Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Cloning, Expression, and Characterization of Protease-resistant Xylanase from Streptomyces fradiae var. k11

  • Li, Ning (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Peilong (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wang, Yaru (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Luo, Huiying (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Meng, Kun (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wu, Nigfeng (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Fan, Yunliu (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yao, Bin (Microbial Engineering Department of Feed Research Institute, Chinese Academy of Agricultural Sciences)
  • Published : 2008.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

Keywords

References

  1. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3: 286-290 https://doi.org/10.1016/0167-7799(85)90004-6
  2. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Caballero, P. A., M. Gomez, and C. M. Rosell. 2007. Improvement of dough rheology, bread quality and bread shelf-life by enzymes combination. J. Food Eng. 81: 42-53 https://doi.org/10.1016/j.jfoodeng.2006.10.007
  4. Collins, T., C. Gerday, and G. Feller. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23 https://doi.org/10.1016/j.femsre.2004.06.005
  5. Fernandez-Abalos, J. M., V. Reviejo, M. Diaz, S. Rodriguez, F. Leal, and R. I. Santamaria. 2003. Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases. Microbiology 149: 1623-1632 https://doi.org/10.1099/mic.0.26113-0
  6. Fontes, C. M. G. A., J. Hall, B. H. Hirst, G. P. Hazlewood, and H. J. Gilbert. 1995. The resistance of cellulases and xylanases to proteolytic inactivation. Appl. Microbiol. Biotechnol. 43: 52-57 https://doi.org/10.1007/BF00170622
  7. Fujimoto, Z., A. Kuno, S. Kaneko, S. Yoshida, H. Kobayashi, I. Kusakabe, and H. Mizuno. 2000. Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain. J. Mol. Biol. 300: 575-585 https://doi.org/10.1006/jmbi.2000.3877
  8. Gussow, D. and T. Glackson. 1989. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17: 4000 https://doi.org/10.1093/nar/17.10.4000
  9. Hall, J., S. Ali, M. A. Surani, G. P. Hazlewood, A. J. Clark, J. P. Simons, B. H. Hirst, and H. J. Gilbert. 1993. Manipulation of the repertoire of digestive enzymes secreted into the gastrointestinal tract of transgenic mice. Biotechnology 11: 376-379 https://doi.org/10.1038/nbt0393-376
  10. Jiang, Z. Q., W. Deng, Q. J. Yan, Q. Zhai, L. T. Li, and I. Kusakabe. 2006. Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86. J. Biotechnol. 126: 304-312 https://doi.org/10.1016/j.jbiotec.2006.05.006
  11. Kaneko, S., A. Kuno, M. Muramatsu, S. Iwamatsu, I. Kusakabe, and K. Hayashi. 2000. Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86. Biosci. Biotechnol. Biochem. 64: 447-451 https://doi.org/10.1271/bbb.64.447
  12. Kuno, A., D. Sbimizu, S. Kaneko, Y. Koyama, S. Yoshida, H. Kohayashi, K. Hayashi, K. Taira, and I. Kusakabe. 1998. PCR cloning and expression of the F/10 family xylanase gene from Streptomyces olivaceoviridis E-86. J. Ferment. Bioeng. 86: 434-439 https://doi.org/10.1016/S0922-338X(98)80147-5
  13. Li, J., P. J. Shi, W. Z. Zhang, X. Y. Han, L. L. Xu, H. T. Zhang, B. Yao, and Y. L. Fan. 2005. Gene cloning and expression of serine protease sfp2 from Streptomyces fradiae var. k11. Chin. J. Biotechnol. 21: 782-788
  14. Li, J., P. J. Shi, X. Y. Han, K. Meng, P. L. Yang, Y. R. Wang, H. Y. Luo, N. F. Wu, B. Yao, and Y. L. Fan. 2007. Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris. Protein Expr. Purif. 54: 79-86 https://doi.org/10.1016/j.pep.2007.02.012
  15. Meng, K., J. Li, Y. N. Cao, P. J. Shi, B. Wu, X. Y. Han, Y. G. Bai, N. F. Wu, and B. Yao. 2007. Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var. k11. Can. J. Microbiol. 53: 186-195 https://doi.org/10.1139/W06-122
