Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Characterization of a New Xylanase from Alkalophilic Paenibacillus sp. 12-11

  • Zhao, Yanyu (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Meng, Kun (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Luo, Huiying (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Peilong (Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Shi, Pengjun (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Huang, Huoqing (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Bai, Yingguo (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yao, Bin (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
  • Received : 2011.02.16
  • Accepted : 2011.05.19
  • Published : 2011.08.28
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Abstract

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

Keywords

References

  1. Bajpai, P. 1999. Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15: 147-157. https://doi.org/10.1021/bp990013k
  2. Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal. 2001. Microbial xylanases and their industrial applications: A review. Appl. Microbiol. Biotechnol. 56: 326-338. https://doi.org/10.1007/s002530100704
  3. Bradford, M. 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
  4. Caballero, P. A., M. Gómez, and C. M. Rosell. 2007. Improvement of dough rheology, bread quality and bread shelf-life by enzymes combination. J. Food Process Eng. 81: 42-53. https://doi.org/10.1016/j.jfoodeng.2006.10.007
  5. Christov, L. P., G. Szakacs, and H. Balakrishnan. 1999. Production, partial characterization and use of fungal cellulase-free xylanases in pulp bleaching. Process Biochem. 34: 511-517. https://doi.org/10.1016/S0032-9592(98)00117-4
  6. 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
  7. Gallardo, O., P. Diaz, and F. I. J. Pastor. 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
  8. Gibbs, M. D., R. A. Reeves, and P. L. Bergquist. 1995. Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B. 1 and activity of the enzyme on fiber-bound substrate. Appl. Environ. Microbiol. 61: 4403-4408.
  9. Guo, B., X. Chen, C. Sun, B. Zhou, and Y. Zhang. 2009. Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-$\beta$-1,4-xylanase from marine Glaciecola mesophila KMM 241. Appl. Microbiol. Biotechnol. 84: 1107-1115. https://doi.org/10.1007/s00253-009-2056-y
  10. Henrissat, B. and A. Bairoch. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316: 695-696. https://doi.org/10.1042/bj3160695
  11. Huang, J., G. Wang, and L. Xiao. 2006. Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli. Bioresour. Technol. 97: 802-808. https://doi.org/10.1016/j.biortech.2005.04.011
  12. Hwang, I. T., H. K. Lim, H. Y. Song, S. J. Cho, J. S. Chang, and N. J. Park. 2010. Cloning and characterization of a xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL- 001. Biotechnol. Adv. 28: 594-601. https://doi.org/10.1016/j.biotechadv.2010.05.007
  13. Kim, D. Y., M. K. Han, H. W. Oh, K. S. Bae, T. S. Jeong, S. U. Kim, et al. 2010. Novel intracellular GH10 xylanase from Cohnella laeviribosi HY-21: Biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues. Bioresour. Technol. 101: 8814-8821. https://doi.org/10.1016/j.biortech.2010.06.023
  14. Ko, C. H., W. L. Chen, C. H. Tsai, W. N. Jane, C. C. Liu, and J. Tu. 2007. Paenibacillus campinasensis BL11: A wood materialutilizing bacterial strain isolated from black liquor. Bioresour. Technol. 98: 2727-2733. https://doi.org/10.1016/j.biortech.2006.09.034
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680- 685. https://doi.org/10.1038/227680a0
  16. Lee, H., D. Shin, N. Cho, H. Kim, S. Shin, S. Im, H. Blaise Lee, S. Chun, and S. Bai. 2000. Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp. Biotechnol. Lett. 22: 387-392. https://doi.org/10.1023/A:1005676702533
  17. Li, N., K. Meng, Y. Wang, P. Shi, H. Luo, Y. Bai, P. Yang, and B. Yao. 2008. Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9. Appl. Microbiol. Biotechnol. 80: 231-240. https://doi.org/10.1007/s00253-008-1533-z
  18. Lineweaver, H. and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658-666. https://doi.org/10.1021/ja01318a036
  19. Liu, L., X. Li, and W. Shao. 2004 Computational analysis of responsible dipeptides for optimum pH in G/11 xylanase. Biochem. Biophys. Res. Commun. 321: 391-396. https://doi.org/10.1016/j.bbrc.2004.06.156
  20. Liu, Y. and R. F. Whittier. 1995. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674-681. https://doi.org/10.1016/0888-7543(95)80010-J
