Engineering of a truncated alpha-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities.
BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.
Chi
Meng-Chun
MC
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan.
Chen
Yan-Hung
YH
Wu
Tai-Jung
TJ
Lo
Huei-Fen
HF
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability; D006207:Half-Life; D006868:Hydrolysis; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017384:Sequence Deletion; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003902:Detergents; D013213:Starch; D000516:alpha-Amylases; D007930:Leucine