Purification of the nicotinic acid hydroxylase system of Pseudomonas fluorescens KB1

The nicotinic acid hydroxylase system of Pseudomonas fluorescens KB 1 has previously been shown to be an adaptive enzyme which hydroxylates nicotinic acid in the C-6 position with the uptake of one molecule of oxygen for two molecules of nicotinic acid (Hughes, 1955). The reaction initiates the breakdown of nicotinic acid to ammonia and formic, acetic, lactic and malic acid according to the reactions described by Behrman & Stanier (1957). In cell-free extracts nicotinic acid is con- verted quantitatively into 6-hydroxynicotinic acid and this compound is not further metabolized. The hydroxyl group added is derived from water and not molecular oxygen, indicating that the reaction probably involves the addition of water followed by dehydrogenation with molecular oxygen as hydro- gen acceptor (Hunt, Hughes & Lowenstein, 1957, 1958). The enzyme is attached to insoluble particulate components of the cell. This paper is concerned with the purification of the. enzyme, together with the components of an associated electron-transport chain, by deoxycholate extraction of the particles, followed by ammonium sulphate fractionation. Preliminary findings of this work have been com- municated (Hunt, 1958). METHODS Growth of organisms. P. fluore8cens KB 1 was grown with aeration at 250 in an inorganic salt medium containing KH2PO4, 6-8 g., NH4C1, 1.0 g., mineral supplement (Behrman & Stanier, 1957), l O ml., MgSO4,7H20, 0-5 g., yeast extract (Oxo Ltd.), 0-2 g. and nicotinic acid, 2-5 g. per litre of demineralized water. The solution was adjusted to pH 6-8 with solid NaOH. Subcultures were maintained on slopes containing in addition 2 % of agar. For the pre- paration of large quantities of cells 401. batches of medium were inoculated with 41. of a logarithmic-phase culture, and growth was allowed to continue under forced aeration in the stainless-steel container of a commercial washing machine (Hoover Ltd.) fitted with a baffle plate and a stainless-steel cover. The airflow was maintained at levels suboptimum for maximum rates of growth. Small quanti- ties of tributyl citrate were added to minimize foaming.