且細胞存活率皆高於 78%。6 種藻類樣品均呈現降低日本腦炎病毒北京一型感染 BHK-21 細胞的現象,且硫酸根含量較高之海菜樣品降低感染的能力優於龍鬚菜樣品。龍鬚菜或海菜萃取多醣經 A. salmonicida MAEF108 粗酵素液或 Agarase AS-II 降解後,均可測得較多的多酚類化合物及硫酸根含量之產物。龍鬚菜樣品 Gra-C108 與 Gra-AS-II 分別具有 93.10% 和 78.49% 的螯合能力,高於 Gra (62.52%) 以及 Mon (21.25%)。
Agarase AS-II was purified from the four fractions having agarase activities of marine bacteria Aeromonas salmonicida MAEF108 agarases, and was used to digest Gracilaria sp. or Monostroma nitidum extracted polysaccharides to evaluate its bioactive properties. Culture suspension of A. salmonicida MAEF108 was centrifuged (30,700 x g), ultrafiltration (30 kDa MWCO), then applied to ion-exchange chromatography to obtain Agarase AS-II which was 44.03-fold purified and was estimated as a 95.4 kDa single protein. The optimal temperature and pH to degrade 0.2% agarose were 45oC and 7.0, respectively. It maintained 60% residual activity when Agarase AS-II was stored below 40oC for 2 hours; the stability of Agarase AS-II was better when stored in pH 6.0-8.0 phosphate buffer. The addition of 5 mM CaCl2 in assay buffer could contribute 30% higher relative activity than no metal ions were added. Activation energy and Km of Agarase AS-II were 46.24 J mol-1 K-1 and 4.39% of agarose, respectively. The purified agarase has substrate specificity on Rhodophyta and its refined products, and could degrade agarose into final products of neoagarohexaose (N6) and neoagarotetraose (N4), indicating that Agarase AS-II could be regarded as a beta-agarase which would directly degrade agarose to produce N6 and N4. In addition, Agarase AS-II has the abilities to biosynthesize N4 and unidentified galactose oligomers from neoagarobiose (N2) and galactose, respectively. 2 mg/mL of (1) either Gracilaria sp. extracted polysaccharides (Gra) or M. nitidum extracted polysaccharides (Mon), Gra or Mon digested by A. salmonicida MAEF108 crude agarases (Gra-C108 and Mon-C108), or (3) Gra or Mon digested by Agarase AS-II (Gra-AS-II or Mon-AS-II) was tested as a carbon source for bifidobacteria growth to evaluate prebiotic potentials. Viable bacteria counts of Bifidobacterium pseudolongum subsp. pseudolongum BCRC 14673 could increase more than 3 log cfu/mL in 24 hour-incubation when Gra-C108 was utilized. M. nitidum extracted polysaccharides and its agarase lysates were also able to be utilized by bifidobacteria as carbon source. B. adolescentis BCRC 14608 and B. pseudolongum subsp. pseudolongum BCRC 14673 could utilize 6 algal samples to lower pH. B. longum BCRC 11847 performed better pH lowering ability when utilizing Gracilaria sp. based samples. Different concentrations of 6 algal samples (Gra, Mon, Gra-C108, Mon-C108, Gra-AS-II, or Mon-AS-II) were cultured with different cell densities of BHK-21 kidney cells in which the algal samples showed no cytotoxicity and the cell viability was higher than 78%. The 6 algal samples showed ability to decrease the effects caused by Japanese Encephalitis Virus (JEV, Beijing-1 strain) infection. It was found that M. nitidum based samples performing better antiviral activity than Gracilaria sp. based samples was due to having higher sulfated group contents. Gra or Mon digested by either A. salmonicida MAEF108 crude agarases or Agarase AS-II, could obtain products containing higher amount of polyphenolic compounds and sulfated group. Gra-C108 and Gra-AS-II performed 93.10 and 78.49% chelating effects on Fe2+, respectively, which were higher than those of Gra (62.52%) or Mon (21.25%).
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M94320030
http://ntour.ntou.edu.tw/ir/handle/987654321/15042
Appears in Collections:[食品科學系] 博碩士論文

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