|Kuan YC, Kao CH, Chen CH, Chen CC, Hu HY, Hsu WH
|Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725
|A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His6-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828 ± 97 U/mg. This enzyme also racemized arginine with a specific activity of 568 ± 28 U/mg but not other amino acids. The optimal conditions for Lyr activity to l-lysine were pH 8.0–9.0 and 50 °C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5′-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5–1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine.