Identification of glutamate residues important for catalytic activity or thermostability of a truncated Bacillus sp. strain TS-23 alpha-amylase by site-directed mutagenesis.
The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 alpha-amylase (BACdeltaNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACdeltaNC. A decreased half-life at 70 degrees C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan.
Chen
Pei-Jing
PJ
Liu
Jai-Shin
JS
Wang
Wen-Ching
WC
Lo
Huei-Fen
HF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D018698:Glutamic Acid; D000516:alpha-Amylases