Peters Matthew W, Meinhold Peter, Glieder Anton, Arnold Frances H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
J Am Chem Soc. 2003 Nov 5;125(44):13442-50. doi: 10.1021/ja0303790.
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.
利用定向进化和定点诱变相结合的方法，对巨大芽孢杆菌的细胞色素P450 BM-3进行改造，使其能够以大气中的氧气作为氧化剂，对直链烷烃进行区域选择性和对映选择性羟基化反应。BM-3变体9-10A-A328V在2-位将辛烷羟基化，形成S-2-辛醇（对映体过量40%）。另一个变体1-12G也主要在2-位将大于己烷的烷烃羟基化，但形成R-2-醇（对映体过量40-55%）。这些生物催化剂具有高活性（速率高达400 min⁻¹），并支持数千次产物转化。在大肠杆菌的全细胞生物转化中，区域选择性和对映选择性得以保留，在这种情况下，改造后的P450能够高水平表达，并且辅因子由内源性提供。
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