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  • 學位論文

脂解酶與beta-澱粉酶的交互作用

Interaction of Lipase and Beta-amylase

指導教授 : 王淑美
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摘要


脂解酶(Lipase)為分解脂肪的重要酵素,植物在發芽初期光合作用系統尚未健全時,必須利用脂解酶或澱粉酶來分解種子內的能源提供本身的初期發育。目前已知脂解酶在大豆子葉中含有大量的脂解酶抑制劑,最早被鑑定的抑制劑為Lipoxygenase。但Satouchi等人在2002年指出脂解酶在lipoxygenase(lipase inhibitor)缺失的大豆種子中依舊會受到抑制,分離此抑制物經由MALDI-TOF MS分析結果,得知此抑制性蛋白質即為beta澱粉酶。而目前beta澱粉酶在萌發玉米穀粒中,其表現位置與澱粉所在的位置顯然不同。目前已知在萌發後的玉米和水稻的糊粉層中,beta澱粉酶活性極高,可是此處幾乎沒有澱粉的存在,因此,beta澱粉酶在糊粉層中扮演的生理功能仍是未知。因此推測玉米的beta澱粉酶可能和大豆beta澱粉酶同樣具有抑制脂解酶的能力,其存在可以部分抑制脂解酶活性。由於脂解酶可能有分解細胞膜的能力,所以beta澱粉酶對細胞膜也許有保護作用,使糊粉層細胞延遲進入細胞凋亡。本實驗利用Prep Cell (製備式電泳)純化玉米萌芽穀粒(台農一號)和大豆子葉中的beta-澱粉酶,分別將兩種不同物種的beta-澱粉酶與胰脂解酶預先反應後,再加入脂解酶反應液中觀察活性的變化,結果發現脂解酶的活性未如預期,沒有抑制的效果產生,將甘薯beta-澱粉酶以同樣方法與胰脂解酶作用,結果同上述兩種beta澱粉酶依舊沒有抑制的現象。更改蛋白質放入的順序,先將beta-澱粉酶加入脂解酶反應液中反應一段時間後,再加入脂解酶觀察活性的變化,結果與Satouchi等人結果相同,玉米和大豆的beta-澱粉酶會使胰脂解酶產生被抑制的效果。將玉米胚盤中的脂解酶部分純化後與玉米的beta-澱粉酶作用,不論是抑制劑先與脂解酶混合或是先置於反應液中,玉米脂解酶的活性皆不會被抑制,所以在玉米中beta-澱粉酶並非為脂解酶的抑制劑,其功能尚有待釐清。

並列摘要


Degradation and mobilization of reserve food in germinating seeds are essential processes for establishing a healthy seedling. Lipase is the crucial enzyme responsible for hydrolysis of triacyglycerol into fatty acids leading the conversion of lipid to carbohydrate. Proteinaceous inhibitors of lipase have been demonstrated in many plant species. Lipoxygenase isolated from germinating soybean cotyledon can compete for lipid substrate and show the inhibitory effect on pancreatic lipase in vitro. Satouchi et al. (2002) reported that a protein existing in lipoxygenase-deficient soybean seeds also acted as lipase inhibitor. The inhibitor was further identified as a beta-amylase by MALDI-MS analysis. In a germinating maize kernel, beta-amylase activity is synthesized in aleurone cells where no starch exists, and unlike beta–amylase, beta–amylase is not secreted to starchy endosperm. Owing to the inaccessibility of substrate, the physiological function of aleurone beta-amylase is unknown. Some lipases reported possess both lipase and phospholipase activities. We suspected that aleurone beta-amylase may serve as a lipase inhibitor to block phospholipase activity and delay the senescence of aleurone cells. To examine the hypothesis, we test whether maize beta-amylase can serve as a lipase inhibitor. In order to conduct the interaction assay of beta-amylase and lipase, we adopted a preparative electrophoresis method with a Prep Cell to purify beta-amylase proteins from germinating maize endosperms and soybean cotyledons. We pre-mixed tested proteins with lipid substrate prior to the addition of lipase preparation and assayed the lipid hydrolytic activity. Although maize beta-amylase did not affect the activity of partially purified maize lipase, both soybean and maize beta-amylases showed the inhibitory effect on the enzyme activity of pancreatic lipase. For testing if there were any physical interactions between lipase and beta-amylase molecules, we pre-incubated lipase with beta-amylase before adding substrate to the reaction mixture. No inhibitory effects of beta-amylases on either pancreatic or maize lipases could be detected. The data suggested that the inhibitory effect of beta-amylase on lipase is not due to the direct interaction of these two protein molecules.

並列關鍵字

Lipase beta-amylase protein purification

參考文獻


Ohto, M.A., Nakamura Kito, K., and Nakamura, K. (1992). Induction of expression
Wang, S.M., Lue, W.L., Wu, S.Y., Huang, H.W., and Chen, J. (1997).
Niittyla, T., Messerli, G., Trevisan, M., Chen, J., Smith, A.M., and Zeeman, S.C.
degradation of endosperm starch during seed germination of maize. Taiwania 49,
Baker, A., Graham, I.A., Holdsworth, M., Smith, S.M., and Theodoulou, F.L.

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