  21. Maat, J., M. Roza, J. Verbakel, H. Stam, M. J. Santos Da Silva, M. Bosse, et al. 1992. Xylanases and their application in bakery, pp. 349-360. In J. Visser, M. A. Kusters van Someren, G. Beldman, and A. G. J. Voragen (eds.). Xylans and Xylanases. Progress in Biotechnology, No. 7. Elsevier Science Publishers, Amsterdam, The Netherlands.
  22. Mamo, G., R. Hatti-Kaul, and B. Mattiasson. 2006. A thermostable alkaline active endo-$\beta$-1-4-xylanase from Bacillus halodurans S7: Purification and characterization. Enzyme Microb. Technol. 39: 1492-1498. https://doi.org/10.1016/j.enzmictec.2006.03.040
  23. Mamo, G., M. Thunnissen, R. Hatti-Kaul, and B. Mattiasson. 2009. An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation. Biochimie 91: 1187-1196. https://doi.org/10.1016/j.biochi.2009.06.017
  24. Manikandan, K., A. Bhardwaj, N. Gupta, N. K. Lokanath, A. Ghosh, V. S. Reddy, and S. Ramakumar. 2006. Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: Structural insights into alkalophilicity and implications for adaptation to polyextreme conditions. Protein Sci. 15: 1951-1960. https://doi.org/10.1110/ps.062220206
  25. Mchunu, N. P., S. Singh, and K. Permaul. 2009. Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli. J. Biotechnol. 141: 26- 30. https://doi.org/10.1016/j.jbiotec.2009.02.021
  26. 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
  27. Monis, P. T., S. Giglio, and C. P. Saint. 2005. Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal. Biochem. 340: 24-34. https://doi.org/10.1016/j.ab.2005.01.046
  28. 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
  29. Qiu, Z., P. Shi, H. Luo, Y. Bai, T. Yuan, P. Yang, S. Liu, and B. Yao. 2010. A xylanase with broad pH and temperature adaptability from Streptomyces megasporus DSM 41476, and its potential application in brewing industry. Enzyme Microb. Technol. 46: 506-512. https://doi.org/10.1016/j.enzmictec.2010.02.003
  30. Roberge, M., F. Shareck, R. Morosoli, D. Kluepfel, and C. Dupont. 1998. Site-directed mutagenesis study of a conserved residue in family 10 glycanases: Histidine 86 of xylanase A from Streptomyces lividans. Protein Eng. 11: 399-404. https://doi.org/10.1093/protein/11.5.399
  31. Schmidt, A., A. Schlacher, W. Steiner, H. Schwab, and C. Kratky. 1998. Structure of the xylanase from Penicillium simplicissimum. Protein Sci. 7: 2081-2088. https://doi.org/10.1002/pro.5560071004
  32. Subramaniyan, S. and P. Prema. 2000. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiol. Lett. 183: 1-7. https://doi.org/10.1111/j.1574-6968.2000.tb08925.x
  33. Subramaniyan, S. and P. Prema. 2002. Biotechnology of microbial xylanases: Enzymology, molecular biology, and application. Crit. Rev. Biotechnol. 22: 33-64. https://doi.org/10.1080/07388550290789450
  34. Sudo, M., M. Sakka, T. Kimyra, K. Ratanakhanokchai, and K. Sakka. 2010. Characterization of Paenibacillus curdlanolyticus intracellular xylanase Xyn10B encoded by the xyn10B gene. Biosci. Biotechnol. Biochem. 74: 2358-2360. https://doi.org/10.1271/bbb.100555
  35. Sun, H. J., S. Yoshida, Y. Kawabata, N. H. Park, and I. Kusakabe. 2002. Separation of two functional domains of the family F/10 $\beta$-xylanase from Streptomyces olivaceoviridis E-86 limited proteolysis with papain and some of their properties. Biotechnol. Lett. 24: 595-601. https://doi.org/10.1023/A:1015055624662
  36. Wang, G., Y. Wang, P. Yang, H. Luo, H. Huang, P. Shi, K. Meng, and B. Yao. 2010. Molecular detection and diversity of xylanase genes in alpine tundra soil. Appl. Microbiol. Biotechnol. 87: 1383-1393. https://doi.org/10.1007/s00253-010-2564-9
  37. Wang, J., Y. Bai, P. Yang, P. Shi, H. Luo, K. Meng, H. Huang, J. Yin, and B. Yao. 2010. A new xylanase from thermoalkaline Anoxybacillus sp. E2 with high activity and stability over a broad pH range. World J. Microbiol. Biotechnol. 26: 917-924. https://doi.org/10.1007/s11274-009-0254-5
  38. Wang, Q., X. Fan, W. Gao, and J. Chen. 2006. Scouring of knitted cotton fabrics with compound enzymes. J. Text. Res. 27: 27-30.
  39. Wood, P. J., J. D. Erfle, and R. M. Teather. 1988. Use of complex formation between Congo red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods Enzymol. 160: 59-74.
  40. Yu, E. K. C., L. U. L. Tan, M. K. H. Chan, L. Deschatelets, and J. N. Saddler. 1987. Production of thermostable xylanase by a thermophilic fungus, Thermoascus aurantiacus. Enzyme Microb. Technol. 9: 16-24. https://doi.org/10.1016/0141-0229(87)90044-5

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