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研究生: 李柏青
Li, Bo-Qing
論文名稱: 蕹菜種子胚乳預存性澱粉水解每的純化及性質分析
Purification and characterization of pre-existing amylases in the endosperm of water spinach(Ipomoea aquatica Forsk)
指導教授: 童武夫
Tong, Wu-Fu
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
畢業學年度: 84
語文別: 中文
論文頁數: 73
中文關鍵詞: 生物學蕹菜種子胚乳澱粉每異構每
英文關鍵詞: BIOLOGY
論文種類: 學術論文
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  • 本研究之主題為蕹菜種子胚乳預存生澱粉每純化及性質分析。蕹菜種子具有子葉及胚
    乳,胚乳組織富含澱粉,於萌芽初期迅速分解使組織萎縮而逐漸消失。此時浸液可測
    得澱粉每活性,且此每活性不受外加cycloheximide 所影響,顯示此澱粉每為預存於
    種子中者,而非萌發時所合成。種子浸液之澱粉每經活性電泳碘液染色可觀察到三個
    同功異構每,利用超過濾濃縮法(ultrafiltration, Amicon YM 30) 濃縮粗萃取液、
    streptomycin sulfate離心沉澱、hydroxylapatite column、Mono Qcolumn等純化步
    驟將種子浸液中的澱粉水解每加以純化分離,得到純化倍率為47.17倍,recovery 為
    35%。
    純化過程中所獲得活性較高的異構每,分別命名為amy I-a、amy I-b。經10%SDS 電
    泳分析,估計amy I-a的分子量為47.5kD ,amy I-b 的分子量為46.3kD。依酵素對
    soluble starch及potato starch 水解產物的分析結果推論:純化出的amy I-a 、
    amy I-b 皆為α-澱粉水解酵素。依受質專一性顯示:amy I-a、amy I-b 對potato
    starch、β-limit dextrin 有很高的專一性,其次為soluble starch 及starch
    azure,但對肝糖(glycogen)及dextrin 均無反應。
    amy I-a及amy I-b 作用最適溫度範圍為40℃~50℃,最適pH值範圍為5.5-7.5。
    純化的amy I-a對熱具有耐受性,但amy I-b 對熱則較敏感;amy I-a 在60℃加熱一
    個小時仍具有90%以上的活性但amy I-b於60℃加熱5 分鐘則活性僅剩原來活性的55
    %。純化酵素對受質的親和性以amy I-a較amy I-b 為佳;以potato starch 當受質
    時amy I-a 的Km值為2.1×10-4g/ml,amy I-b 的Km值則為3.2×10-3g/ml,而其對受
    質的親和性以potato starch 大於soluble starch。還原劑2-mercaptoethanol、
    dithiothreitol對amy I-b 的活性有明顯的穩定作用,尤其在室溫的狀態下,有加還
    原劑的酵素活性則明顯比未加還原劑的酵素活性來的高。但還原劑對amy I-a 的酵素
    活性則無明顯的影響作用;無論在室溫或4 ℃的狀況下amy I-a 的活性皆可穩定數小
    時之久而不會迅速消失。所以相對的是否加入還原劑並不影響其穩定性。

    The properties of pre-existing amylolytic enzyme in the
    endosperm of water sp-inach were investigated.water spinach is a dicotyledon plant,and the seed contains cotyledon and a starch rich endosperm. During germination , the endospermtissue is progressively collapsed and disappeared. Meanwhile a fraction of am-ylase activity which could not effect by cycloheximide treatment was detectedin the soaking solution. Thus the enzyme seems likely to be pre-existing inthe seed rather than de novo synthesis durin germination.After electrophoresis,three amylolytic-activity bands were presented in the 6.5% native polyacryla-mide gels containing 0.1 % (w/v) poato starch. The enzymes in socking solutionwere purified by the serial steps of ultrafiltration , streptomycin sulfateprecipitation, hydroxylapatite chromatography and fast protein purificationliquid chromatography (FPLC). Two isoenzymes were purified and named as amy I-a and amy I-b. The molecular weight of both enzymes were closely and they wereestimated at the value of 47.5 KD and 46.3 KD respectively by 10% SDS-PAGE.The purified enzyme were classified as alpha-amylase by substrate specificity,digested products analysis . According to the assay of enzyme-substrate speci-ficity,we could know that purified enzyme were very high substrate specificitywith potato starch beta-limit dextrin , and low substrate specificity withglycogen、dextrin . Both enzymes exhibit the optimum pH and temperature are atthe range of pH 5.5-7.5 and 40C-50C respectively. In addition , amyI-a exhibithigher the theromostability and sub strate affinity than amy I-b. The purifiedamy I-b were reversibly inactivated by oxidation in the absence of reducingagents. The activity was restorD by adding reductant,such as dithiothreitoland 2-mercaptoethanol.
    While the activity of amy I-a is stable from oxidation and will not affect by the present of reductant.

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