藤仓赤霉菌中蟾毒灵7β-羟化酶的发现与功能鉴定

Identification and functional characterization of a bufalin 7β⁃hydroxylase from Fusarium fujikuroi

目的挖掘能催化蟾蜍内酯类化合物特异性羟化修饰的酶,以促进名贵中药材蟾酥功效组分的开发与应用。方法采用同源序列比对挖掘藤仓赤霉菌(Fusariumfujikuroi)中的甾体羟化酶,通过毕赤酵母异源表达、工程菌株细胞转化以及代谢产物的分离与鉴定,从而确定酶的功能。结果以蟾蜍内酯类化合物蟾毒它灵、蟾毒灵为底物,毕赤酵母重组菌株KM71H-FF294转化蟾毒它灵没有产生明显的产物,而转化蟾毒灵时产生了羟化产物7β-羟基蟾毒灵。结论从藤仓赤霉菌中发现能催化蟾毒灵发生7β-羟化修饰的甾体羟化酶FF294,是目前文献报道首个能催化蟾蜍内酯类化合物羟基化反应的酶。

Objective To explore enzymes that catalyze specific hydroxylation modification of bufadienolides so as to promote the development and application of the efficacy components of the rare traditional Chinese medicine Chansu(Bufonis Venenum).Methods The steroid hydroxylase from Fusarium fujikuroi was discovered by sequence alignment,and its function was determined by heterologous expression in Pichia pastoris and isolation and identification of the metabolites transformed by the engineered strains.Results The activity of the strain KM71H-FF294 was investigated by using bufalin and bufotaline as substrates.No product was produced when KM71H-FF294 transformed bufotaline,but the hydroxylation product 7β-OH bufalin was produced when bufalin was transformed.Conclusion Steroid hydroxylase FF294 from Fusarium fujikuroi could catalyze the conversion of bufalin into 7β-hydroxylated products,and this is the first report of an enzyme that catalyzes the hydroxylation of bufadienolides.