研究生: |
張郁莘 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 Engineered fructosyl peptide oxidase expression, purification and its application for the determination of HbA1c in blood |
指導教授: |
李振綱
Cheng-Kang Lee |
口試委員: |
王孟菊
Meng-Jiy Wang 周秀慧 Shiu-Huey Chou |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
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: | HbA1c, FPOX, FV, Neutral protease, DA-64, Trehalose |
相關次數: | 點閱:234 下載:1 |
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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.
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