Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Enhanced Expression and Substrate Specificity Changes of Barley $\alpha$-Amylase Isozyme 2 in E. coli by Substitution of the $42^{nd}$ Alanine Residue with Proline

42번째 alanine 잔기의 proline 치환에 의한 보리 $\alpha$-amylase isozyme 2의 대장균 내 발현 증가 및 기질특이성 변화

  • Choi, Seung-Ho (Department of Food Science and Technology, Chungbuk National University) ;
  • Jang, Myoung-Uoon (Department of Food Science and Technology, Chungbuk National University) ;
  • Lee, Hong-Gyun (Department of Food Science and Technology, Chungbuk National University) ;
  • Svensson, Birte (Department of Systems Biology, Technical University of Denmark) ;
  • Kim, Tae-Jip (Department of Food Science and Technology, Chungbuk National University)
  • Received : 2009.11.17
  • Accepted : 2010.01.08
  • Published : 2010.04.30
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Abstract

Although barley $\alpha$-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.

보리 맥아에는 2종의 $\alpha$-amylase isozyme(AMY1, AMY2)이 존재하며, 이들 효소는 80% 이상의 높은 아미노산 서열 상동성을 보이지만, calcium 의존성 등 효소의 작용특성은 서로 매우 다르다. 따라서 본 연구에서는 AMY2의 활성부위 중 2번째 $\beta\rightarrow\alpha$ loop에 존재하는 42번째 alanine 잔기를 saturation mutagenesis를 이용하여 다양한 아미노산으로 치환하고, 전분 분해활성이 증가한 돌연변이를 선발하였다. 결과적으로 alanine이 proline으로 치환된 AMY2-A42P의 경우에서만 발현도가 2배 증가하는 것을 확인하였으며, 특히 정제 과정에서의 회수율 또한 4배 증가하므로 향후 효소의 생산 및 활용에 유리할 것으로 판단하였다. 이 돌연변이 효소의 calcium 의존성 및 pH 안정성 등은 AMY2와 유사한 것으로 나타났으나, 각종 전분에 대한 기질특이성은 AMY1과 AMY2의 중간적인 특성으로 변화되었다. 결국 42번째 아미노산 잔기의 proline 치환에 의해 상대적으로 발현율이 높고 기질특이성이 변화된 AMY2 유사효소의 생산이 가능하였으며, 향후 이를 이용하여 분자진화기술 등 최신 효소공학적 방법론을 적용한 다양한 연구가 가능할 것으로 기대한다.

Keywords

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