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Research article

Biotechnology and Bioengineering (BB)  |  Enzyme, Protein Engineering, and Metabolic Engineering

Improvement of the Thermostability of Xylanase from Thermobacillus composti through Site-Directed Mutagenesis

Yong-Sheng Tian 1, 2, 3*, Jing Xu 1, Lei Chen 1, Xiao-Yan Fu 1, Ri-He Peng 1 and Quan-Hong Yao 1

1Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China, 2Shanghai Ruifeng Agricultural Science and Technology Co., Ltd, Shanghai 201106, P.R. China, 3College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R. China

Received: May 10, 2017; Accepted: August 28, 2017

J. Microbiol. Biotechnol. 2017; 27(10): 1783-1789

Published October 28, 2017 https://doi.org/10.4014/jmb.1705.05026

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium, was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life of xylanase at 65°C increased from 10 to 50 min after a disulfide bridge was introduced between the α-helix and its adjacent β-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this α-helix enhanced the half-life of xylanase for 60 min at 65°C. Therefore, the mutant may be utilized for industrial applications.

Keywords

Xylanase thermostability, Thermobacillus composti, disulfide bridge, structure

References

  1. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23.
    Pubmed CrossRef
  2. Shi H, Zhang Y , Zhong H , Huang Y , Li X, Wang F. 2 014. Cloning, over-expression and characterization of a thermotolerant xylanase from Thermotoga thermarum. Biotechnol. Lett. 36: 587-593.
    Pubmed CrossRef
  3. Han HJ, Fu XY, Zhu B, Zhao W, Tian YS, Peng RH, et al. 2015. Characterization and high expression of recombinant Ustilago maydis xylanase in Pichia pastoris. Biotechnol. Lett. 37: 697-703.
    Pubmed CrossRef
  4. Davies G, Henrissat B. 1995. Structures and mechanisms of glycosyl hydrolases. Structure 3: 853-859.
    CrossRef
  5. Jeffries TW. 1996. Biochemistry and genetics of microbial xylanases. Curr. Opin. Biotechnol. 7: 337-342.
    CrossRef
  6. Zhou C , Bai J, Deng S, Wang J, Zhu J , Wu M, et al. 2008. Cloning of a xylanase gene 44 from Aspergillus usamii and its expression in Escherichia coli. Bioresour. Technol. 99: 831-838.
    Pubmed CrossRef
  7. Wang Y , Fu Z, Huang H , Zhang H , Yao B , Xiong H , et al. 2012. Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge. Bioresour. Technol. 112: 275-279.
    Pubmed CrossRef
  8. Fukunaga N , Iwasaki Y , Kono S, Kita Y , Izumi Y. 1 998. Thermostable xylanase. US Patent 5, 916,795.
  9. Kumar PR, Eswaramoorthy S, Vithayathil PJ, Viswamitra MA. 2000. The tertiary structure at 1.59 A resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti Bainier. J. Mol. Biol. 295: 581-593.
    Pubmed CrossRef
  10. Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KK, Allison RW, et al. 1998. Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Appl. Environ. Microbiol. 64: 1759-1765.
    Pubmed PMC
  11. Paloheimo M, Mäntylä A, Vehmaanperä J, Hakola S, Lantto R, Lahtinen T, et al. 1998. Thermostable xylanases produced by recombinant Trichoderma reesei for pulp bleaching, pp. 255-264. In Claeyssen M, Nerinkx W, Piens K (eds.), Carbohydrate from Trichoderma reesei and Other Microorganisms. Royal Society of Chemistry, Cambridge, UK.
  12. Samain E, Debeire P, Debeire-Gosselin M, Touzel JP. 1991. Xylanase, souches de Bacillus productrices de xylanase et leurs ytilisation. Patent FR-9101191.
  13. Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H. 1996. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J. Biotechnol. 49: 211-218.
    CrossRef
  14. Fenel F, Leisola M, Janis J, Turunen O. 2004. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II. J. Biotechnol. 108: 137-143.
    Pubmed CrossRef
