Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Domain Function and Relevant Enzyme Activity of Cycloinulooligosaccharide Fructanotransferase from Paenibacillus polymyxa

Paenibacillus polymyxa Cycloinulooligosaccharide Fructanotransferase의 효소 활성에 미치는 각 Domain의 역할

  • You Dong-Ju (Department of Material and Life science, Graduate School of Engineering, Osaka University) ;
  • Park Jung-Ha (Department of Life Science and Biotechnology, Dongeui University) ;
  • You Kyung-Ok (Department of Life Science and Biotechnology, Dongeui University) ;
  • Nam Soo-Wan (Department of Biotechnology and Bioengineering, Dongeui University) ;
  • Kim Kwang-Hyeon (Department of Life Science and Biotechnology, Dongeui University) ;
  • Kim Byung-Woo (Department of Life Science and Biotechnology, Dongeui University) ;
  • Kwon Hyun-Ju (Department of Life Science and Biotechnology, Dongeui University)
  • 유동주 (오사카대학 물질생명공학) ;
  • 박정하 (동의대학교 생명응용학과) ;
  • 유경옥 (동의대학교 생명응용학과) ;
  • 남수완 (동의대학교 생명공학과) ;
  • 김광현 (동의대학교 생명응용학과) ;
  • 김병우 (동의대학교 생명응용학과) ;
  • 권현주 (동의대학교 생명응용학과)
  • Published : 2006.09.01
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Abstract

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cycloinulooligosaccharides (cyclofructan, CF) of ${\beta}-(2{\to}1)$-linked D-fructofuranose as well as hydrolysis of cyclofructan. Sequences analysis indicated that CFTase was divided into five distinct regions containing three repeated sequences (R1, R3, and R4) at the N-terminus and C-terminus. Each domain function was investigated by comparison of wild type CFTase enzyme (CFT148) and deletion mutant proteins (CFT108: R1 and R3 deletion; CFT130: R4 deletion; and CFT88: R1, R3, and R4 deletion) of CFTase. The CFT108 mutant had both CFTase and CF hydrolyzing activity as CFT148 did. CFTase activities and CF hydrolysing activities were disappeared in CFT130 and CFT88 mutants. These results indicated that the C-terminal R4 region of P. polymyxa CFTase is necessary for cyclization and hydrolyzing activity.

토양으로부터 분리한 P. polymyxa 균주가 생산하는 CFTase의 각 repeat region의 결손 변이체를 제작하였으며 그에 따른 야생형 효소와 변이 효소의 활성변화 및 효소특성을 비교 검토하였다. 야생형 CFTase를 CFT148로, N-말단의 R1과 R3를 제거한 결손변이체 CFTase를 CFT108로, C-말단의 R4를 제거한 결손변이체는 CFT130, 모든 R 영역을 제거한 것은 CFT88로 명명하였다. 각각 정제된 재조합 단백질은 148 kDa, 108 kDa, 130 kDa, 그리고 88 kDa의 크기를 나타내었다. Inulin을 기질로 각 재조합 단백질의 활성을 검토한 결과 CFT108은 CF생성, CF분해 반응을 모두 가지고 있었으며 CFT130, CFT88의 경우에는 CF생성 활성을 나타내지 않았으며 endo-inulinae와 동일한 inulin 분해활성을 나타내었다. 따라서 N-말단의 repeat region인 R1과, R3의 역할은 균체 외로의 분비를 담당하며 C-말단의 R4영역은 CFTase의 중요 활성인 cyclization반응을 담당하고 있는 것으로 확인되었다. Repeat region의 모두 제거한 결손 변이체는 기질 inulin을 F2-F4 사이의 중합도를 가진fructooligosaccharide를 생산하는 endo-inulinase의 활성을 가지고 있었다. CFTase는 다기능 효소로 여러 domain으로 구성되어 있으며 각 domain의 결손에도 단백질의 활성을 유지하였으며, 특히 결손 변이체 CFT108은 고효율의 CF 생산이 가능하여 산업적으로 유용하게 사용될 수 있는 효소이다.

