Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of Xylanase from Bacillus sp. A-6

Bacillus sp. A-6의 Xylanase의 정제와 특성

  • Choi, Suk-Ho (Division of Animal Science and Life Resources, Sangji University)
  • 최석호 (상지대학교 동물생명자원학부)
  • Received : 2009.03.02
  • Accepted : 2009.04.16
  • Published : 2009.06.28
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Abstract

A xylanase was purified from the culture supernatant of Bacillus sp. A-6 by using ultrafiltration and ion exchange chromatography on the column of SP-Sepharose using 5 mM acetate buffer, pH 5.0. The xylanase was eluted from the column at the concentration less than 0.05 M NaCl. The eluted xylanase was shown to be a single protein band in SDS-PAGE. Zymogram analysis indicated that the protein band in SDS-PAGE had the enzyme activity to hydrolyze oat spelt xylan. The molecular weights of the xylanase were 15,000 based on SDS-PAGE and 14,100 based on gel filtration chromatography. Thin layer chromatography showed that the xylanase hydrolyzed oat spelt xylan into xylobiose and high-molecular-weight xylooligosaccharides. The relative activities of the heated xylanase decreased to 80% at $40^{\circ}C$ after 7 hr and less than 40% at $60^{\circ}C$ after 1 hr.

Bacillus sp. A-6의 배양액의 상등액으로부터 한외여과와 5 mM sodium acetate, pH 5.0 용액으로 평형화된 SP-Sepharose column을 사용한 이온교환 크로마토그래피에 의해 xylanase를 정제하였다. Column에 흡착된 xylanase는 0.05 M NaCl 이하의 농도에서 용출되었다. 용출된 xylanase가 SDS-PAGE에서 단일 펩티드 밴드로 분리되어 순수함을 확인하였으며 oat spelt xylan을 기질로한 zymogram에서 xylan을 분해하는 밴드로 나타났다. Xylanase의 분자량은 SDS-PAGE에서 15,000이었고 겔여과 크로마토그래피에서 14,100 이었다. 박층막 크로마토그래피에서 xylanase가 oat spelt xylan을 xylobiose와 xylooligosaccharide로 분해함을 보였다. Xylanase를 가열할 때에 상대활성도가 $40^{\circ}C$에서 7시간 후에 80%로 감소하였으며 $60^{\circ}C$에서는 1시간 후에 40% 이하로 감소하였다.

Keywords

References

  1. Adeola, O. and M. R. Bedford. 2004. Exogenous dietary xylanase ameliorates viscosity-induced anti-nutritional effects in wheat-based diets for White Pekin ducks (Anas platyrinchos domesticus). Br. J. Nutr. 92: 87-94 https://doi.org/10.1079/BJN20041180
  2. Aspinali, G. O. 1959. Structural chemistry of the hemicelluloses. Adv. Carbohydr. Chem. 14: 429-468 https://doi.org/10.1016/S0096-5332(08)60228-3
  3. Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondai. 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56: 326-338 https://doi.org/10.1007/s002530100704
  4. Biely. P. 1985. Microbial xylanolytic systems. Trends Biotechonol. 3: 286-290 https://doi.org/10.1016/0167-7799(85)90004-6
  5. Collins, T., G. Gerday, and G. Feller. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Mirobiol. Rev. 29: 3-23 https://doi.org/10.1016/j.femsre.2004.06.005
  6. Cowieson, S. J., M. Hruby, and M. Faurschou Isaksen. 2005. The effect of conditioning temperature and exogenous xylanase addition on the viscosity of wheat-based diets and the performance of broiler chickens. Br. Poult. Sci. 46: 717-24 https://doi.org/10.1080/00071660500392506
  7. Esteben, R., J. R. Villanueva, and T. G. Villa. 1992. β-DXylanases of Bacillus circulans WL-12. Can. J. Microbiol. 28: 733-793 https://doi.org/10.1139/m82-112
  8. Graham, H., P. H. Simmins, and J. Sands. 2003. Reducing environmental pollution using animal feed enzymes. Commun. Agric. Appl. Bio. Sci. 68: 285-289
  9. Honda, H., T. Kudo, and K. Horikoshi. 1985. Two types of xylanases of alkalophilc Bacillus sp. No. C-125. Can. J. Microbiol. 31: 538-542 https://doi.org/10.1139/m85-100
  10. Hong, H. A., L. H. Duc, and S. M. Cutting. 2005. The use of bacterial spore formers as probiotics. FEMS Microbiol. Rev. 29: 813-835 https://doi.org/10.1016/j.femsre.2004.12.001
  11. Kim, K. C., S.-S. Yoo, Y.-A. Oh, and S.-J. Kim. 2003. Isolation and characteristics of Trichoderma harzianum FJ1 producing cellulases and xylanase. J. Microbiol. Biotechnol. 13: 1-8
  12. Kulkarni, N., A. Shendye, and M. Rao. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23: 411-456 https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature(London) 227: 680-685 https://doi.org/10.1038/227680a0
  14. Lee, J.-H. and S. H. Choi. 2006. Xylanase production by Bacillus sp. A-6 isolated from rice bran. J. Microbiol. Biotechnol. 16: 1856-61
  15. 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
  16. Okazaki, W., T. Akiba, K. Horikosh, and R. Akahoshi 1985. Purification and characterization of xylanases from an alkalophilic thermophilic Bacillus spp. Agric. Biol. Chem. 49: 2033-2039 https://doi.org/10.1271/bbb1961.49.2033
  17. Polizeli, M. L., A. C. Rizzatti, R. Monti, H. f. Terenzi, J. A. Jorge, and D. S. Amorim. 2005. Xylanases from fungi: properties and industrial applications. Appl. Microbiol. Biotechnol. 67: 577-591 https://doi.org/10.1007/s00253-005-1904-7
  18. Ratanakhanokchal, K., K. L. Kyu, and M. Tanticharoen. 1999. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. Strain K-1. Appl. Environ. Microbiol. 65: 694-697
  19. Sunna, A. and G. Antranikian. 1997. Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotecchnol. 17: 39-67 https://doi.org/10.3109/07388559709146606
  20. Wang, Z. R., S. Y. Qiao, and W. Q. Lu, and D. F. Li. 2005. Effects of enzyme supplementation on performance, nutrient digestibility, gastrointestinal morphology, and volatile fatty acid profiles in the hindgut of broilers fed wheat-based diets. Poult. Sci. 84: 875-881 https://doi.org/10.1093/ps/84.6.875
  21. Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganisms: Functions and Applications. Mcirobiol. Rev. 52: 305-317