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KMID : 0380620100420020198
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Korean Journal of Food Science and Technology
2010 Volume.42 No. 2 p.198 ~ p.203
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Enhanced Expression and Substrate Specificity Changes of Barley α-Amylase Isozyme 2 in E. coli by Substitution of the 42nd Alanine Residue with Proline
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Choi Seung-Ho
Jang Myoung-Uoon
Lee Hong-Gyun
Birte Svensson
Kim Tae-Jip
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Abstract
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Although barley α-amylase isozyme 1 (AMY1) and 2 (AMY2) share up to 80% of amino acid sequence identity, their enzymatic properties differ remarkably. In this study, the 42nd alanine residue of AMY2 was replaced with another random amino acid via saturation mutagenesis. Eight out of 370 recombinant E. coli cells showing enhanced starch-hydrolyzing activity were characterized as possessing the same proline residue instead of alanine. Even though the specific activity of AMY2-A42P is reduced to 81% of wild-type, its expression level and purification yield were enhanced by approximately 2 and 4 times that of AMY2, respectively. Characterization of its enzymatic properties confirmed that AMY2-A42P is similar to that of wild-type. However, its specificity to starch substrates is likely to be intermediate between AMY1 and AMY2.
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KEYWORD
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barley α-amylase 2, saturation mutagenesis, enhanced expression, substrate specificity, Escherichia coli
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