ISOLATION, PURIFICATION, AND FURTHER CHARACTERIZATION OF AN L-PHENYLALANINE OXIDASE FROM MORGANELLA-MORGANII

A L-amino acid oxidase was isolated, purified, and characterized from Morganella morganii 53187, a bacterium formerly known as Proteus morga nii. The synthesis of the enzyme by this bacterial strain was growth-a ssociated and decreased sharply when the culture just reached the stat ionary phase. Based on this finding, the preparation of spheroplast by lysozyme-ethylene-diaminetetra-acetic acid (EDTA) disruption was carr ied out using the cells harvested during the exponential growth phase. Among several detergents tested, at the detergent-to-protein ratio of 2.5, olamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) was v ery effective in solubilizing most of the enzyme attached to the membr anes while still preserving the activity of the solubilized enzyme. Th e resulting enzyme solution was then purified by hydrophobic interacti on chromatography, followed by ion exchange chromatography and gel per meation. The enzyme was purified 19-fold with an overall recovery yiel d of 12%, corresponding to a specific activity of 252.2 U/mg protein. The selectivity of the purified enzyme toward L-amino acids was PH-dep endent. At pH 6.35, the enzyme was very specific to L-leucine, whereas the selectivity for L-phenylalanine could be improved at PH 7.4. The enzyme exhibited a wide optimum temperature range 35-43 degrees C and exhibited 1, 1'-dimethylferricinium reductase capability in the presen ce of L-phenylalanine.