Paraburkholderia caffeinilytica CF1咖啡因降解关键酶CdnB的特性

Characterization of CdnB,a key enzyme for caffeine degradation in Paraburkholderia caffeinilytica CF1

CdnB是在Paraburkholderia caffeinilytica CF1咖啡因降解途径中催化3,7-二甲基黄嘌呤生成7-甲基黄嘌呤的N3-脱甲基酶。本实验通过基因重组在大肠杆菌中表达CdnB-His6,利用亲和层析法进行纯化。经SDS-PAGE结合Native-PAGE,测定其分子质量为58 ku。CdnB的最适底物为3,7-二甲基黄嘌呤,最适反应温度为30℃,最适反应pH为8.0,在pH 6.5~8.5酶活力保持稳定。除Fe^2+对该酶活力有促进作用外,其他金属离子对该酶活力有不同程度的抑制作用,其中Co^2+及Cu^2+对酶活性抑制程度最为显著。

A N3-demethylase named CdnB is a key enzyme in the caffeine degradation of Paraburkholderia caffeinilytica CF1,which is in charge of catalyzing N3-demethylation of 3,7-dimethylxanthine to produce 7-methylxanthine.CdnB-His6 was heterologously over-expressed and purified to homogeneity as His-tagged proteins.SDS-PAGE and Native-PAGE analysis were used to determine the molecular weight of the enzyme to be 58 ku.The optimal substrate for CdnB-His6 was 3,7-dimethylxanthine.The purified CdnB-His6 showed the highest activity at pH 8.0 with 30℃,and the stable pH range in 6.5 to 8.5.The activity of CdnB-His6 was inhibited by metal ions(except for Fe^2+),especially with addition of Co^2+and Cu^2+.