title: First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor.
abstract: 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.
authors.affiliation: College of Chemistry and Chemical Engineering, Shenzhen University, Shenzhen, 518060 Guangdong, People's Republic of China.
authors.forename: Liqing
authors.initials: L
authors.lastname: Zhao
authors.forename: Güray
authors.initials: G
authors.lastname: Güven
authors.forename: Yin
authors.initials: Y
authors.lastname: Li
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010647:Phenyl Ethers / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D004800:Enzymes, Immobilized; D050505:Mutant Proteins; D010647:Phenyl Ethers; C077432:1-methyl-3-phenoxybenzene; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases