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Paenibacillus amylolyticus 유래 xylanase GH10 및 GH30의 xylan 가수분해 특성

Enzymatic characterization of Paenibacillus amylolyticus xylanases GH10 and GH30 for xylan hydrolysis

  • 남경화 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 장명운 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 김민정 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 이정민 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 이민재 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공) ;
  • 김태집 (충북대학교 대학원 축산.원예.식품공학부 식품공학전공)
  • Nam, Gyeong-Hwa (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Jang, Myoung-Uoon (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Kim, Min-Jeong (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Lee, Jung-Min (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Lee, Min-Jae (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University) ;
  • Kim, Tae-Jip (Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University)
  • 투고 : 2016.11.29
  • 심사 : 2016.12.19
  • 발행 : 2016.12.31

초록

Xylan의 효소적 가수분해는 고부가가치 기능성 물질 또는 바이오에너지 생산을 위한 발효성 당을 얻는 가장 유용한 방법 중 하나이다. endo-${\beta}$-Xylanase는 xylan 주사슬 내부의 ${\beta}$-1,4-결합을 가수분해하여 xylobiose, xylotriose를 포함한 다양한 XOS를 생산하는 핵심 효소이다. 이들 효소 중에서 glucuronoxylanase GH30은 methylglucuronic acid가 측쇄에 수식된 xylan에 특이적으로 작용한다. 본 연구에서는 Paenibacillus amylolyticus KCTC 3005에서 유래한 2종의 xylan 가수분해효소(PaXN_10과 PaGuXN_30) 유전자를 클로닝하고, Escherichia coli에서 각각 발현시켰다. PaXN_10 (38.7 kDa)은 ${\beta}$-xylanase GH10 계열, PaGuXN_30 (58.5 kDa)은 glucuronoxylanase GH30에 해당하는 효소이며, $50^{\circ}C$와 pH 7.0에서 최대 활성을 나타내었다. 가수분해 특성 연구를 통해 P. amylolyticus가 목질계 glucuronoxylan을 분해하는 효소 시스템을 제안하였다. 세포 외로 분비되는 PaGuXN_30은 glucuroxylan을 가수분해하여 methylglucuronic acid 측쇄를 가지는 다양한 aldouronic acid mixtures를 생성하며, 이러한 분해산물은 세포 내로 이동하여 PaXN_GH10에 의해 xylose, xylobiose와 같은 저분자 XOS로 분해되어 세포 내 대사경로에 이용될 수 있다. 또한 이들 효소의 가수분해특성을 이용하여 다양한 탄수화물 소재 생산이 가능할 것으로 기대한다.

The enzymatic degradation of xylans is the most versatile way to obtain the high value-added functional compounds or the fermentable sugars for renewable energy. The endo-${\beta}$-xylanases are the major enzymes which hydrolyze the internal ${\beta}$-1,4-linkages of xylan backbones to produce the mixtures of xylooligosaccharides including xylobiose and xylotriose. Among them, glucuronoxylanase GH30 can exclusively hydrolyze the internal ${\beta}$-1,4-linkages of xylans decorated with methylglucuronic acid branches. In the present study, two xylanolytic enzyme (PaXN_10 and PaGuXN_30) genes were cloned from Paenibacillus amylolyticus KCTC 3005, and expressed in Escherichia coli, respectively. PaXN_10 (38.7 kDa) belongs to the endo-${\beta}$-xylanases GH10 family, while PaGuXN_30 (58.5 kDa) is a member of glucuronoxylanase GH30. They share the same optimal reaction conditions at $50^{\circ}C$ and pH 7.0. Enzymatic characterization proposed that P. amylolyticus can utilize the hardwood glucuronoarabinoxylans via the cooperative actions of xylanases GH10 and GH30. The extracellular PaGuXN_30 is secreted into the medium and hydrolyzes glucuronoarabinoxylans to release a series of aldouronic acid mixtures with a methylglucuronic acid branch. The resultant products being transported into the microbial cell are successively degraded into the smaller xylooligosaccharides by the intracellular PaXN_10, which will be utilized for the cellular metabolism.

