The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site-directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative-resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild-type (WT) enzyme (pH 7.011.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50 degrees C of the WT to <40 degrees C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (kcat/Km) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween-60 and sodium dodecyl sulfate. When incubated with 500 mM H2O2 at 35 degrees C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative-resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012