Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Expression and Optimum Production of Cyclodextrin Glucanotransferase Gene of Paenibacillus sp. JB-13 in E. coli

Paenibacillus sp. JB-13 Cyclodextrin Glucanotransferase 유전자의 E. coli 에서의 발현 및 최적 생산

  • Kim, Hae-Yun (Division of Biological Science, Pusan National University) ;
  • Lee, Sang-Hyeon (Department of Bioscience and Biotechnology, Silla University) ;
  • Kim, Hae-Nam (Department of Beauty Care, Masan College) ;
  • Min, Bok-Kee (Department of Biomedical Laboratory Science, Eulji University) ;
  • Baik, Hyung-Suk (Division of Biological Science, Pusan National University) ;
  • Jun, Hong-Ki (Division of Biological Science, Pusan National University)
  • 김해윤 (부산대학교 생명과학부) ;
  • 이상현 (신라대학교 생명공학과) ;
  • 김해남 (마산대학 뷰티케어과) ;
  • 민복기 (을지의과대학교 임상병리학과) ;
  • 백형석 (부산대학교 생명과학부) ;
  • 전홍기 (부산대학교 생명과학부)
  • Published : 2008.03.31
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Abstract

The purpose of this study is to clone cgt gene from Paenibacillus sp. JB-13 and to overexpress the protein in E. coli. For this purpose, the cgt gene was amplified from Paenibacillus sp. JB-13 genomic DNA by PCR using degenerate oligonucleotide primers. The sequence analysis results showed that the cgt gene from Paenibacillus sp. JB-13 has 98% homology with the cgt gene of Bacillus sp. To overexpress the protein, the cgt gene was cloned into pEXP7 expression vector and transformed into E. coli. The production of CGTase by recombinant E. coli was optimized under following conditions: 0.5% glucose, 3.0% polypeptone, 0.3% $K_2HPO_4$, 0.5% NaCl, and 7.0 of initial pH, 2.0% of inoculum, $37^{\circ}C$ of culture temperature for 14 hr. And the optimal agitation was found at 0.1 vvm. The synthesis of 2-O-${\alpha}$-D-Glucopyranosyl L-Ascorbic acid (AA-2G) using the CGTase expressed in E. coli was identified as AA-2G by HPLC and HPLC confirmed that treating AA-2G made by cloned CGTase with ${\alpha}$-glucosidase substantially produced AA and glucose.

L-ascorbic acid (AA)의 2번 위치의 수산기에 부위 특이적 활성을 갖는 Paenibacillus sp. JB-13 유래의 cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) 유전자(cgt gene)를 pEXP7 발현 vector에 클로닝하여 재조합 균주를 구축하였다. 재조합 균주의 CGTase생산 최적 조건을 검토하여 본 결과 LB 배지에 0.5% glucose, 3.0% polypeptone, 0.3% $K_2HPO_4$, 0.5% NaCl이 되도록 추가하고, 초기 pH 7.0, 접종량 2%, 통기량 0.1 vvm, 배양 온도 $37^{\circ}C$, 배양 시간 14시간의 조건에서 최대 활성을 나타내었다. 재조합 균주와 기존 균주의 CGTase 활성을 비교한 결과 재조합 균주는 배양 14시간째 640 units/ml의 활성을 가져 기존 균주에 비해 70%의 활성을 나타내지만 배양 시간을 1/4로 단축시킬 수 있다는 이점이 있음을 확인하였다. 재조합 균주가 생산한 CGTase를AA-2G합성에 적용하여 AA-2G를 합성하고 HPLC로 분석한 결과 단일 peak를 확인할 수 있었고 ${\alpha}$-glucosidase를 처리하여 확인한 결과 AA와 glucose로 분리됨을 확인할 수 있었다.