키워드

참고문헌

  1. Ahmed, S., Riaz, S., and Jamil, A. 2009. Molecular cloning of fungal xylanases: An overview. Appl. Microbiol. Biotechnol. 84, 19-35. https://doi.org/10.1007/s00253-009-2079-4
  2. Andrews, S.R., Taylor, E.J., Pell, G., Vincent, F., Ducros, V.M.A., Davies, G.J., Lakey, J.H., and Gilbert, H.J. 2004. The use of forced protein evolution to investigate and improve stability of family 10 xylanases: The production of $Ca^{2+}$-independent stable xylanases. J. Biol. Chem. 279, 54369-54379. https://doi.org/10.1074/jbc.M409044200
  3. Blanco, A., Diaz, P., Martinez, J., Lbpez, O., Soler, C., and Pastor, F.I.J. 1996. Cloning of a Bacillus sp. BP-23 gene encoding a xylanase with high activity against aryl xylosides. FEMS Microbiol. Lett. 137, 285-290. https://doi.org/10.1111/j.1574-6968.1996.tb08120.x
  4. Chaikumpollert, O., Methacanon, P., and Suchiva, K. 2004. Structural elucidation of hemicelluloses from Vetiver grass. Carbohydr. Polym. 57, 191-196. https://doi.org/10.1016/j.carbpol.2004.04.011
  5. Collins, T., Gerday, C., and Feller, G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29, 3-23. https://doi.org/10.1016/j.femsre.2004.06.005
  6. Do, T.T., Quyen, D.T., Nguyen, T.N., and Nguyen, V.T. 2013. Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger. Protein Expr. Purif. 92, 196-202. https://doi.org/10.1016/j.pep.2013.09.011
  7. Falck, P., Precha-Atsawanan, S., Grey, C., Immerzeel, P., Staislbrand, H., Adlercreutz, P., and Karlsson, E.N. 2013. Xylooligosaccharides from hardwood and cereal xylans produced by a thermostable xylanase as carbon sources for Lactobacillus brevis and Bifidobacterium adolescentis. J. Agric. Food Chem. 61, 7333-7340. https://doi.org/10.1021/jf401249g
  8. Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. 2004. Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86. J. Biol. Chem. 279, 9606-9614. https://doi.org/10.1074/jbc.M312293200
  9. Fukuda, M., Watanabe, S., Yoshida, S., Itoh, H., Itoh, Y., Kamio, Y., and Kaneko, J. 2010. Cell surface xylanases of the glycoside hydrolase family 10 are essential for xylan utilization by Paenibacillus sp. W-61 as generators of xylo-oligosaccharide inducers for the xylanase genes. J. Bacteriol. 192, 2210-2219. https://doi.org/10.1128/JB.01406-09
  10. Fukumura, M., Sakka, K., Shimada, K., and Ohmiya, K. 1995. Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product. Biosci. Biotechnol. Biochem. 59, 40-46. https://doi.org/10.1271/bbb.59.40
  11. Gallardo, O., Diaz, P., and Pastor, F.I.J. 2003. Characterization of a Paenibacillus cell-associated xylanase with high activity on aryl-xylosides: a new subclass of family 10 xylanases. Appl. Microbiol. Biotechnol. 61, 226-233. https://doi.org/10.1007/s00253-003-1239-1
  12. Gallardo, O., Pastor, F.I.J., Polaina, J., Diaz, P., Lysek, R., Vogel, P., Isorna, P., Gonzalez, B., and Sanz-Aparicio, J. 2010. Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution. J. Biol. Chem. 285, 2721-2733. https://doi.org/10.1074/jbc.M109.064394
  13. Harada, K.M., Tanaka, K., Fukuda, Y., Hashimoto, W., and Murata, K. 2008. Paenibacillus sp. strain HC1 xylanases responsible for degradation of rice bran hemicellulose. Microbiol. Res. 163, 293-298. https://doi.org/10.1016/j.micres.2006.05.011
  14. Ihsanawati, Kumasaka, T., Kaneko, T., Morokuma, C., Yatsunami, R., Sato, T., Nakamura, S., and Tanaka, N. 2005. Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8. Proteins Struct. Funct. Genet. 61, 999-1009. https://doi.org/10.1002/prot.20700
