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|標題:||Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725||作者:||Kuan, Y.C.
|關鍵字:||Proteus mirabilis;Lysine racemase;Lysine racemization;Arginine;racemization;PLP-dependent enzyme;Substrate specificity;amino-acid racemases;bacterial-cell wall;alanine racemase;crystal-structure;bacillus-stearothermophilus;glutamate racemase;pseudomonas-putida;escherichia-coli;d-aspartate;gene||Project:||Process Biochemistry||期刊/報告no：:||Process Biochemistry, Volume 46, Issue 10, Page(s) 1914-1920.||摘要:||
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 Hiss-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 degrees C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxy-lamine 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. (C) 2011 Elsevier Ltd. All rights reserved.
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