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KMID : 0545120020120060921
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 6 p.921 ~ p.928
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Cloning and Characterization of Cycloinulooligosaccharide Fructanotransferase (CFTase) from Bacillus polymyxa MGL21
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JEON, SUNG-JONG
YOU, DONG-JU/KWON, HYUN-JU/KANAYA, SHIGENORI/KUNIHIRO, NAMIO/KIM, KWANG-HYEON/KIM, YOUNG-HEE
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Abstract
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Microorganism producing extracellular CFTase was isolated from soil and designed as Bacillus polymyxa MGL21. The gene encoding and CFTase (CFT) from B. polymyxa MGL21 was cloned and sequenced. The oRF of the cft 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℃ and 9.0 respectively. The enzyme activity was completely inhibited by 10 mM Ag^+ and Cu^2+. Thin-payer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction but also disproportionaion and hydrolysis reactions as well.
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