Keywords

References

  1. Abe, S., Y. Nagamine, K. Omichi, and T. Ikenaka. 1991. Investigation of the active site of Bacillus macerans cyclodextrin glucanotransferase by use of modified maltooligosaccharides. J. Biochem. 110 ,756-761 https://doi.org/10.1093/oxfordjournals.jbchem.a123654
  2. Ahn, J.H., J.B. Hwang, and S.H. Kim. 1990. Cyclodextrin glucanotransferase from Bacillus stearothermophilus : Purification by affinity chromatography and its properties. Kor. J. Appl. Microbiol. Biotechnol. 18, 585-590
  3. Bae, K.M., Y. Kang, and H.K. Jun. 2001. Production of 2-O-${\alpha}$-D-glucopyranosyl L-ascorbic acid by cyclomaltodextrin glucanotransferase from Paenibacillus sp. JB-13. Kor. J. Appl. Microbiol. Biotechnol. 29, 31-36
  4. Bovetto, L.J., D.P. Backer, J.R. Villette, P.J. Sicard, and S.J. Bouquelet. 1992. Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192. 1. Purification and characterization of the enzyme. Biotechnol. Appl. Biochem. 15, 48-58 https://doi.org/10.1111/j.1470-8744.1992.tb00196.x
  5. Frei, B., L. England, and B.N. Ames. 1998. Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl. Acad. Sci. USA 86, 6377-6381
  6. Gawande, B.N., A. Goel, A.Y. Patkar, and S.N. Nene. 1999. Purification and properties of a novel raw starch degrading cuclomaltodextrin glucanotransferase from Bacillus firmus. Appl. Microbiol. Biotechnol. 51, 504-509 https://doi.org/10.1007/s002530051424
  7. Hajime, A., Y. Masaru, S. Shuzo, and Y. Itaru. 1991. Synthesis of 2-O-${\alpha}$-D-glucopyranosyl L-ascorbic acid by cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus. Agric. Biol. Chem. 55, 1751-1756 https://doi.org/10.1271/bbb1961.55.1751
  8. Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23-28 https://doi.org/10.1016/0378-1119(90)90336-P
  9. Jun, H.K., K.M. Bae, and S.K. Kim. 2001. Production of 2-O-${\alpha}$-D-glucopyranosyl L-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp. Biotechnol. Lett. 23, 1793-1797 https://doi.org/10.1023/A:1012413301061
  10. Kelly, R.M., H. Leemhuis, and L. Dijkhuizen. 2007. Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies. Biochem. 46, 11216-11222 https://doi.org/10.1021/bi701160h
  11. Kitahata, S. and S. Okada. 1982. Comparison of action of cyclodextrin glucanotransferase from Bacillus megaterium, B. circulans, B. stearothermophilus and B. macerans. J. Jpn. Soc. Starch Sci. 29, 13-18 https://doi.org/10.5458/jag1972.29.13
  12. Lee, Y.H., S.H. Lee, and H.D. Shin. 1991. Evaluation of immobilization methods for cyclodextrin glucanotransferase and characterization of its enzymatic properties. J. Microbiol. Biotechnol. 1, 54-62
  13. Makoto, O., M. Hiroshi, and O. Takahisa. 1990. Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme. J. Biochem. 107, 21-26 https://doi.org/10.1093/oxfordjournals.jbchem.a123004
  14. Mandai, T., M. Yoneyama, S. Sakai, N. Muto, and I. Yamamoto. 1992. The crystal structure and physicochemical properties of L-ascorbic acid 2-glucoside. Carbohydr. Res. 232, 197-205 https://doi.org/10.1016/0008-6215(92)80054-5
  15. Marechal, L.R., A.M. Rosso, M.A. Marechal, N. Krymkiewicz, and S.A. Ferrarotti. 1996. Some properties of a cyclomaltodextrin gluconotransferase from Bacillus circulans DF 9 R type. Cell Mol. Biol. 42, 659-664
  16. Nakamura, A. and K. Yamane. 1994. Four aromatic residues in the active center of cyclodextrin glycanotransferase from an alkalophilic Bacillus sp. 1011. Effect of the replacement on substrate binding and cyclization characteristics. Biochemistry 33, 929-936
  17. Nelson, N. 1950. A photometric adaption of the Somogyi method for the determination of glucose. J. Biol. Chem. 166, 444-449
  18. Rimphanitchayakit, V., T. Takashi, and S. Yoshiyuki. 2005. Construction of chimeric cyclodextrin glucanotransferases from Bacillus circulans A11 and Paenibacillus macerans IAM1243 and analysis of their product specificity. Carbohyd. Res. 340, 2279-2289 https://doi.org/10.1016/j.carres.2005.07.013
  19. Tanaka, M., N. Muto, and I. Yamamoto. 1991. Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-${\alpha}$-glucoside formation. Biochem. Biophys. Acta. 1078, 127-132 https://doi.org/10.1016/0167-4838(91)99000-1
  20. Turnes, R.E. and S. Bahar. 1996. Production of cyclomaltodextrin glucanotransferase from an alkalophilic Bacillus sp. in rice and corn flakes. Acta. Cient. Venez. 47, 133-137
  21. Vetter, D. and W. Thorn. 1992. Directed enzymatic synthesis of linear and branched glucooligosaccharide, using cyclodextrin glucanotransferase. Carbohydrate Res. 223, 61-69 https://doi.org/10.1016/0008-6215(92)80006-M
  22. Yamamoto, I., N. Muto, K. Murakami, and J. Akiyama. 1992. Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-O-${\alpha}$-D-glucopyranosyl L-ascorbic acid. J. Nutr. 122, 871-877 https://doi.org/10.1093/jn/122.4.871
  23. Yamamoto, I., N. Muto, E. Nagato, T. Nakamura, and Y. Suzuki. 1990. Formation of a stable L-ascorbic acid ${\alpha}$-glucoside by mammalian ${\alpha}$-glucosidase catalyzed transglucosylation. Biophys. Acta. 1035, 44-51 https://doi.org/10.1016/0304-4165(90)90171-R
  24. Yamamoto, I., S. Suga, Y. Mitoch, M. Tanaka, and N. Muto. 1990. Antiscorbutic activity of L-ascorbic acid 2-glucoside and its availability as a vitamin C supplement in normal rats and guinea pigs. J. Pharmacobiodyn. 13, 688-695 https://doi.org/10.1248/bpb1978.13.688
  25. Yamamoto, I., M. Tanaka, and N. Muto. 1993. Enhancement of in vitro antibody production of murine splenocytes by ascorbic acid 2-O-alpha-glucoside. Int. J. Immunopharmacol. 15, 319-325 https://doi.org/10.1016/0192-0561(93)90042-W