한국응용과학기술학회지 (Journal of the Korean Applied Science and Technology)

  • 제33권2호
  • /
  • Pages.286-292
  • /
  • 2016
  • /
  • 1225-9098(pISSN)
  • /
  • 2288-1069(eISSN)
한국응용과학기술학회 (The Korean Society of Applied Science and Technology)
권호 표지
DOI QR코드

DOI QR Code

대장균 베타-갈락토시데이즈를 이용하여 합성된 1, 2-Hexanediol Galactoside의 NMR Spectroscopy 및 Mass spectrometry

NMR Spectroscopy and Mass Spectrometry of 1, 2-Hexanediol Galactoside synthesized using Escherichia coli β-Galactosidase

  • 김이옥 (한국교통대학교 생명공학과) ;
  • 이향렬 (한국교통대학교 생명공학과) ;
  • 정경환 (한국교통대학교 생명공학과)
  • Kim, Yi-Ok (Department of Biotechnology, Korea National University of Transportation) ;
  • Lee, Hyang-Yeol (Department of Biotechnology, Korea National University of Transportation) ;
  • Jung, Kyung-Hwan (Department of Biotechnology, Korea National University of Transportation)
  • 투고 : 2016.05.16
  • 심사 : 2016.06.16
  • 발행 : 2016.06.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

화장품용 방부제로 사용하고 있는 1, 2-hexanediol (HD)에 높은 농도의 lactose (300 g/l)를 넣고, 재조합 대장균 ${\beta}$-galactosidase (${\beta}$-gal)를 이용하여 galactose 한 분자를 결합시키는 transgalactosylation 반응을 시켜서, 1, 2-hexanediol galactoside (HD-gal)을 합성하였다. 그리고, 합성된 HD-gal 분자를 확인하기 위하여, HD-gal에 대한 NMR ($^1H$- and $^{13}C$-) 스펙트럼 분석과 mass 스펙트럼 분석을 실시하였다. HD-gal의 $^1H$ NMR 스펙트럼에서 HD에 갈락토실화가 되었음을 보여주는 다양한 피크를 확인하였다. $^1H$ NMR 스펙트럼의 다운필드인 ${\delta}_H$ 4.44 ppm과 ${\delta}_H$ 3.96~3.58 ppm에서 나타나는 다양한 피크들은 HD에 갈락토실화가 되었다는 것을 잘 암시하고 있으며, 또한 $^1H$ NMR 스펙트럼의 업필드에서 나타나는 ${\delta}_H$ 1.60~1.35 ppm과 0.92 ppm의 피크는 HD의 $CH_2$와 $CH_3$ 작용기로부터 나타나는 피크로써 HD가 본 물질에 존재한다는 것을 나타내고 있다. $^{13}C$ NMR 스펙트럼에서는 HD-gal의 알파-아노머와 베타-아노머의 구조에서 기인하는 총 21의 카본피크가 나타났고, 각 아노머마다 12개의 카본이 존재하는데 이중 ${\delta}_C$ 68.6, 60.9 and 13.2 ppm에 보이는 3개의 카본은 겹쳐서 나타나 총 24개의 피크 중 21개가 나타났다. 또한, 질량스펙트럼 분석에서는 protonated HD-gal인 281.1601 (m/z)의 peak를 확인할 수 있었다. 이를 종합하면, NMR ($^1H$- and $^{13}C$-) 스펙트럼 분석 결과와 질량분석 결과들은 우리가 기대했던 HD-gal의 구조와 매우 잘 일치하고 있다는 것을 알 수 있었다. 앞으로 추가적으로, 세균에 대한 minimum inhibitory concentrations (MICs) 조사와 human skin cell에 대한 독성연구를 추가적으로 진행할 예정이며, 이러한 결과를 근거로 항균력을 유지하면서 피부세포에 대한 독성이 감소된 화장품용 방부제의 연구/개발이 계속되기를 기대하고 있다.

