The present invention provides a method for preparing a hybrid gene. The method includes a step of amplifying a P450 gene fragment contained in a sample using primers designed on the basis of regions of a plurality of P450 in which amino acid sequences are highly conserved and a step of preparing the hybrid gene using the amplified fragments and a known P450 gene. The method includes no culturing step or a step of normalizing extracted DNAs and is useful in isolating a P450 gene from various microbial resources.
The present invention further provides a fused cytochrome P450 monooxygenase containing a peptide which is linked to the C-terminus of a P450 protein with a linker portion disposed therebetween and which has the same function as that of a reductase domain contained in a cytochrome P450 monooxygenase originating from Rhodococcus sp. strain NCIMB 9784. This enables the construction of a high-efficiency electron transfer system useful for various P450 proteins and also enables the production of an active P450 monooxygenase.
本发明提供了一种制备杂交
基因的方法。该方法包括使用根据多种 P450 的
氨基酸序列高度保守的区域设计的引物扩增样品中所含的 P450
基因片段的步骤,以及使用扩增片段和已知 P450
基因制备杂交
基因的步骤。该方法不包括培养步骤或将提取的 DNA 归一化的步骤,可用于从各种微
生物资源中分离 P450
基因。
本发明进一步提供了一种融合的细胞色素 P450 单加氧酶,该融合的细胞色素 P450 单加氧酶含有一个
多肽,该
多肽与 P450 蛋白的 C 端相连,中间有一个连接部分,其功能与源自 Rhodococcus sp.这样,就能构建一个对各种 P450 蛋白有用的高效电子传递系统,还能生产一种活性 P450 单加氧酶。