Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3
Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3
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DOI：
10.1021/ja0303790
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发表时间：
2003-11-05
影响因子：
15
通讯作者：
Arnold, FH
中科院分区：
文献类型：
--
作者：
Peters, MW;Meinhold, P;Arnold, FH
使用定向进化和定点诱变的组合来工程化来自巨大芽孢杆菌的细胞色素P450 BM-3，以使用大气中的分子氧作为氧化剂区域选择性地和对映选择性地羟基化直链烷烃。BM-3变体9- 10A-A328 V在2位羟基化辛烷以形成S-2-辛醇（40%ee）。另一种变体1- 12 G也主要在2-位羟基化大于己烷的烷烃，但形成R-2-醇（40- 55%ee）。这些生物催化剂具有高活性（速率高达400 min（-1）），并支持数千次产品周转。区域选择性和对映体选择性保留在大肠杆菌的全细胞生物转化中，其中工程化的P450可以高水平表达，并且辅因子是内源性提供的。
Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers. The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.