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KMID : 0545120130230010007
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Journal of Microbiology and Biotechnology
2013 Volume.23 No. 1 p.7 ~ p.14
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Role of the Salt Bridge Between Arg176 and Glu126 in the Thermal Stability of the Bacillus amyloliquefaciens α-Amylase (BAA)
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Roseata Zonouzi
Khosro Khajeh
Majid Monajjemi
Naser Ghaemi
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Abstract
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In the Bacillus amyloliquefaciens α-amylase (BAA), the loop (residues 176-185; region I) that is the part of the calcium-binding site (CaI, II) has two more amino acid residues than the α-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region II (residues 118-130), but this interaction is lost in BLA owing to substitution of R176Q and E126V. The goal of the present work was to quantitatively estimate the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of the corresponding salt bridge, Glu126 was deleted (ΔE126) and converted to Val (E126V), Asp (E126D), and Lys (E126K) by site-directed mutagenesis. Kinetic constants, thermodynamic parameters, and structural changes were examined for the wild-type and mutated forms using UV-visible, atomic absoption, and fluorescence emission spectroscopies. Wild type exhibited higher kcat and Km but lower catalytic efficiency than the mutant enzymes. A decreased thermostability and an increased flexibility were also found in all of the mutant enzymes when compared with the wild type. Additionally, the calcium content of the wild type was more than ΔE126. Thus, it may be suggested that ionic interaction could decrease the mobility of the discussed region, prevent the diffusion of cations, and improve the thermostability of the whole enzyme. Based on these observations, the contribution of loop destabilization may be compensated by the formation of a salt bridge that has been used as an evolutionary mechanism or structural adaptation by the mesophilic enzyme.
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KEYWORD
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Bacillus amyloliquefaciens α-amylase, ionic interaction, site-directed mutagenesis, thermal stability
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