10.1002/cctc.201902044
ChemCatChem
FULL PAPER
(Agilent, 3.0 250 mm 5 µm). GC analyses were carried out on a Hewlett
Packard HP6890 GC system connected to a DB-5MS capillary column
(Agilent, 30 m 0.25 mm 0.25 µm). UV-Vis spectra were recorded on a
UV/visible-spectrophotometer Ultrospec 2000, connected to a temperature
control unit, both from Amersham Pharmacia Biotech. Oxygen
consumption experiments were carried out with a Clarke-type electrode
system (Hansatech Oxytherm).
Conflicts of interest
The authors declare no conflict of interest.
Keywords: coenzyme specificity • flavin-containing
monooxygenases • hydroxylases • nicotinamide coenzyme
biomimetics • selective oxidation
Production of enzymes: 3-Hydroxybenzoate 6-hydroxylase from
Rhodococcus jostii RHA1 (3HB6H), p-hydroxybenzoate hydroxylase
Cys116Ser variant from Pseudomonas fluorescens (PHBH) and 2-
hydroxybiphenyl 3-hydroxylase from Pseudomonas azelaica HBP1
(HbpA) were produced from recombinant expression in Escherichia coli
and purified as described previously.[17, 27, 32d] Salicylate hydroxylase from
Pseudomonas putida S-1 was purified as described by Yamamoto et al.[19]
Enzyme concentrations were determined by measuring the absorbance for
protein-bound FAD using the following molar absorption coefficients:
3HB6H, ε453 = 10300 M-1cm-1;[17] PHBH, ε453 = 10200 M-1cm-1;[27] HbpA,
ε452 = 9700 M-1cm-1;[32d] SalH, ε450 = 11300 M-1cm-1.[19]
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This project has received funding from the European Union’s
Horizon 2020 MSCA ITN-EJD program under grant agreement
No 764920. AG, CEP and WJHB are grateful to the VLAG
graduate school at Wageningen University. CEP acknowledges a
Netherlands Organization for Scientific Research VENI grant (No
722.015.011). The authors thank M. van Schie for a preliminary
kinetic assay with 3HB6H, Dr. C. Tolmie and E. Schuiten for
preliminary screenings of BVMOs, and E. van der Klift, M.
Strampraad and S. Eustace for technical assistance.
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