1, 2-Hexanediol galactoside (HD-gal) has been synthesized from 1, 2-hexanediol (HD), a cosmetic preservative, using recombinant Escherichia coli ${\beta}$-galactosidase (${\beta}$-gal) at the high lactose concentration (300 g/l). To confirm the molecular structure of synthesized HD-gal, NMR ($^1H$- and $^{13}C$-) spectroscopy and mass spectrometry of HD-gal were conducted. $^1H$ NMR spectrum of HD-gal showed multiple peaks corresponding to the galactocyl group, which is an evidence of galactocylation on HD. Downfield proton peaks at ${\delta}_H$ 4.44 ppm and multiple peaks from ${\delta}_H$3.96~3.58 ppm were indicative of galactocylation on HD. Up field proton peaks at ${\delta}_H$ 1.60~1.35 ppm and 0.92 ppm showed the presence of $CH_2$ and $CH_3$ protons of HD. $^{13}C$ NMR spectrum revealed the presence of 21 carbons suggestive of ${\alpha}$- and ${\beta}$-anomers of HD-gal. Among 12 carbon peaks from each anomers, the 3 peaks at dC 68.6, 60.9 and 13.2 ppm were assigned to be overlapped showing only 21 peaks out of total 24 peaks. The mass value (protonated HD-gal, m/z = 281.1601) from mass spectrometry analysis of HD-gal, and $^1H$ and $^{13}C$ NMR spectral data were in well agreement with the expecting structure of HD-gal. For further study, the minimum inhibitory concentrations (MICs) of HD-gal against bacteria will be investigated, and, in addition, cytotoxicity to human skin cells of HD-gal will be examined. It is expected that it will eventually be able to develop a new cosmetic preservative, which have low cytotoxicity against human skin cell and maintains antimicrobial effect.

