College of Chemistry and Chemical Engineering, Shenzhen University, Shenzhen, 518060 Guangdong, People's Republic of China.
Appl Microbiol Biotechnol. 2011 Aug;91(4):989-99. doi: 10.1007/s00253-011-3290-7. Epub 2011 May 12.
Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% (v/v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000.
细胞色素 P450s 是具有吸引力的合成羟化催化剂。对于无细胞应用，NAD(P)H 的持续供应可能非常昂贵。介体如 Zn/Co(III)sep 可以替代 NAD(P)H 作为辅助因子系统。我们小组已经获得了几种具有向 Zn/Co(III)sep 传递电子速率提高的细胞色素 P450 BM3 突变体。P450 BM3 M7（F87A V281G M354S R471C A1011T S1016G Q1022R）固定在 DEAE-650S 上，进一步与锌粉一起包埋在κ-卡拉胶中，锌粉作为电子源，而过氧化氢酶可立即去除产生的过氧化氢。用 0.05%（v/v）戊二醛处理固定化 P450 BM3 M7 以增强操作稳定性。P450 BM3 M7 保留了约 76%的活性，在 10 批循环中转化率保持在 80%以上，表明固定化 P450 BM3 M7 具有很高的稳定性。为了探索合成潜力，开发了一个小规模生物反应器来研究 P450 BM3 M9（R47F F87A M238K V281G M354S D363H W575C A595T）的稳定性和效率。由于 P450 BM3 M9 对 3-苯氧基甲苯的活性比用于优化固定化条件的 P450 BM3 M7 高 3 倍，因此 P450 BM3 M9 用于在推流式反应器 (PFR) 中连续转化 3-苯氧基甲苯。该反应器可连续运行 5 天，总转化数（TTN）超过 2000。
