Staggered Extension Process를 통해 제조한 보리 알파아밀라제 Chimera 효소의 특성
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초록
보리 맥아로부터 발견된 서로 다른 알파아밀라제 동질효소(AMY1, AMY2)는 80%에 달하는 높은 아미노산 서열의상동성을 보이지만, 효소적 특성은 서로 매우 다르다. 따라서 본 연구에서는 staggered extension process(StEP) 기술을이용하여 AMY1과 AMY2 유전자가 조합된 5종의 chimera 효소를 제조하고, 각각의 특성을 비교하여 총 8개 부위(I~VIII)의 영향을 확인하였다. 결과적으로, AMY-D2,E12 chimera 효소의 경우, AMY1과 AMY2의 중간적 칼슘의존성을 보였으며, BASI(barley α-amylase/subtilisin inhibitor)단백질에 의한 저해효과는 AMY-E10 효소에서만 관찰되었다. 한편 AMY-C6의 경우, AMY1과 유사한 효소특성을 보였으며, AMY-E10은 AMY2 형태의 칼슘의존성을나타내었다. 따라서 보리 아밀라제의 제 II, III, IV부위가BASI와의 상호작용에 중요한 역할을 담당하며, 제 III, V,VI, VII부위는 칼슘의존성에 부분적인 영향을 미치는 것으로 판단하였다.
Abstract
Barley malt produces two different α-amylase isozymes (AMY1 and AMY2), which share up to 80% of amino acid sequence identity with each other. However, their enzymatic properties differ remarkably. In this study, five chimeric enzymes between AMY1 and 2 were constructed by staggered extension process (StEP) technique,and their enzymatic properties were characterized. According to the results, chimeric AMY-D2, D8, and E12showed the mixed or intermediate types of calcium-dependent activity between AMY1 and 2. Meanwhile,only AMY-E10 chimera could be significantly inhibited by barley α-amylase/subtilisin inhibitor (BASI) protein. Chimera AMY-C6 showed the same calcium-dependency as AMY1, while AMY-E10 was closely similar to AMY2. As a result, it can be proposed that some amino acid residues in the region II, III, and IV of barley α-amylases can play very important roles in the interaction with BASI, and those in III, V, VI, and VII may partly affect on the calcium-dependent activity.
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