放線菌Nonomuraea angiospora Cytochrome P450 Monooxygenase之純化與檢定

Abstract

Cytochrome P450 monooxygenases (P450s) 是由超級家族(superfamily) 基因所表現，普遍存在於生物體中。P450s催化多樣反應，包括carbon hydroxylation、dealkylation、epoxidation及dehalogenation，並且參與藥物的轉化、異種生物化合物的轉換以及生理上重要化合物的生合成。 Nonomuraea angiospora 為放線菌之一株，為探討N. angiospora P450催化之反應，本研究以硫酸銨分劃及DEAE-Sephacel離子交換管柱層析自N. angiospora 中純化P450。然而，純化所得的酵素濃度偏低，不易偵測。因此將N. angiospora之P450基因選殖入表現載體中，轉形至酵母菌Pichia pastioris X-33表現重組P450蛋白質。以TALON® Metal Affinity Resin純化末端帶有His-tag之重組P450蛋白質，可於SDS-PAGE及免疫轉印分析中觀察到重組P450蛋白質色帶。於in vitro的活性分析中未見重組P450蛋白質之轉化活性，可能由於重組P450蛋白質並未與heme結合，或者因為缺少了P450催化反應時所需的適當redox partners所致。

Cytochrome P450 monooxygenases (P450s), which are encoded by a superfamily of genes, are widely distributed in various organisms. They catalyze a broad of versatility reactions such as carbon hydroxylation, dealkylation, epoxidation and dehalogenation, and are involved in biotransformation of drugs, bioconversion of xenobiotics, biosynthesis of physiologically important compounds, and so on. Nonomuraea angiospora belongs to Actinobacteria.To investigate the reactions catalyzed by P450 of N. angiospora, the enzyme was purified by ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography. However, the enzyme activity could not be detected, which might due to the low level of proteins. The plasmid carrying the open reading frame of a P450 gene of N. angiospora was transformed into Pichia pastioris X-33 for expression of recombinant P450. The His- tagged recombinant proteins were purified by TALON® Metal Affinity Resin. A major protein band was observed when the Co2+-based IMAC- purified proteins were analyzed by SDS-PAGE and immunoblotting. The result of in vitro activity assay showed that the recombinant P450 did not exhibit enzyme activity , which might due to the absence of heme group in the recombinant protein and/or the lack of appropriate redox partners for coupling the reaction catalyzed by P450.