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KMID : 0364820100460010080
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Korean Journal of Microbiology
2010 Volume.46 No. 1 p.80 ~ p.85
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Characterization of Barley α-Amylase Chimeric Enzymes Expressed in Pichia pastoris
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Kim Tae-Jip
Yuk Jeong-Bin
Choi Seung-Ho
Jang Myoung-Uoon
Birte Svensson
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Abstract
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Two different α-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley α-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in
Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley α-amylases play very important roles in calcium-dependency and interaction with BASI.
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KEYWORD
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barley α-amylase isozymes, calcium-dependent activity, chimeric enzymes, P. pastoris
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