  15. Juturu, V. and Wu, J.C. 2012. Microbial xylanases: engineering, production and industrial applications. Biotechnol. Adv. 30, 1219-1227. https://doi.org/10.1016/j.biotechadv.2011.11.006
  16. Kang, H.J., Jeong, C.K., Jang, M.U., Choi, S.H., Kim, M.H., Ahn, J.B., Lee, S.H., Jo, S.J., and Kim, T.J. 2009. Expression of cyclomaltodextrinase gene from Bacillus halodurans C-125 and characterization of its multisubstrate specificity. Food Sci. Biotechnol. 18, 776-781.
  17. Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428. https://doi.org/10.1021/ac60147a030
  18. Moon, J.S., Shin, S.Y., Choi, H.S., Joo, W., Cho, S.K., Li, L., Kang, J.H., Kim, T.J., and Han, N.S. 2015. In vitro digestion and fermentation properties of linear sugar-beet arabinan and its oligosaccharides. Carbohydr. Polym. 131, 50-56. https://doi.org/10.1016/j.carbpol.2015.05.022
  19. Saha, B.C. 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279-291. https://doi.org/10.1007/s10295-003-0049-x
  20. St. John, F.J., Rice, J.D., Preston, J.F., and John, F.J.S. 2006a. Paenibacillus sp. strain JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization. Appl. Environ. Microbiol. 72, 1496-1506. https://doi.org/10.1128/AEM.72.2.1496-1506.2006
  21. St. John, F.J., Rice, J.D., and Preston, J.F. 2006b. Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan. J. Bacteriol. 188, 8617-8626. https://doi.org/10.1128/JB.01283-06
  22. Sunna, A. and Antranikian, G. 1997. Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17, 39-67. https://doi.org/10.3109/07388559709146606
  23. Teleman, A., Lundqvist, J., Tjerneld, F., Stalbrand, H., and Dahlman, O. 2000. Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing $^1$H and $^{13}$C NMR spectroscopy. Carbohydr. Res. 329, 807-815. https://doi.org/10.1016/S0008-6215(00)00249-4
  24. Uday, U.S.P., Choudhury, P., Bandyopadhyay, T.K., and Bhunia, B. 2016. Classification, mode of action and production strategy of xylanase and its application for biofuel production from water hyacinth. Int. J. Biol. Macromol. 82, 1041-1054. https://doi.org/10.1016/j.ijbiomac.2015.10.086
  25. Valenzuela, S.V., Diaz, P., and Pastor, F.I.J. 2010. Recombinant expression of an alkali stable GH10 xylanase from Paenibacillus barcinonensis. J. Agric. Food Chem. 58, 4814-4818. https://doi.org/10.1021/jf9045792
  26. Valenzuela, S.V., Diaz, P., and Pastor, F.I.J. 2012. Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydratebinding module. Appl. Environ. Microbiol. 78, 3923-3931. https://doi.org/10.1128/AEM.07932-11
  27. Vrsanska, M., Kolenova, K., Puchart, V., and Biely, P. 2007. Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi. FEBS J. 274, 1666-1677. https://doi.org/10.1111/j.1742-4658.2007.05710.x
  28. Waeonukul, R., Pason, P., Kyu, K.L., Sakka, K., Kosugi, A., and Mori, Y. 2009. Cloning, sequencing, and expression of the gene encoding a multidomain endo-${\beta}$-1,4-xylanase from Paenibacillus curdlanolyticus B-6, and characterization of the recombinant enzyme. J. Microbiol. Biotechnol. 19, 277-285.
  29. Wang, W., Yan, R., Nocek, B.P., Voung, T.V., Leo, R. Di, Xu, X., Cui, H., Gatenholm, P., Toriz, G., Tenkanen, M., Savchenko, A., and Master, E.R. 2016. Biochemical and structural characterization of a five-domain GH115 ${\alpha}$-glucuronidase from the marine bacterium Saccharophagus degradans 2-40T. J. Biol. Chem. 291, 14120-14133. https://doi.org/10.1074/jbc.M115.702944

피인용 문헌

  1. Paenibacillus woosongensis로부터 대장균에 Xylanase 10A의 유전자 클로닝과 정제 및 특성분석 vol.48, pp.2, 2020, https://doi.org/10.4014/mbl.2002.02013