• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.921-928
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• 2002

Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.727-730
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• 2001

Xanthomonas oryzae #5로부터 클로닝 된 CFTase 의 functional domain의 분석을 위해 CFTase의 recombinant deletion mutant를 구성하고, recombinant protein을 분리, 정제하였다. 분리, 정제한 recombinant protein의 활성을 측정한 결과 C-terminal이 deletion 된 mutant는 cyclization 반응이 소실 되었다. 이와 같은 결과로부터 CFTase의 C-terminal 은 cyclization 반응의 중요한 functional domain 이다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.528-530
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• 2002

Xanthmonas oryzae MGL21유래의 CFTase의 repeat영역을 deletion 시킨 ${\triangle}N{\triangle}C$ deletion mutant는 protein 정제결과 약 90kDa 이있으며 pH6.5, $45^{\circ}C$에서 최적 효소 반응을 하였다. 또한 Inulin과 반응시켰을 때 CFTase는 main product가 CF인데 비해 ${\triangle}N{\triangle}C$ deletion mutant는 main product가 fructooligosaccharide였다. 이러한 결과로부터 CFTase의 N말단 repeat영역과 C말단 repeat영역을 제거하였을 경우 endoinulinase와 활성이 유사하며, 유전자 크기 및 아미노산 서열도 유사함을 알 수 있었다.