P. Bouvrette et Jht. Luong, ISOLATION, PURIFICATION, AND FURTHER CHARACTERIZATION OF AN L-PHENYLALANINE OXIDASE FROM MORGANELLA-MORGANII, Applied biochemistry and biotechnology, 48(2), 1994, pp. 61-74
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.