血液中的糖化血紅素濃度可視為體內2-3個月平均血糖的重要指標。分析糖化血素的方法有很多種，本論文選用反應速率較快且不易受變異血紅素影響的酵素呈色法進行分析。利用醣化胜肽氧化酶(fructosyl peptide oxidase, FPOX) 氧化經中性水解酶(Neutral protease)水解HbA1c所得到的FVH (fructosyl valyl histidine)片段產生過氧化氫(H2O2)，再利用酵素呈色法，於過氧化氫酶(horseradish peroxidase, HRP)催化呈色劑DA-64反應，由呈色深淺推算出FPOX活性與HbA1c的濃度。由基因工程合成的fructosyl peptide oxidase (FPOX-C-PnL1)，其對FVH的反應性佳，在大腸桿菌中表現所得的可溶性蛋白含量十分低且活性不穩定，為改善此缺失，因此使用來自熱纖梭菌(Clostridium thermocellum)的纖維素結合功能域(cellulose binding domain, CBD)蛋白的高可溶性表現特性，將之融合於FPOX之N-端；並利用CBD與細菌纖維素(bacterial cellulose, BC)間的高親和性特性進行純化與固定化，藉此增加FPOX可溶性蛋白的表現，為提高其固定化蛋白環境耐受性，將固定化於BC上之CBD-FPOX懸浮液，以抽氣過濾方式進行乾燥成膜，製備出CBD-FPOX-BC膜。此CBD-FPOX-BC膜之活性，不易受環境溫度與pH變異影響，保存於4℃-70℃環境下，活性可維持95%以上，而可承受之pH範圍，也由pH 6-8擴大至pH 5-9。使用CBD-FPOX-BC膜進行酵素呈色法可偵測出血液中5.3-11% HbA1c，所得之檢量線線性回歸值可達0.9834。

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 is one of the methods for detecting HbA1c. It is based on the enzymatic reaction of fructosyl peptide oxidase (FPOX) toward fructosyl valyl histidine (FVH), a fragment obtained by protease digestion of HbA1c. FVH will be oxidized by FPOX to generate hydrogen peroxide which can further oxidize sodium N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine (DA-64) by using horseradish peroxidase (HRP) are catalyst to develop green color. The color intensity can reflect the concentration of HbA1c. To develop an engineered FPOX which has a higher reactivity toward FVH, we constructed the loop-substitution mutants of FPOX-C (FPOX-C-PnL1). However, most of this mutant FPOX expressed in Escherichia coli existed as inclusion bodies. To improve the solubility of FPOX-C-PnL1, cellulose binding domain (CBD) was fused in N-terminus to generate a soluble CBD-FPOX fusion protein. By taking advantage of CBD’s strong affinity toward crystalline cellulose, CBD-FPOX was easily purified from Escherichia coli cell lysate and immobilized on bacterial cellulose (BC) produced from Gluconacetobacter xylinus. CBD-FPOX immobilized on BC not only increased the stability of FPOX but also could be directly applied for HbA1c detection. CBD fusion FPOX significantly improved the solubility of FPOX without affecting its activity. Approximately 0.24 mg/mg of CBD-FPOX could be tightly bound on bacterial cellulose in 10 minutes. The FPOX activity of CBD-FPOX BC film could be stable maintained at 4-70℃ and pH 5-9. The CBD-FPOX BC film prepared could be applied for HbA1c detection in blood sample. A linear correlation between color developed and HbA1c sample concentration of 5.3-11% was observed by using this FPOX film catalyzed colorimetric method.

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