Abstract
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 45kDa 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±97U/mg. This enzyme also racemized arginine with a specific activity of 568±28U/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 5mM 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.
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