Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Functional expression and enzymatic characterization of cyclomaltodextrinase from Streptococcus pyogenes

Streptococcus pyogenes 유래 cyclomaltodextrinase 유전자의 발현 및 효소 특성

  • Jang, Myoung-Uoon (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Kang, Hye-Jeong (Chungcheongbuk-do Agricultural Research and Extesion Services) ;
  • Jeong, Chang-Ku (Advanced Protein Technologies Co.) ;
  • Oh, Gyo Won (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Lee, Eun-Hee (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Son, Byung Sam (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Kim, Tae-Jip (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University)
  • 장명운 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 강혜정 (충청북도 농업기술원 친환경연구과) ;
  • 정창구 ((주)에이피테크놀로지) ;
  • 오규원 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 이은희 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 손병삼 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 김태집 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공)
  • Received : 2017.08.23
  • Accepted : 2017.09.20
  • Published : 2017.09.30
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Abstract

A cyclomaltodextrinase (SPCD) gene was cloned from Streptococcus pyogenes ATCC 700294. Its open reading frame consists of 567 amino acids (66.8 kDa), which shows less than 37% of amino acid sequence identity with the other CDase-family enzymes. The homo-dimeric SPCD with C-terminal six-histidines was expressed and purified from Escherichia coli. It showed the highest activity at pH 7.5 and $45^{\circ}C$, respectively. SPCD has the broad substrate specificities against ${\beta}$-cyclodextrin, starch, and maltotriose to produce mainly maltose, whereas it hydrolyzes pullulan to panose. It can also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. Interestingly, it showed much higher activity on ${\beta}$-cyclodextrin and acarbose than that on starch, pullulan, or maltotriose, which makes SPCD distinguished from common CDase-family enzymes. Although SPCD has significantly high acarbose-hydrolyzing activity, it showed negligible transglycosylation activity.

Streptococcus pyogenes ATCC 700294 유전체로부터 cyclomaltodextrinase (SPCD)로 예상되는 유전자를 발견하였다. SPCD는 총 567개의 아미노산으로 이루어진 66.8 kDa의 효소이며, 기존에 알려진 CDase 계열 효소들과 37% 미만의 아미노산 서열 상동성을 가진다. 본 연구에서는 SPCD 유전자를 클로닝하였으며, 대장균 내에서 카복시 말단에 6개의 histidine 잔기가 결합된 dimer 형태로 발현 및 정제되었다. SPCD는 pH 7.5, $45^{\circ}C$의 반응조건에서 최대의 활성을 나타내었으며, ${\beta}$-cyclodextrin, starch, maltotriose를 기질로 반응하여 maltose를 주산물로 생성하였다. 또한 pullulan을 panose 단위로 분해하며, acarbose를 glucose와 acarviosine-glucose로 가수분해하는 CDase 계열의 효소로 확인되었다. 그러나, SPCD는 다른 효소에 비해 저분자 소당류인 ${\beta}$-cyclodextrin에 대한 활성이 매우 높고, starch 및 pullulan과 같은 고분자 기질에 대해 매우 낮은 활성을 보였다. 또한 maltotriose 분해 활성이 매우 낮은 반면 acarbose에 대해 상대적으로 높은 가수분해 활성을 가지나, 당전이 활성은 매우 낮아 다른 CDase 계열 효소들과 구별된다.

Keywords

References

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