키워드

참고문헌

  1. W. Johnson Jr., W. F. Bergfeld, D. V. Belsito, R. A. Hill, C. D. Klaassen, D. Liebler, J. G. Marks Jr., R. C Shank, T. J. Slaga, P. W. Snyder, and F. A. Andersen, Safety Assessment of 1, 2-Glycols as Used in Cosmetics. Int. J. Toxicol., 31(5 Suppl), 147S (2012). https://doi.org/10.1177/1091581812460409
  2. E. Lee, S. An, S.-A. Cho, Y. Yun, J. Han, Y. K. Hwang, H. K. Kim, and T. R. Lee, The Influence of Alkane Chain Length on the Skin Irritation Potential of 1, 2-Alkanediols, Int. J. Cosmet. Sci., 33(5), 421 (2011). https://doi.org/10.1111/j.1468-2494.2011.00646.x
  3. S. B. Levy, A. M. Dulichan, and M. Helman, Safety of a Preservative System containing 1, 2-Hexanediol and Caprylyl glycol, Cutan. Ocu.l Toxicol., 28(1), 23 (2009). https://doi.org/10.1080/15569520802636082
  4. Y. -O. Kim and K. -H. Jung, Enzymatic Synthesis of 1, 2-Hexanediol Galactoside by Whole cells of ${\beta}$-Galactosidasecontaining Recombinant Escherichia coli. J. Life Sci., 26(5), 608 (2016). https://doi.org/10.5352/JLS.2016.26.5.608
  5. S. -E. Lee, H. -Y. Lee, and K. -H. Jung, Production of Chlorphenesin Galactoside by Whole Cells of ${\beta}$-Galactosidasecontaining Escherichia coli, J. Microbiol. Biotechnol., 23(6), 826 (2013). https://doi.org/10.4014/jmb.1211.11009
  6. H. -Y. Lee and K. -H. Jung, Enzymatic Synthesis of 2-Phenoxyethanol Galactoside by Whole Cells of ${\beta}$-Galactosidasecontaining Escherichia coli, J. Microbiol. Biotechnol., 24(9), 1254 (2014). https://doi.org/10.4014/jmb.1404.04004
  7. S. -E. Lee, T. M. Jo, H. -Y. Lee, J. Lee, and K. -H. Jung, ${\beta}$-Galactosidasecatalyzed Synthesis of Galactosyl Chlorphenesin and its Characterization, Appl. Biochem. Biotechnol., 171(6), 1299 (2013). https://doi.org/10.1007/s12010-013-0213-3
  8. K. -H. Jung and H. -Y. Lee, Escherichia coli ${\beta}$-Galactosidase-catalyzed Synthesis of 2-Phenoxyethanol Galactoside and its Characterization, Bioprocess Biosyst. Eng., 38(2), 365 (2015). https://doi.org/10.1007/s00449-014-1276-4
  9. G. D. Benjamin and M. A. Robinson, Drug Delivery Systems based on Sugar-macromolecule Conjugates, Curr. Opin. Drug Discov. Devel., 5(2), 279 (2002).
  10. J. Huang, F. Gao, X. Tang, J. Yu, D. Wang, S. Liu, and Y. Li, Liver-targeting Doxorubicin-conjugated Polymeric Prodrug with pH-Triggered Drug Release Profile, Polym. Int., 59(10), 1390 (2010). https://doi.org/10.1002/pi.2880
  11. D. Melisi, A. Curcio, E. Luongo, E. Morelli, and M. G. Rimoli, D-Galactose as a Vector for Prodrug Design, Curr. Top. Med. Chem., 11(18), 2288 (2011). https://doi.org/10.2174/156802611797183258
  12. L. F. Tietze and K. Schmuck, Prodrugs for Targeted Tumor Therapies: Recent Developments in ADEPT, GDEPT and PMT, Curr. Pharm. Design, 17(32), 3527 (2011). https://doi.org/10.2174/138161211798194459
  13. L. Q. Yan, N. Li, and M. H. Zong, First Enzymatic Galactosylation of Acyclic Nucleoside Drugs by ${\beta}$-Galactosidase: Synthesis of Water-soluble ${\beta}$-DGalactosidic Prodrugs, Biotechnol. Bioprocess Eng., 19(4), 586 (2014). https://doi.org/10.1007/s12257-013-0823-1
  14. K. -H. Jung, Enhanced Enzyme Activities of Inclusion Bodies of Recombinant ${\beta}$-Galactosidase via the Addition of Inducer Analog after L-Arabinose Induction in the araBAD Promoter System of Escherichia coli, J. Microbiol. Biotechnol., 18(3), 434 (2008).
  15. K. -H. Jung, J. -H. Yeon, S. -K. Moon, and J. -H. Choi, Methyl ${\alpha}$-DGlucopyranoside Enhances the Enzymatic Activity of Recombinant ${\beta}$-Galactosidase Inclusion Bodies in the araBAD Promoter System of Escherichia coli. J. Ind. Microbiol. Biotechnol., 35(7), 695 (2008). https://doi.org/10.1007/s10295-008-0329-6
  16. N. Bridiau, S. Taboubi, N. Marzouki, M. D. Legoy, and T. Maugard, ${\beta}$ -Galactosidase Catalyzed Selective Galactosylation of Aromatic Compounds, Biotechnol. Prog., 22(1), 326 (2006). https://doi.org/10.1021/bp050230n
  17. C. Scheckermann, F. Wagner, and L. Fischer, Galactosylation of Antibiotics using the ${\beta}$-Galactosidase from Aspergillus oryae, Enzyme Microb. Technol., 20(8), 629 (1997). https://doi.org/10.1016/S0141-0229(96)00211-6

피인용 문헌

  1. 대장균 β-Galactosidse를 이용한 1, 2-Hexanediol galactoside의 합성과 Ethyl Acetate 추출 및 Silica Gel Chromatography를이용한 정제 vol.33, pp.3, 2016, https://doi.org/10.12925/jkocs.2016.33.3.498
  2. 베타-갈락토시데이즈를 이용하여 합성된 Benzyl Alcohol Galactoside의 NMR Spectroscopy 및 Mass spectrometry vol.36, pp.1, 2019, https://doi.org/10.12925/jkocs.2019.36.1.84
  3. 대장균 베타-갈락토시데이즈를 이용한 Phenylethanol Galactoside 합성 조건의 최적화 vol.38, pp.1, 2016, https://doi.org/10.12925/jkocs.2021.38.1.99
  4. 1, 2-Octanediol과 1, 2-Octanediol Galactoside의 항균력 및 세포독성 비교연구 vol.38, pp.3, 2016, https://doi.org/10.12925/jkocs.2021.38.3.629
  5. Mass spectrometry와 NMR Spectroscopy를 이용한 1, 2-Octanediol Galactoside의 효소합성 확인 vol.38, pp.3, 2016, https://doi.org/10.12925/jkocs.2021.38.3.824