Lactococcus lactis subsp. lactis 유래 cyclomaltodextrinase 유전자의 대장균 내 발현 및 효소 특성
Enzymatic Characterization of Lactococcus lactis subsp. lactis Cyclomaltodextrinase Expressed in E. coli.
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초록
본 연구에서 584개의 아미노산(68.7 kDa)으로 구성된 cyclomaltodextrinase (LLCD)의 유전자를 Lactococcus lactis subsp. lactis KCTC 3769 (ATCC 19435)로부터 클로닝하였다. LLCD는 일반적인 CDase 계열 효소들과 약 40% 전후의 아미노산 서열 상동성을 나타내었다. C-말단에 6개의 히스티딘 잔기를 가진 재조합 효소는 dimer의 형태로 대장균에서 발현되고 정제되었다. LLCD는 pH 7.0 및 37oC에서 최대의 β-CD 가수분해 활성을 나타내었다. 특히, 이 효소는 starch 및 pullulan에 대해 극히 낮은 활성을 보였으나, 반면에 CD에 대한 가수분해 활성은 starch에 비해 약 80배 이상 높았다. 이처럼 높은 CD에 대한 활성을 근거로 LLCD는 CDase 계열 효소로 분류될 수 있으나, starch, pullulan, 그리고 acarbose에 대한 매우 낮은 활성은 다른 유사효소와 비교하여 차별화되는 특징이다.
Abstract
A putative cyclomaltodextrinase (LLCD) gene was cloned from the genome of Lactococcus lactis subsp. lactis KCTC 3769(ATCC 19435), which encodes 584 amino acids with the predicted molecular mass of 68.7 kDa. KCTC 3769 shares approximately 40% of amino acid sequence identity with the CDase-family of enzymes. The dimeric enzyme with C-terminal six-histidines was heterologously expressed and purified from recombinant E. coli. LLCD showed the highest activity against β-cyclodextrin (CD) at pH 7.0 and 37oC. In particular, LLCD exhibited extremely low activity against starch and pullulan, while its CD-hydrolyzing activity was about 80 times higher than starch. Due to its much higher activity on CD over starch, LLCD has been identified as a member of CDases. However, LLCD can be distinguished from the other common CDases on the basis of its extremely low hydrolyzing activity against starch, pullulan, and acarbose.
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