  15. Jeong MY, Kim S , Yun C W, Choi YJ, Cho SG. 2 007. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. J Biotechnol. 127: 300-309.
    Pubmed CrossRef
  16. Li YY, Zhong KX, Hu AH, Liu DN, Chen LZ, Xu SD. 2015. High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris. Protein Expr. Purif. 108: 90-96.
    Pubmed CrossRef
  17. Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu X, Rouvinen J, et al. 2001. A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism. J. Biotechnol. 88: 37-46.
    CrossRef
  18. Song L, Dumon C , Siguier B , Andre I , Eneyskaya E , Kulminskaya A, et al. 2014. Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus. J. Biotechnol. 174: 64-72.
    Pubmed CrossRef
  19. Xiong A S, Yao QH, Peng RH, Li X, Fan H Q, Cheng ZM, et al. 2004. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32: e98.
    Pubmed PMC CrossRef
  20. Peng RH, Xiong AS, Yao QH. 2006. A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl. Microbiol. Biotechnol. 73:234-240.
    Pubmed CrossRef
  21. Xiong AS, Peng RH, Li X, Fan HQ, Yao QH, Guo MJ, et al. 2003. [Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris]. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 35: 154-160.
  22. Miao S, Ziser L, Aebersold R, Withers SG. 1994. Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry 33: 7027-7032.
    Pubmed CrossRef
  23. Bradford MM. 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.
    CrossRef
  24. Miller GL Jr. 1959. Measurement of methods for assay of xylanase activity. Anal. Biochem. 2: 127-132.
  25. Sun JY, Zhao D, Wang W, Liu J, Cheng J, Li Y, Jia YN. 2007. Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Food Chem. 104: 1055-1064.
    CrossRef
  26. Wakarchuk WW, Campbell RL, Sung WL, Davoodi J, Yaguchi M. 1994. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 3: 467-475.
    Pubmed PMC CrossRef
  27. Bray MR, Clarke AJ. 1994. Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune. Eur. J. Biochem. 219: 821-827.
    Pubmed CrossRef
  28. Krengel U, Dijkstra BW. 1996. Three-dimensional structure of endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum. J. Mol. Biol. 263: 70-78.
    Pubmed CrossRef
  29. Qiu J , Han H , Sun B , Chen L, Yu C, Peng R, et al. 2016. Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH. Microbiol. Res. 182: 1-7.
    Pubmed CrossRef
  30. Sapre M P, Jha H , Patil M B. 2 005. P urification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn. J. Gen. Appl. Microbiol. 51:327-334.
    Pubmed CrossRef
  31. Wang K, Luo H, Tian J. 2014. Thermostability improvement of a Streptomyces xylanase by introducing proline and glutamic acid residues. Appl. Environ. Microbiol. 80: 2158-2165.
    Pubmed PMC CrossRef
  32. Li H, Kankaanpää A, Xiong H. 2013. Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid on the enzymatic performance. Enzyme Microb. Technol. 53: 414-419.
    Pubmed CrossRef
  33. Yin X , Yao Y , Wu M C, 2014. A unique d isulfide b ridge of the thermophilic xylanase SyXyn11 plays a key role in its thermostability. Biochemistry (Mosc.) 79: 531-537.
    Pubmed CrossRef
  34. Facchiano AM, Colonna G, Ragone R. 1998. Helix stabilizing factors and stabilization of thermophilic proteins: an X-ray based study. Protein Eng. 11: 753-760.
    Pubmed CrossRef
  35. Davoodi J, Wakarchuk WW, Carey PR, Surewicz WK. 2007. Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds. Biophys. Chem. 125: 453-461.
    Pubmed CrossRef
  36. Betz SF. 1993. Disulfide bonds and the stability of globular proteins. Protein Sci. 2: 1551-1558.
    Pubmed PMC CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(10): 1783-1789

Published online October 28, 2017 https://doi.org/10.4014/jmb.1705.05026

Copyright © The Korean Society for Microbiology and Biotechnology.

