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|標題:||Generating oxidation-resistant variants of Bacillus kaustophilus leucine aminopeptidase by substitution of the critical methionine residues with leucine||作者:||Chi, M.C.
|關鍵字:||Bacillus kaustophilus;chemical oxidation;directed mutagenesis;leucine;aminopeptidase;methionine;x-ray crystallography;hydrogen-peroxide;alpha-amylase;active-site;proteins;mechanism;complex;ion||Project:||Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology||期刊/報告no：:||Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Volume 86, Issue 4, Page(s) 355-362.||摘要:||
Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K-m value, more than 67.8% increase in the value of k(cat)/K-m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H2O2 most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues.
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