  16. 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
  17. Mondou, F., F. Sharek, R. Morosoli, and D. Kluepful. 1986. Cloning of the xylanase gene of Streptomyces lividans. Gene 49: 323-329 https://doi.org/10.1016/0378-1119(86)90368-9
  18. Moreau, A., M. Roberge, C. Manin, F. Shareck, D. Kluepfel, and R. Morosoli. 1994. Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans. Biochem. J. 302: 291-295 https://doi.org/10.1042/bj3020291
  19. Morosoli, R., J. L. Bertrand, F. Mondou, F. Shareck, and D. Kluepfel. 1986. Purification and properties of a xylanase from Streptomyces lividans. Biochem. J. 239: 587-592 https://doi.org/10.1042/bj2390587
  20. Ohmiya, K., K. Sakka, S. Karita, and T. Kimura. 1997. Structure of cellulases and their applications. Biotechnol. Genet. Eng. Rev. 14: 365-414 https://doi.org/10.1080/02648725.1997.10647949
  21. Ossola, M. and Y. M. Galante. 2004. Scouring of flax rove with the aid of enzymes. Enzyme Microb. Technol. 34: 177-186 https://doi.org/10.1016/j.enzmictec.2003.10.003
  22. Pettersson, D. and P. Aman. 1989. Enzyme supplementation of a poultry diet containing rye and wheat. Br. J. Nutr. 62: 139-149 https://doi.org/10.1079/BJN19890014
  23. Prade, R. A. 1996. Xylanases: From biology to biotechnology. Biotechnol. Genet. Eng. Rev. 13: 101-131 https://doi.org/10.1080/02648725.1996.10647925
  24. Ruiz-Arribas, A., J. M. Fernandez-Abalos, P. Sanchez, A. L. Garda, and R. I. Santamaria. 1995. Overproduction, purification, and biochemical characterization of a xylanase (Xysl) from Streptomyces halstedii JM8. Appl. Environ. Microbiol. 61: 2414-2419
  25. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  26. Shareck, F., C. Roy, M. Yaguchi, R. Morosoli, and D. Kluepfel. 1991. Sequences of three genes specifying xylanases in Streptomyces lividans. Gene 107: 75-82 https://doi.org/10.1016/0378-1119(91)90299-Q
  27. Sinha, U., S. A. Wolz, and P. J. Lad. 1991. Two new extracellular serine proteases from Streptomyces fradiae. Int. J. Biochem. 23: 979-984 https://doi.org/10.1016/0020-711X(91)90133-8
  28. Tsujibo, H., K. Miyamoto, T. Kuda, K. Minami, T. Sakamoto, T. Hasegawa, and Y. Inamori. 1992. Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520. Appl. Environ. Microbiol. 58: 371-375
  29. Tsujibo, H., T. Ohtsuki, T. Iio, I. Yamazaki, K. Miyamoto, M. Sugiyama, and Y. Inamori. 1997. Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520. Appl. Environ. Microbiol. 63: 661-664
  30. Vats-Mehta, S., P. Bouvrette, F. Shareck, R. Morosoli, and D. Kluepfel. 1990. Cloning of a second xylanase-encoding gene of Streptomyces lividans 66. Gene 86: 119-122 https://doi.org/10.1016/0378-1119(90)90123-9
  31. van de Vyver, W. F. J., K. A. Dawson, N. H. Casey, and J. M. Tricarico. 2004. Effect of glycosylation on the stability of fungal xylanase exposed to proteases or rumen fluid in vitro. Anim. Feed Sci. Tech. 116: 259-269 https://doi.org/10.1016/j.anifeedsci.2004.07.012
  32. Wang, Q., X. R. Fan, W. D. Gao, and J. Chen. 2006. Scouring of knitted cotton fabrics with compound enzymes. J. Text. Res. 27: 27-30
  33. Wood, P. J., J. D. Erfle, and R. M. Teather. 1988. Use of complex formation between Congo red and polysaccharide in detection and assay of polysaccharide hydrolases. Methods Enzymol. 160: 59-74 https://doi.org/10.1016/0076-6879(88)60107-8
  34. Yang, P. L., P. J. Shi, Y. R. Wang, Y. G. Bai, K. Meng, H. Y. Luo, T. Z. Yuan, and B. Yao. 2007. Cloning and overexpression of a Paenibacillus $\beta$-glucanase in Pichia pastoris: Purification and characterization of the recombinant enzyme. J. Microbiol. Biotechnol. 17: 58-66
  35. Zhang, H. L., B. Yao, T. Z. Yuan, Y. R. Wang, S. S. Cao, and Y. L. Fan. 2002. Purification and properties of 43 kD xylanase XYNA from Streptomyces olivaceoviridis A1. J. Agric. Biotechnol. 10: 10-14