建立快速檢測生物胺類化合物之系統

Abstract

中文摘要 生物胺類化合物，例如組織胺、酪胺及5-羥色胺等，在生物體內的多種生理作用中扮演著重要的角色，如神經的傳導、胚胎的發展、細胞的增生與分化等。當食品腐敗時，由於微生物的代謝作用產生一些生物胺類化合物，會引起生物體的過敏、發炎反應。因此，發展具有輕便、快速與經濟等優點的檢測方法對於食品品質及大眾健康相當重要。首先，針對雙酵素偶聯之最適化反應條件進行試驗，結果發現，在100 mM Tris-HCl，pH 8.5，0.5 mg/ml (0.115 U/ml) HRP及55 mM luminol的反應條件下可偵測0.1 mM-100 mM之H2O2；在100 mM Tris-HCl，pH 8.5，0.5 mg/ml (0.115 U/ml) HRP，0.1 mg/ml (0.007 mUml) DAO及10 mM luminol之反應條件下可偵測0.1-200 mM之組織胺。我們的研究方向在於利用基因重組與蛋白質工程的技術，將可針對組織胺反應的Arthrobacter globiformis之組織胺氧化酵素及Coprinus cinereus之過氧化酵素進行選殖，並將此二酵素與可做生物素修飾的生物素梭基載體蛋白加以剪接而形成融合酵素，所建構而成之融合酵素表現載體於大腸桿菌BL21/RP中可大量表現且為可溶態，而對於活性方面的測試發現其具有催化反應之功能。未來，我們將進行融合酵素之大量表現及純化。

Abstract Biogenic amines, e.g., histamine, putrescine, tyramine and serotonin, play important roles in many physiological functions, including neuron transmission, embryo development, cell proliferation and cell differentiation. In spoiled food, the accumulated microorganisms produce lots of biogenic amines, which may induce hypersensitive and inflammatory responses in human. Hence, the development of a handy, quick and economic method for the detection of biogenic amines will be very useful for the food quality monitoring and public health promotion. In this study, we developed a bi-enzyme assay system by coupling diamine oxidase and peroxidase, for the determination of biogenic amines. The study of reaction condition optimization showed that at pH 8.5, in the presence of 100 mM Tris-HCl, and 55 mM luminol, 0.115 U/mL horseredish peroxidase (HRP) can detect as low as 0.1 mM H2O2. The linear range for H2O2 determination is from 0.1 nM to 100 mM. Further more, under the condition of 100 mM Tris-HCl, pH 8.5, 0.5 mg/mL (0.115 U/mL) HRP, 0.1 mg/mL (0.007 mU/mL) DAO and 10 mM luminol the detection of 0.1-200 mM range histamine was feasible. In this project, we also subcloned histamine oxidase from A. globiformis and peroxidase from C. cinereus and BCCP from E. coli K12 by polymerase chain reaction. Two experssion vectors encoding fusion proteins, termed BCCPt-AGDAO and CIP-BCCPts were generated by inserting corresponding gene into the expression vector pTrc99A. We found that both CIP-BCCPts and BCCPt-AGDAO could be expressed in E. coli strains BL21(DE3)-RP as a soluble form, although the expression level for CIP-BCCPts was low. The activity assay suggests that these fusion proteins are functional. In conclusion, we have obtain valuble information about the reaction condition for amine oxidase-peroxidase bienzyme assay system. Further more, we constructed BCCPt-AGDAO and CIP-BCCPts fusion protein genes. The expression of two fusion protein in E. coli strain BL21(DE3)-RP were successful.