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研究生:張郁莘
研究生(外文):Yu-Hsin Chang
論文名稱:基改醣化胜肽氧化酶(FPOX)之表現純化及測定糖化血紅素(HbA1c)之應用
論文名稱(外文):Engineered fructosyl peptide oxidase expression, purification and its application for the determination of HbA1c in blood
指導教授:李振綱李振綱引用關係
指導教授(外文):Cheng-Kang Lee
口試委員:李振綱
口試委員(外文):Cheng-Kang Lee
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:81
中文關鍵詞:中性蛋白水解酶糖化血紅素糖化纈草胺酸醣化胜肽氧化酶DA-64海藻糖
外文關鍵詞:HbA1cFPOXFVNeutral proteaseDA-64Trehalose
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血液中的葡萄糖與血紅素會結合形成糖化血紅素(HbA1c),一般紅血球平均壽命約為120天,因此糖化血紅素的濃度可以作為體內近2~3個月的平均血糖指標。分析HbA1c含量的方法有許多種,本研究是以FPOX (fructosyl peptide oxidase)酵素氧化經Neutral protease水解HbA1c所得到的FVH (fructosyl valyl histidine)片段而產生的H2O2,再利用酵素呈色法與電化學法進行偵測H2O2濃度,進而推算出HbA1c的濃度。酵素呈色法是利用水解所產生之H2O2經過氧化酶 (horseradish peroxidase,HRP)催化使呈色劑DA-64呈色,由呈色深淺推算出HbA1c的濃度。電化學法是以碳為工作與輔助電極,Ag/AgCl為參考電極製備而成的三極式電極作為過氧化氫感測試片,於施加電位0.4V下,利用鉑-多層壁奈米碳管(PtCNT)觸媒催化H2O2所產生之電流反推出HbA1c的濃度。FPOX已在許多微生物中被發現,例如Phaeosphaeria nodorum SN15 (XP_001798711) (PnFPOX)及Coniochaeta sp. NISL 9330 (BAD00186) (FPOX-C),本論文將PnFPOX與loop置換突變株PnFPOX-CL2以及FPOX-C-PnL1於大腸桿菌中進行大量表達,在低溫下(4℃)進行固定化金屬螯合層析法(IMAC)純化,當基質為FV (fructosyl valine)時,三酵素的比活性差異不大,但以FVH為基質時,FPOX-C-PnL1酵素之比活性為32.3 mU/mg,約為其餘兩酵素比活性的4倍。PnFPOX與兩loop置換突變株在4~30℃環境下皆能維持70~80%的活性,但在50℃的環境下皆失去其活性,若將酵素保存於Trehalose溶液中50℃環境下仍可維持80~90%活性,而PnFPOX於pH 7之環境中能維持最佳的活性,PnFPOX-CL2與FPOX-C-PnL1則為pH 6環境下能維持其最佳活性。使用FPOX-C-PnL1酵素進行酵素呈色法可偵測出5.2~10.7%HbA1c的校正檢量線而電化學法可偵測出5.1~12.5%的HbA1c校正檢量線。
The glycated hemoglobin (HbA1c) level in blood is an important indicator for evaluating long-term control of glycemic status in diabetic patients. Enzymatic colorimetric assay and electrochemical method are two of the methods to measure HbA1c level. Enzymatic colorimetric assay is based on the enzymatic reaction of fructosyl peptide oxidase (FPOX) toward fructosylvalyl-histidine (FVH). FVH is the hydrolysis product released from the β-chain of HbA1c by neutral protease. The FVH is oxidized by FPOX and generate hydrogen peroxide which will oxidize sodium N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine (DA-64) via horseradish peroxidase (HRP) to develop green color. Electrochemical method detects HbA1c by using H2O2 electrochemical sensor. A single-use H2O2 electrochemical sensor was fabricated by assembling a PtCNT-carbon modified working electrode, an Ag/AgCl reference electrode, and a carbon counter electrode. The working potential for the amperometric detection was fixed at +0.4V versus the reference electrode. FPOX have been found in various microorganisms such as Phaeosphaeria nodorum SN15 (XP_001798711) (PnFPOX), Coniochaeta sp. NISL 9330 (BAD00186) (FPOX-C). PnFPOX and the loop-substitution mutants of PnFPOX and FPOX-C were expressed in Escherichia coli. After immobilized metal affinity chromatography (IMAC) purification, the wild-type PnFPOX and loop-substitution mutants showed no significant different in specific activity with fructosyl valine (FV) as substrate, while the loop-substitution mutants reacted with FVH with much higher rate. The higher specific activity toward FVH of loop-substitution mutants was resulted from the combination of the first loop of PnFPOX and the second loop of FPOX-C. The specific activity of FPOX-C-PnL1 enzyme is 32.3 mU/mg. It is about 4 fold higher than the other two enzymes. The wild-type PnFPOX and loop-substitution mutants have 70-80% residual activity after 30 minutes incubation at 30℃ and no activity at 50℃, but it can be stabilized by trehalose. In trehalose solution, enzymes can retain about 80-90% activity at 50℃. The optimum pH for activity of wild-type PnFPOX is around 7, while optimum pH of loop-substitution mutants shifts to about 6. HbA1c concentration range from 5.2 to 10.7% were detected by enzymatic colorimetric assay, and HbA1c concentration range from 5.1 to 12.5% can be detected by electrochemical method by using loop substituted FPOX.
