Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Optimization of Glycosyl Aesculin Synthesis by Thermotoga neapolitana β-Glucosidase Using Response-surface Methodology

반응표면분석법을 이용한 Thermotoga neapolitana β-glucosidase의 당전이 활성을 통한 glycosyl aesculin 합성 최적화

  • Park, Hyunsu (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Park, Young-Don (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Cha, Jaeho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • Received : 2016.08.03
  • Accepted : 2016.09.26
  • Published : 2017.01.30
Download PDF
⟨ Previous Next ⟩

Abstract

Glycosyl aesculin, a potent anti-inflammatory agent, was synthesized by transglycosylation reaction, catalyzed by Thermotoga neapolitana ${\beta}-glucosidase$, with aesculin as an acceptor. The key reaction parameters were optimized using response-surface methodology (RSM) and $2{\mu}g$ of the enzyme. As shown by a statistical analysis, a second-order polynomial model fitted well to the data (p<0.05). The response surface curve for the interaction between aesculin and other parameters revealed that the aesculin concentration and reaction time were the primary factors that affected the yield of glycosyl aesculin. Among the tested factors, the optimum values for glycosyl aesculin production were as follows: aesculin concentration of 9.5 g/l, temperature of $84^{\circ}C$, reaction time of 81 min, and pH of 8.2. Under these conditions, 61.7% of glycosyl aesculin was obtained, with a predicted yield of 5.86 g/l. The maximum amount of glycosyl aesculin was 6.02 g/l. Good agreement between the predicted and experimental results confirmed the validity of the RSM. The optimization of reaction conditions by RSM resulted in a 1.6-fold increase in the production of glycosyl aesculin as compared to the yield before optimization. These results indicate that RSM can be effectively used for process optimization in the synthesis of a variety of biologically active glycosides using bacterial glycosidases.

강한 항 염증 활성을 갖는 glycosyl aesculin이 Thermotoga neapolitana ${\beta}-glucosidase$의 당전이 활성을 통하여 aesculin을 수용체로 이용하여 합성되었다. 약 $2{\mu}g$의 효소를 이용하여 반응표면분석법을 통한 주요 반응 매개변수들의 최적화가 시도되었다. 각 반응 변수들의 통계분석 결과 2차 다항식모델이 적용된 유의값(p<0.05)에 잘 맞았다. Aesculin과 다른 매개변수사이의 상호관계를 의미하는 반응표면곡선 그래프는 glycosyl aesculin의 수율이 주로 aesculin의 농도와 반응시간에 영향을 받음을 나타내었다. Glycosyl aesculin의 생산을 위한 반응최적조건은 aesculin의 농도 9.5 g/l, 온도 $84^{\circ}C$, 반응시간 81분, 그리고 pH 8.2로 나타났으며, 이러한 조건하에서 효소반응시 61.7%의 전환율로 5.86 g/l 의 수율이 예상되었다. 실험을 통한 실질적인 수율은 6.02 g/l으로 나타났다. 실질적인 수율과 가까운 값을 예측 가능하다는 것을 통하여 반응표면분석법이 효소반응의 전환율을 최적화하는데 타당하다는 것이 입증되었다. 본 연구에서는 반응표면분석법을 활용하여 최적화 이전에 비하여 약 1.6배의 glycosyl aesculin을 얻을 수 있었다. 이러한 결과들은 반응표면분석법이 미생물유래 당화효소를 이용한 생물학적 활성을 갖는 배당체 합성의 생산 최적화에 효과적으로 활용할 수 있다는 것을 보여준다.

