A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: determination of toluene-4-monooxygenase activity.
A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (epsilon) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was determined experimentally to be 12,827 M(-1) cm(-1) with a linear range of quantification between 2.5 and 75 microM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as determined spectrophotometrically, were 61.8+/-4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9+/-4.6 nmol/min/mg P. mendocina KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6+/-1.4 nmol/min/mg P. mendocina KR1 protein.
Nolan
Louise C
LC
Department of Industrial Microbiology, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and Biomedical Research, National University of Ireland, University College Dublin, Belfield, Dublin 4, Republic of Ireland.
O'Connor
Kevin E
KE
D014442:Monophenol Monooxygenase / Q000378:metabolism; D010105:Oxygenases / Q000032:analysis*; D010636:Phenols / Q000378:metabolism*; D044203:Pseudomonas mendocina / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods
D010636:Phenols; C106880:4-fluorophenol; D010105:Oxygenases; C068610:toluene-4-monooxygenase; D014442:Monophenol Monooxygenase