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Characterization of Barley ${\alpha}$-Amylase Chimeric Enzymes Expressed in Pichia pastoris  

Kim, Tae-Jip (Department of Food Science and Technology, Chungbuk National University)
Yuk, Jeong-Bin (Department of Food Science and Technology, Chungbuk National University)
Choi, Seung-Ho (Department of Food Science and Technology, Chungbuk National University)
Jang, Myoung-Uoon (Department of Food Science and Technology, Chungbuk National University)
Svensson, Birte (Department of Systems Biology, Technical University of Denmark)
Publication Information
Korean Journal of Microbiology / v.46, no.1, 2010 , pp. 80-85 More about this Journal
Abstract
Two different ${\alpha}$-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 ${\alpha}$-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 ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.
Keywords
barley ${\alpha}$-amylase isozymes; calcium-dependent activity; chimeric enzymes; P. pastoris;
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