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標題: | 玉米β-澱粉?的純化輿定性 Purification and Characterization of maize β-amylase |
作者: | 吳淑媛 |
出版年 : | 1991 |
學位: | 碩士 |
摘要: | 玉米種子在萌芽時,出現了五個澱粉水解?,其中四個為α-澱粉?,只有一個是β-澱粉?。利用超高速離心、硫酸銨沉澱、離子交換及膠體過濾層析等方法純化β-澱粉?,純化率為13倍。分析所純化的酵素液與澱粉作用後終產物,確定所得為β-澱粉?。經SDS-PAGE分析計算,β-澱粉?分子量為64KD,而經膠體過濾層析法所得的分子量為76KD,為單元體,經檢測β-澱粉?具下列特性:pI為4.25,以可溶性澱粉為受質反應的最適pH值是6.5;對熱敏感,65℃加熱5分鐘即完全失去活性。活性受重金屬離子、pCMB嚴重的抑制,活性部位(active site)可能有SH基直接參與催化作用。 將純化的酵素製備液經SDS-PAGE再淨化後,注入兔子體內誘導產生抗體,以Western blot鑑定抗玉米β-澱粉?抗體,抗體可以有效的辨認變性的β-澱粉?,但與原態β-澱粉?的反應則很微弱。 收集不同時期的玉米種子(發育中及萌芽時),抽取RNA進行離體轉譯(in vitro translation),接著用抗體進行免疫沉澱、SDS-PAGE分析及螢光顯像,結果顯示發育中的玉米穀粒沒有β-澱粉?mRNA活性,而萌芽玉米糊粉層的部分,第2天即出現β-澱粉?mRNA活性,但內子葉中一直沒有β-澱粉? mRNA出現,因此證明玉米的β-澱粉?是在萌芽時於糊粉層中de novo合成的。而利用活體同位素標定(in vivo labeling)β-澱粉?與離體轉譯所合成的β-殿粉?在SDS-PAGE上比較,結果並無明顯差異。 The endosperm of maize kernel contains large amount of starch. Upon seed germination, amylolytic enzymes are synthesized and secreted into endosperm by scutellum and aleurone layer to hydrolyze starch. Five amylolytic isozymes, namely Amyl-1, 2, 3, 4, 5 were separated and identified on native gel by activity staining after electrophoresis. Amyl-1, 2, 3, 4 are α-amylases, while Amyl-5 is β-amylase. Maizeβ-amylase (Amyl-5) was purified from endosperm of germinating seeds by ultracentrifugation, ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The specific activity was increased by 13-fold. The purified Amyl-5 was identified to be a β-amylase by end product analysis. The molecular weight of maize β-amylase was determined to be 64 KD by SDS-PAGE, and 76 KD by gel filtration. The isoelectric point of the purified enzyme was 4.25. The cplimal pH for catalysis was 6.5. Beta-amylase as rapidly inactived at by heating at 65°C. The enzyme was strongly inhibitad by heavy metals, p-chloromercoriphenylsulfonic acid, that indicated cysteinyl sulfhydryls are directly involved in catalytic site(s). A polyclonal antiserum against maize β-amylase was obtained by injecting a highly purified enzyme preparation into a rabbit. The identity of β-amylase specific antibodies was confirmed by Western blot analysis. The antiserum efficiently recognized the denatured β-amylase molecule, but weakly crossreacted with the native form of β-amylase. By using rabbit reticulocyte lysate system, in vitro translation directed by RNA isolated from kernels at various developing stages was conducted. Based on the results from in vitro translation and immunoprecipitaton, I concluded that β-amylase is de nova synthesized during seed germination but not developing stage. The β-amylase molecules (64KD) synthesized in vitro and in vivo co-migrated on SDS-PAGE, and there was no apparent co-or post-translational processing. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75842 |
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顯示於系所單位: | 植物科學研究所 |
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