title: Generating oxidation-resistant variants of Bacillus kaustophilus leucine aminopeptidase by substitution of the critical methionine residues with leucine.
abstract: 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.
authors.affiliation: Graduate Institute of Biotechnology, National Chiayi University, 60083 Chiayi, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Wei-Mou
authors.initials: WM
authors.lastname: Chou
authors.forename: Chih-Hsien
authors.initials: CH
authors.lastname: Wang
authors.forename: Wenlung
authors.initials: W
authors.lastname: Chen
authors.forename: Wen-Hwei
authors.initials: WH
authors.lastname: Hsu
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*
chemical_list: D008715:Methionine; D006861:Hydrogen Peroxide; D007931:Leucyl Aminopeptidase; D007930:Leucine; D010100:Oxygen