HbA1c (Hemoglobin A1c) 在總糖化血紅素中含量最多，且可代表二至三個月體內平均血糖濃度，因此HbA1c的指數一直是糖尿病患者的平均血糖指標。分析HbA1c含量的方法有許多種，本論文是以FPO (fructosyl peptide oxidase) 酵素氧化HbA1c中valine殘基上之fructose (稱為fructosyl valine, FV) 產生H2O2，再利用過氧化酶 (horseradish peroxidase, HRP) 催化呈色劑呈色，由呈色的深淺推算HbA1C的濃度。本研究首先將FPO表現質體在大腸桿菌中進行大量表現，發現在37℃、LB培養基的培養下，過量表現之FPO異源性蛋白質多以不具活性的內涵體形式存於胞內，若降低培養溫度為26℃，可大幅增加具活性和可溶性FPO的含量。以固定化金屬螯合層析法 (IMAC) 純化大腸桿菌所表現生產之FPO氧化酵素，其比活性可達26.01U/mg，為市售之FAO的五倍。此FPO酵素在溫度4℃~40℃、pH 5~10 環境下，仍能保有相當之活性。
此外，本論文也探討使用兩種不同的呈色劑ODA與DA-64對偵測FV靈敏度之差異，結果顯示DA-64之靈敏度為ODA的6.3倍，可偵測到0.02 mM之FV。最後將FPO氧化酵素利用多巴胺 (dopamine) 能自我氧化聚合的效應固定在PP微孔膜中，製作出能偵測FV濃度之酵素膜，此酵素膜可以重覆使用至8次仍能保有約20% 之活性。

HbA1c (Hemoglobin A1c) is the major glycated hemoglobin in human blood, the amount of the HbA1c reflects the average blood glucose concentration of the past few months. HbA1c is recognized as an important diagnostic indicator of diabetes mellitus.
HbA1c is a hemoglobin molecule in which the N-terminal valine residue of its β－subunit has been modified by blood glucose. In this study, the enzymatic method was developed to detect HbA1c concentration in blood. It consists three steps: (1) proteolysis of the HbA1c β－subunits to release fructosyl valine (FV) (2) oxidation of the released FV by FPO (fructosyl peptide oxidase) to produce H2O2 (3) detection of the produced H2O2
FPO was expressed by a genetic engineered E.coli pET23a－FPO in this work. The recombinant E.coli strain cultured at 26℃ was found out can produce more FPO activity than at 37℃. The specific activity of purified FPO protein is 26.01 U/mg after IMAC purification. It is about 5 fold higher than that of commercially available FAO. The FPO protein can well maintain its activity in the range of 4℃~ 40℃ and pH 5~10.
In addition, the FV detection sensitivity using the dye of ODA and DA-64 has been investigated. The results showed that the sensitivity of the DA-64 was 6.3 fold higher than that of ODA. FV concentration as low as 0.02Mm could be detected by using this enzymatic colorimetric method.

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