Keywords

References

  1. Eder, J., and A. R. Fersht. 1995. Pro-sequence-assisted protein folding. Mol. Microbiol. 16: 609-614 https://doi.org/10.1111/j.1365-2958.1995.tb02423.x
  2. Eom, S. J., Y. M. Kwon, and Y. J. Choi. 1995. Molecular cloning of Pseudomonas sp. inulinase gene and its expression in E. coli. Kor. J. Appl. Microbiol. Biotechnol. 23: 550-555
  3. Jeon, S. J., D. J. You, H. J. Kwon, K. Shigenori, K. Namio, K. H. Kim, Y. H. Kim, and B. W. Kim. 2002. Cloning and characterization of Cycloinulooligosaccharide Fructanotrans-ferase (CFTase) from Bacillus polymyxsa MGL21. J. Microbiol. Biotechnol. 12: 921-928
  4. Kanai, T., N. Ueki, T. Kawaguchi, Y. Teranishi, H. Atomi, C. Tomorbaatar, M. Ueda, and A. Tanaka. 1997. Recombinant thermostable cycloinulooligosaccharide fructanotrans- ferase produced by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 63: 4956-4960
  5. Kawamura, M. and T. Uchiyama. 1993. Reaction catalyzed by cycloinulooligosaccha- ride fructanotransferase, Biosci. Biotechnol. Biochem. 57: 343
  6. Kawamura, M. and T. Uchiyama. 1994. Purification and some properties of cycloinulo- oligosaccharide fructanotrans-ferase from Bacillus circulans OKUMZ31B. Carbohydr. Res. 260: 297-304 https://doi.org/10.1016/0008-6215(94)84047-4
  7. Kawamura, M., T. Uchiyama, T. Kuramoto, Y. Tamura, and K. Mizutani. 1989. Formation of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ31B. Carbohydr. Res. 192: 83-90 https://doi.org/10.1016/0008-6215(89)85167-5
  8. Kim, D. W., S. J. Lin, S. Morita, I. Terada, and H. Matsuzawa. 1997. A carboxy-terminal prosequence of aqualysin I presents proper folding of the protease domain on its secretion by Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 231: 535-539 https://doi.org/10.1006/bbrc.1996.5899
  9. Kim, H. Y. and Y. J. Chio. 2001. Moledular characterization of cycloinulooligosaccharide fructanotransferase(CFTase) from Bacillus macerans. Appl. Environ. Microbiol. 67: 995-1000 https://doi.org/10.1128/AEM.67.2.995-1000.2001
  10. Klein, C. and G E. Schulz. 1991. Structure of cycledextrin glycosyltransferase refined at $2.0{\AA}$ resolution. J. Mol. Biol. 217: 737-750 https://doi.org/10.1016/0022-2836(91)90530-J
  11. Kubota, M., Y. Matsuura, S. Sakai, and Y. Katsube. 1991. Molecular structure of B. stearothermophilus cyclodextrin glucanotrasferase and analysis of substrate binding site. Denpun Kagaku. 38: 141-146
  12. Kushibe, S., K. Mitsui, M. Yamagishi, K. Yamada, and Y. Morimoto. 1995. Purification and characterization of cycloinulooligosaccharide fructanotransferase (CFTase) from Bacillus circulans MCI-2554. Biosci. Biotechnol. Biochem. 59: 31-34
  13. Kushibe, S., R. Sashida, and Y. Morimoto. 1994. Production of cyclofructan from inulin by Bacillus circulans MCI-2554. Biosci. Biotechnol. Biochem. 58: 1136-1138
  14. Kwon, H. J., S. J. Jeon, D. J. You, K. H. Kim, Y. G. Jeong, Y. H. Kim, Y. M. Kim, and B. W. Kim. 2002. Cloning and characterization of exoinulinase from Bacillus polymyxa. Biotechnol. Lett. 25: 155-159
  15. Kwon, Y. M., H. Y. Kim, and Y. J. Choi. 2000. Cloning and characterization of Pseudomonas mucidolens exoinulinase. J. Microbiol. Biotechnol. 10: 238-243
  16. Lawson, C. L., R. Vanmontfort, B. Strokopytov, H. J. Rozeboom, K. H. Kalk, G. E. Devries, D. Penninga, L. Dijkhuizen, and B. W. Dijkstra. 1994. Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J. Mol. Biol. 236: 590-600 https://doi.org/10.1006/jmbi.1994.1168
  17. Park, J. H., H. J. Kwon, and B. W. Kim. 2006. High yield production of cyclofructan by deletion mutant enzyme of cycloinulooligosaccharide fructanotransferase. J. Life Sci. 16: 1-5 https://doi.org/10.5352/JLS.2006.16.1.001
  18. Sawada, M., T. Tanaka, Y. Takai, T. Hanafusa, T. Taniguchi, M. kawamura, and T. Uchiyama. 1991. The crystal structure of cycloinulohexaose produced from inulin by cycloinulooligosaccharide fructanotransferase. Carbohydr. Res. 217: 7-17 https://doi.org/10.1016/0008-6215(91)84112-R
  19. Schmid, G. 1989. Cyclodextrin glycosyltransferase production: yield enhancement by overexpression of cloned genes. Trends Biotechnol. 7: 244-248 https://doi.org/10.1016/0167-7799(89)90015-2
  20. Takai, Y., Y. Okumura, S. Takahashi, M. Sawada, M. Kawamuta, and T. Uchiyama. 1993. A permethylated cyclic fructo-oligosaccharide host that can bind cations in solution. J. Chem. Soc. Chem. Commun. 1: 53-54
  21. Takata, H., Kuriki, T., Okada, S., Takesada, Y., Iizuka, M., Minamiura, N., Imanaka, T. 1992. Action of neopillilanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1-4)-and alpha-(1-6)-glucosidic linkages. J. Biol. Chem. 267: 18447-18452
  22. Uchiyama, T., M. Kawamura, T. Uragami, and H. Okuno. 1993. Complexing of cycloinulo-oligosaccharides with metal ions. Carbohydr. Res. 241: 245-248 https://doi.org/10.1016/0008-6215(93)80111-Q
  23. Wetmore, D. R, S. L, Wong, and R. S. Roche. 1992. The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus. Mol. Microbiol. 6: 1593-604 https://doi.org/10.1111/j.1365-2958.1992.tb00884.x