Replacement of methionine 208 in a truncated Bacillus sp. TS-23 alpha-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide.
The methionine residues at positions 17, 104, 208, 214, 292, 315, 324, and 446 in the primary amino acid sequence of a truncated Bacillus sp. TS-23 alpha-amylase (His(6)-tagged BLADeltaNC) was changed to oxidative-resistant leucine by site-directed mutagenesis. The mutant enzymes with an apparent molecular mass of approximately 54 kDa were overexpressed in recombinant Escherichia coli. The specific activity for Met315Leu and Met446Leu was decreased by more than 76%, while Met17Leu, Met104Leu, Met208Leu, Met214Leu, Met292Leu, and Met324Leu showed 247, 128, 37, 260, 232, and 241%, respectively, higher activity than the wild-type enzyme. In comparison with wild-type enzyme, a lower K(m) value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (k(cat)/K(m)) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in K(m) values. Wild-type enzyme was sensitive to chemical oxidation, but Met208Leu was stable even in the presence of 500 mM H(2)O(2). Except for Met214Leu, which was quite sensitive to H(2)O(2), the other mutants showed a profile of oxidative inactivation similar to that of the wild-type enzyme. These observations indicate that the oxidative stability of His(6)-tagged BLADeltaNC can be improved by replacement of the critical methionine residue with leucine.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 60083, Taiwan. llin@mail.ncyu.edu.tw
Lo
Huei-Fen
HF
Chiang
Wen-Ying
WY
Hu
Hui-Yu
HY
Hsu
Wen-Hwei
WH
Chang
Chen-Tien
CT
D019943:Amino Acid Substitution; D001407:Bacillus / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000235:genetics*; D008715:Methionine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D000516:alpha-Amylases / Q000235:genetics*
D008715:Methionine; D006861:Hydrogen Peroxide; D000516:alpha-Amylases; D007930:Leucine