Oxidative Stabilizzation of an Alkaliphilic Bacillus α-Amylase by Replacing a Single Specific Methionine Residue by Site-directed Mutagenesis

Abstract

The oxidative stabilization of α-amylase from Bacillus sp . strain KSM-1378 (AmyK) was achievedby replacing Met202 with non-oxidizable amino acid residues. Computer-aided modeling of the enzyme suggested that Met202 among a total of eleven Met residues was spatially the closest to, although 10-14 Å away from, the catalytic site. The recombinant mutant enzymes were purified from cultures of Bacillus subtilis cells harboring the respective mutated genes. The results indicated that 1) the wild -type enzyme was readily inactivated by incubation with H₂O₂; 2) the change from Met to non-oxidizable Thr, Len, Ile, Ser, or Ala at posi-ti on 202 conferred high oxidative stability against H₂O₂; and 3) the increases in the substrate affinity (1/K_m) and catalytic efficiency (k_cat/K_m) for borohydride-reduced amylose occurred with enlargement of the position 202 side chain in the mutant enzymes. These results suggest that oxidation of Met202 to the sulfoxide deriva tive interfered with substrate binding, thereby abolishing the enzyme activity .