Keywords

References

  1. Box, G. E. P. and Wilson, K. B. 1951. On the experimental attainment of optimum conditions (with discussion). J. R. Aust. Hist. Soc. Series B 13, 1-45.
  2. Choi, K. W., Park, K. M., Jun, S. Y., Park, C. S., Park, K. H. and Cha, J. 2008. Modulation of the regioselectivity of a Thermotoga neapolitana ${\beta}$-glucosidase by site-directed mutagenesis. J. Microbiol. Biotechnol. 18, 901-907.
  3. Gurme, S. T., Surwase, S. N., Patil, S. A., Jadhav, S. B. and Jadhav, J. P. 2013. Optimization of biotransformation of l-tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 using response surface methodology. Ind. J. Microbiol. 53, 194-198. https://doi.org/10.1007/s12088-012-0346-z
  4. Jun, S. Y., Park, K. M., Choi, K. W., Jang, M. K., Kang, H. Y., Lee, S. H., Park, K. H. and Cha, J. 2008. Inhibitory effects of arbutin-${\beta}$-glycosides synthesized from enzymatic transglycosylation for melanogenesis. Biotechnol. Lett. 30, 743-748. https://doi.org/10.1007/s10529-007-9605-1
  5. Kim, K. H., Park, H., Park, H. J., Choi, K. H., Sadikot, R. T., Cha, J. and Joo, M. 2016. Glycosylation enables aesculin to activate Nrf2. Sci. Rep. 6, 29956. https://doi.org/10.1038/srep29956
  6. Kim, K. H., Park, Y. D., Park. H., Moon, K. O., Ha, K. T., Baek, N. I., Park, C. S., Joo, M. and Cha, J. 2014. Synthesis and biological evaluation of a novel baicalein glycoside as an anti-inflammatory agent. Eur. J. Pharmacol. 744, 147-156. https://doi.org/10.1016/j.ejphar.2014.10.013
  7. Ko, J. A., Ryu, Y. B., Park, T. S., Jeong, H. J., Kim, J. H., Park, S. J., Kim, J. S., Kim, D., Kim, Y. M. and Lee, W. S. 2012. Enzymatic synthesis of puerarin glucosides using Leuconostoc dextransucrase. J. Microbiol. Biotechnol. 22, 1224-1229. https://doi.org/10.4014/jmb.1202.02007
  8. Park, T. H., Choi, K. W., Park, C. S., Lee, S. B., Kang, H. Y., Shon, K. J., Park, J. S. and Cha, J. 2005. Substrate specificity and transglycosylation catalyzed by a thermostable ${\beta}$-glucosidase from marine hyperthermophile Thermotoga neapolitana. Appl. Microbiol. Biotechnol. 69, 411-422. https://doi.org/10.1007/s00253-005-0055-1
  9. Raab, T., Barron, D., Vera, F. A., Crespy, V., Oliveira, M. and Williamson, G. 2010. Catechin glucosides: occurrence, synthesis, and stability. J. Agric. Food Chem. 58, 2138-2149. https://doi.org/10.1021/jf9034095
  10. Rodriguez-Nogales, J. M., Roura, E. and Contreras, E. 2005. Biosynthesis of ethyl butyrate using immobilized lipase: a statistical approach. Process Biochem. 40, 63-68. https://doi.org/10.1016/j.procbio.2003.11.049
  11. Schmid, G. and Wandrey, C. 1987. Purification and partial characterization of a cellodextrin glucohydrolase (${\beta}$-glucosidase) from Trichoderma reesei strain QM 9414. Biotechnol. Bioeng. 30, 571-585. https://doi.org/10.1002/bit.260300415
  12. Tanyildizi, M., Ozer, D. and Elibol, M. 2005. Optimization of ${\alpha}$-amylase production by Bacillus sp. using response surface methodology. Process Biochem. 40, 2291-2296. https://doi.org/10.1016/j.procbio.2004.06.018
  13. Thangavel, P., Balaraman, M. and Soundra, D. 2009. A response surface methodological study on prediction of glucosylation yields of thiamin using immobilized ${\beta}$-glucosidase. Process Biochem. 44, 251-255. https://doi.org/10.1016/j.procbio.2008.10.017
  14. Thuong, P. T., Pokharel, Y. R., Lee, M. Y., Kim, S. K., Bae, K., Su, N. D., Oh, W. K. and Kang, K. W. 2009. Dual anti-oxidative effects of fraxetin isolated from Fraxinus rhinchophylla. Biol Pharm Bull. 32, 1527-1532. https://doi.org/10.1248/bpb.32.1527
  15. Tianzhu, Z. and Shumin, W. 2015. Esculin inhibits the inflammation of LPS-induced acute lung injury in mice via regulation of TLR/NF-${\kappa}B$ pathways. Inflammation 38, 1529-1536. https://doi.org/10.1007/s10753-015-0127-z
  16. Turner, P., Svensson, D., Adlercreutz, P. and Karlsson, E. N. 2007. A novel variant of Thermotoga neapolitana ${\beta}$-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation. J. Biotechnol. 130, 67-74. https://doi.org/10.1016/j.jbiotec.2007.02.016
  17. Velickovic, D., Dimitrijevic, A., Bihelovic, F., Bezbradica, D., Jankov, R. and Milosavic, N. 2011. A highly efficient diastereoselective synthesis of ${\alpha}$-isosalicin by maltase from Saccharomyces cerevisiae. Process Biochem. 46, 1698-1702. https://doi.org/10.1016/j.procbio.2011.05.007
  18. Witaicenis, A., Seito, L. N., da Silveira Chagas, A., de Almeida, L. D. Jr, Luchini, A. C., Rodrigues-Orsi, P., Cestari, S. H. and Di Stasi, L. C. 2014. Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives. Phytomedicine 21, 240-246. https://doi.org/10.1016/j.phymed.2013.09.001
  19. Witaicenis, A., Seito, L. N. and Di Stasi, L. C. 2010. Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis. Chem. Biol. Interact. 186, 211-218. https://doi.org/10.1016/j.cbi.2010.03.045
  20. Woo, H. J., Kang, H. K., Nguyen, T. T., Kim, G. E., Kim, Y. M., Park, J. S., Kim, D., Cha, J., Moon, Y. H., Nam, S. H., Xia, Y. M., Kimura, A. and Kim, D. 2012. Synthesis and characterization of ampelopsin glucosides using dextransucrase from Leuconostoc mesenteroides B-1299CB4: glucosylation enhancing physicochemical properties. Enzyme Microb. Technol. 51, 311-318. https://doi.org/10.1016/j.enzmictec.2012.07.014
  21. Woodward, J. and Wiseman, A. 1982. Fungal and other ${\beta}$-glucosidases-their properties and applications. Enzyme Microb. Technol. 4, 73-79. https://doi.org/10.1016/0141-0229(82)90084-9