Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
    8.
    发明授权
    Regio- and enantioselective alkane hydroxylation with modified cytochrome P450 有权
    修饰的细胞色素P450的区域和对映选择性烷烃羟基化

    公开(公告)号:US09145549B2

    公开(公告)日:2015-09-29

    申请号:US14293465

    申请日:2014-06-02

    CPC classification number: C12N9/0071 C12N9/0077 C12P7/04 C12Y114/14001

    Abstract: 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. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-position to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.

    Abstract translation: 使用定向进化和定点诱变的组合将来自巨大芽孢杆菌的细胞色素P450 BM-3设计为使用大气二氧氧化物作为区域和对映选择性的羟基化线型烷烃。 突变体9-10A-A328V主要在2-位羟基化辛烷,形成S-2-辛醇(40%ee)。 另一个突变体1-12G,主要在2-位羟基化烷烃大于己烷,但形成R-2-醇(40-55%ee)。 这些生物催化剂对烷烃底物具有高活性,并支持数千种产品周转。 这些区域和对映体选择性保留在与大肠杆菌的全细胞生物转化中,其中工程化的P450可以以高水平表达并且昂贵的辅因子是内源性提供的。

    REGIO- AND ENANTIOSELECTIVE ALKANE HYDROXYLATION WITH MODIFIED CYTOCHROME P450
    10.
    发明申请
    REGIO- AND ENANTIOSELECTIVE ALKANE HYDROXYLATION WITH MODIFIED CYTOCHROME P450 有权
    具有改良的细胞色素P450的REGIO-和选择性碱性羟化

    公开(公告)号:US20150010976A1

    公开(公告)日:2015-01-08

    申请号:US14293465

    申请日:2014-06-02

    CPC classification number: C12N9/0071 C12N9/0077 C12P7/04 C12Y114/14001

    Abstract: 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. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-position to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.

    Abstract translation: 使用定向进化和定点诱变的组合将来自巨大芽孢杆菌的细胞色素P450 BM-3设计为使用大气二氧氧化物作为区域和对映选择性的羟基化线型烷烃。 突变体9-10A-A328V主要在2-位羟基化辛烷,形成S-2-辛醇(40%ee)。 另一个突变体1-12G,主要在2-位羟基化烷烃大于己烷,但形成R-2-醇(40-55%ee)。 这些生物催化剂对烷烃底物具有高活性,并支持数千种产品周转。 这些区域和对映体选择性保留在与大肠杆菌的全细胞生物转化中,其中工程化的P450可以以高水平表达并且昂贵的辅因子是内源性提供的。

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