School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Institute of Bioengineering, Seoul National University, Seoul 151-742, South Korea.
Biotechnol Bioeng. 2010 Mar 1;105(4):697-704. doi: 10.1002/bit.22582.
Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.
阿维链霉菌 MA4680 来源的 CYP105D7 对大豆苷元进行区域选择性 3'-羟化反应。在存在 1 μM 的 CYP105D7、putidaredoxin（CamB）和 putidaredoxin reductase（CamA）的情况下，CYP105D7 对大豆苷元的表观 K(m)和 k(cat)值分别为 21.83±6.3 μM 和 15.01±0.6 min(-1)。当 CYP105D7 在阿维链霉菌 MA4680 中表达时，使用全细胞提取物通过 450nm 处的 CO 差光谱证实其细胞色素 P450 活性。当用 100μM 的大豆苷元在 100mM 的磷酸盐缓冲液（pH7.5）中进行大豆苷元 3'-羟化反应的全细胞反应时，在 R2YE 培养基中过表达 CYP105D7 和 ferredoxin FdxH（SAV7470）的重组阿维链霉菌比相应的野生型细胞（6.7%）显示出 3.6 倍更高的转化率（24%）。在 7L（工作体积 3L）搅拌釜发酵罐中，在 R2YE 培养基中生长的重组阿维链霉菌从 381mg 的大豆苷元中生产出 112.5mg 的 7,3',4'-三羟基异黄酮（即 29.5%的转化率），在 15h 内。
