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.

Hydrolysis of Amides

“…This last enzyme, which belongs to the family of M17 leucine aminopeptidases (LAPs), has been studied in great detail, including several investigations to identify the roles of conserved residues that are not directly involved in catalysis, metal ion binding, and/or substrate binding [187][188][189]. The thermostability of the B. kaustophilus LAP could be further enhanced by immobilization in Ca-alginate/k-carrageenan beads (ID 1270), whereas its resistance against oxidative damage was improved by replacement of the critical methionine residues with leucine [190]. This engineering study resulted in the generation of more active mutants as well.…”