摘要I
AbstractII
誌謝III
目錄IV
圖目錄VI
表目錄VIII
第一章 緒論1
1.1前言1
1.2研究目的及內容簡介2
第二章 文獻回顧4
2.1糖化血紅素 (Glycated hemoglobin) 之來源與組成4
2.1.1 HbA1c 糖化血紅素6
2.2糖化血紅素之測定與分析9
2.2.1陽離子交換樹指層析法 ( Cation exchange HPLC)9
2.2.2硼酸親和性層析法 (Boronate affinity HPLC)10
2.2.3免疫比濁法 (Immunoassay)11
2.2.4電化學法12
2.2.5酵素比色法12
2.3酵素比色法所使用之氧化酵素13
2.3.1 FAOX (Fructosyl amino acid oxidase)14
2.3.2 FPOX (Fructosyl peptide oxidase)15
2.4海藻糖 (Trehalose)19
2.5酵素比色法測試糖化血紅素HbA1c所使用之呈色劑20
2.6電化學分析方法21
2.6.1循環伏安法 (Cyclic Voltammetry)21
2.6.2計時安培法 (Amperometric i-t)24
第三章 實驗材料與方法26
3.1實驗流程26
3.2實驗材料27
3.2.1實驗菌株27
3.2.2質體27
3.2.3其它27
3.3實驗藥品28
3.4溶液配置30
3.5實驗儀器與設備34
3.6實驗方法35
3.6.1 E.coli重組基因菌株培養及FPOX重組蛋白之生產35
3.6.2 FPOX重組蛋白之純化36
3.6.3蛋白質電泳分析38
3.6.4 FPOX重組蛋白質之濃度分析40
3.6.5 FPOX活性分析41
3.6.6 FPOX之酵素動力學測定42
3.6.7 FPOX專一性測試43
3.6.8溫度與pH值對FPOX活性的影響43
3.6.9加入不同濃度Trehalose後溫度對FPOX活性的影響43
3.6.10溶血與偵測HbA1c中fructosyl valyl histidine(FVH)的含量44
3.6.11合成糖化纈草胺酸(FV)45
3.6.12過氧化氫感測試片製備45
第四章 結果與討論49
4.1含pET23a – FPOX質體之大腸桿菌表現生產FPOX49
4.1.1 培養條件49
4.2 DA-64之消光係數(ε)與FPOX活性分析及純化51
4.2.1消光係數(ε)51
4.2.2 FPOX活性分析及室溫下純化52
4.2.3 FPOX活性分析及冰浴下純化54
4.2.4對基質FV與FVH酵素FPOX之活性影響比較57
4.3溫度與pH值對FPOX活性之影響61
4.3.1 酵素之熱穩定性61
4.3.2 酵素之pH穩定性62
4.4 FPOX酵素存放之穩定性63
4.5 Trehalose對FPOX之熱穩定性影響64
4.6 FPOX酵素於50℃下之溫度半衰期66
4.7三種 FPOX 酵素與市售FPO之酵素動力學67
4.8 FPOX對基質反應之專一性72
4.9糖化血紅素(HbA1c)之偵測73
4.9.1酵素呈色法偵測糖化血紅素 (HbA1c)73
4.9.2以電化學法偵測糖化纈草胺酸 (FV)74
4.9.3以電化學法偵測糖化血紅素 (HbA1c)76
第五章 結論77
參考資料79
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