Separation and partial characterization of soluble fatty acid alpha-hydroxylase from Sphingomonas paucimobilus

The crude extracts from Sphingomonas paucimobilus containing 2-hydroxy fatty acid-rich sphingolipids were found to oxidize [1-14C]-myristate to 2-hydroxymyristate in the presence of NADH. The myristate-oxidation activity was partially purified about 290-fold from the cell-free extracts by ammonium sulfate fractionation and hydroxylapatite chromatography. When laurate, myristate, and palmitate were used as the substrates, the reaction products were identified as the corresponding 2-hydroxy fatty acids by gas-chromatography (GC), HPLC, and GC-mass spectrometry. The enzyme preparation required NADH and molecular oxygen for its alpha-hydroxylation activity, suggesting that the bacterial alpha-hydroxylation is typical of monooxygenase reactions. Of fatty acids tested, myristate was the most efficient substrate.