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研究生: 葉偉鑫
Wily
論文名稱: 木鱉果假種皮冷凍乾燥萃取物的潛在蛋白酶與脂肪酶之鑑定
Identification of Potent Bioactive Protease and Lipase from Lyophilized Aril Extract of Momordica Cochichinensis Fruit
指導教授: 徐志宏
Douglas J. H. Shyu
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
論文出版年: 2022
畢業學年度: 111
語文別: 英文
論文頁數: 59
中文關鍵詞: 木鱉果蛋白酶假種皮脂肪酶酵素
外文關鍵詞: Gac fruit, Protease, Aril, Lipase, Enzyme
研究方法: Extraction 、 Enzymatic activity 、 Identifications 、 Carotenoids content 、 Antioxidant 、 Scratch assay
DOI URL: http://doi.org/10.6346/NPUST202200461
相關次數: 點閱:126下載:14
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  • 東南亞超級水果木鱉果的假種皮富含番茄紅素和其他大量類胡蘿蔔素。 已經對苦瓜的假種皮進行了大量研究,但還沒有對酶活性或蛋白質組學進行研究。 在這項研究中,液氮被用來研磨假種皮,然後進行超聲波輔助提取和冷凍乾燥。 CAP 提取物通過水解棕櫚酸 4-硝基苯酯並在 37 °C 下孵育 20 分鐘期間產生 0.0478 U/ml 的 4-硝基苯酚來顯示脂解活性。 CAP 還通過水解酪蛋白並產生 2.18 U/ml 的酪氨酸而具有蛋白水解活性。 使用凝膠 LC-MS/MS 檢測可能的蛋白酶,該蛋白質被鑑定為類奇異果甜蛋白,它是一種與在丹氏奇異球菌中發現的奇異果甜蛋白相關的半胱氨酸蛋白酶。 進行初步實驗以確定 CAP 對正常人真皮成纖維細胞 (HFC) (CG1476) 遷移的影響。

    The arils of the extraordinary Southeast Asian superfruit Momordica cochinchinensis are rich with lycopene and other enormous carotenoids. Numerous investigations have been done on the aril of Momordica cochinchinensis, but no research has been conducted on enzymatic activity or proteomics. In this investigation, liquid nitrogen was used to grind the aril, followed by ultrasound-assisted extraction and freeze-drying. CAP extracts revealed lipolytic activity by hydrolyzing 4-nitrophenyl palmitate and generating 0.0478 U/ml of 4-nitrophenol during a 20-minute incubation at 37 °C. CAP also contains proteolytic activity by hydrolyzing casein and generating 2.18 U/ml of tyrosine. Using gel LC-MS/MS to detect the probable protease enzyme, the protein was identified as a thaumatin-like protein, which is a cysteine protease associated with the thaumatin discovered in Thaumatococcus daniellii. A preliminary experiment was undertaken to determine the effect of CAP on the migration of normal human dermal fibroblasts (HFCs) (CG1476).

    Table of Contents
    中華民ll1年12月15日 I
    摘要 I
    Abstract II
    Acknowledgement III
    List of Figures VI
    Lists of Equations VIII
    Lists of Tables IX
    I. INTRODUCTION 1
    1.1 Background 1
    1.2 Purpose of Study 3
    II. LITERATURE REVIEW 4
    2.1 M. cochichinensis 4
    2.2. Antioxidant scavenging 6
    2.3 Plant lipase 6
    2.4 Protease 7
    2.4.1 Cysteine protease 8
    2.4.2 Thaumatin Like-Protein 9
    2.4.3 Heat shock protein 10
    2.5 Wound healing potential 11
    III. MATERIALS AND METHODS 13
    3.1 Conceptual Framework 13
    3.2 Materials 14
    3.2.1 Fruit 14
    3.2.2 Cell lines 14
    3.2.3 Chemicals 14
    3.2.4 Apparatus 15
    3.3 Methods 18
    3.3.1 Ultrasound-Assisted Extraction (UAE) of Gac aril 18
    3.3.2 Determination of Carotenoids analysis. 18
    3.3.3 Determination of Antioxidant capacity. 19
    3.3.4 Proteomic analysis 19
    3.3.5 Scratch Assay 25
    IV. RESULTS 26
    4.1 Crude Aril Powder (CAP) extraction 26
    4.2 Determination of Lycopene and β-Carotene content. 26
    4.3 Antioxidant activity 27
    4.4 Enzymatic assay 28
    4.4.1 Determination of lipolytic activity using P-Nitrophenyl Palmitate 28
    4.4.2 Hydrolysis Effect of CAP using Casein. 32
    4.5 Scratch assay 42
    V. DISCUSSION 44
    VI. CONCLUSIONS 49
    REFERENCES 50

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