Improvement of the Thermostability of Xylanase from Thermobacillus composti through Site-Directed Mutagenesis

Yong-Sheng Tian 1, 2, 3*, Jing Xu 1, Lei Chen 1, Xiao-Yan Fu 1, Ri-He Peng 1 and Quan-Hong Yao 1

1Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China, 2Shanghai Ruifeng Agricultural Science and Technology Co., Ltd, Shanghai 201106, P.R. China, 3College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R. China

Received: May 10, 2017; Accepted: August 28, 2017

Abstract

Thermostability is an important property of xylanase because high temperature is required for
its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this
study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium,
was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable
xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life
of xylanase at 65°C increased from 10 to 50 min after a disulfide bridge was introduced
between the α-helix and its adjacent β-sheet at S98 and N145. Further mutation at the side of
A153E named XynB-CE in the C-terminal of this α-helix enhanced the half-life of xylanase for
60 min at 65°C. Therefore, the mutant may be utilized for industrial applications.

Keywords: Xylanase thermostability, Thermobacillus composti, disulfide bridge, structure

References

  1. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23.
    Pubmed CrossRef
  2. Shi H, Zhang Y , Zhong H , Huang Y , Li X, Wang F. 2 014. Cloning, over-expression and characterization of a thermotolerant xylanase from Thermotoga thermarum. Biotechnol. Lett. 36: 587-593.
    Pubmed CrossRef
  3. Han HJ, Fu XY, Zhu B, Zhao W, Tian YS, Peng RH, et al. 2015. Characterization and high expression of recombinant Ustilago maydis xylanase in Pichia pastoris. Biotechnol. Lett. 37: 697-703.
    Pubmed CrossRef
  4. Davies G, Henrissat B. 1995. Structures and mechanisms of glycosyl hydrolases. Structure 3: 853-859.
    CrossRef
  5. Jeffries TW. 1996. Biochemistry and genetics of microbial xylanases. Curr. Opin. Biotechnol. 7: 337-342.
    CrossRef
  6. Zhou C , Bai J, Deng S, Wang J, Zhu J , Wu M, et al. 2008. Cloning of a xylanase gene 44 from Aspergillus usamii and its expression in Escherichia coli. Bioresour. Technol. 99: 831-838.
    Pubmed CrossRef
  7. Wang Y , Fu Z, Huang H , Zhang H , Yao B , Xiong H , et al. 2012. Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge. Bioresour. Technol. 112: 275-279.
    Pubmed CrossRef
  8. Fukunaga N , Iwasaki Y , Kono S, Kita Y , Izumi Y. 1 998. Thermostable xylanase. US Patent 5, 916,795.
  9. Kumar PR, Eswaramoorthy S, Vithayathil PJ, Viswamitra MA. 2000. The tertiary structure at 1.59 A resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti Bainier. J. Mol. Biol. 295: 581-593.
    Pubmed CrossRef
  10. Morris DD, Gibbs MD, Chin CW, Koh MH, Wong KK, Allison RW, et al. 1998. Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp. Appl. Environ. Microbiol. 64: 1759-1765.
    Pubmed KoreaMed
  11. Paloheimo M, Mäntylä A, Vehmaanperä J, Hakola S, Lantto R, Lahtinen T, et al. 1998. Thermostable xylanases produced by recombinant Trichoderma reesei for pulp bleaching, pp. 255-264. In Claeyssen M, Nerinkx W, Piens K (eds.), Carbohydrate from Trichoderma reesei and Other Microorganisms. Royal Society of Chemistry, Cambridge, UK.
  12. Samain E, Debeire P, Debeire-Gosselin M, Touzel JP. 1991. Xylanase, souches de Bacillus productrices de xylanase et leurs ytilisation. Patent FR-9101191.
  13. Schlacher A, Holzmann K, Hayn M, Steiner W, Schwab H. 1996. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J. Biotechnol. 49: 211-218.
    CrossRef
  14. Fenel F, Leisola M, Janis J, Turunen O. 2004. A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II. J. Biotechnol. 108: 137-143.
    Pubmed CrossRef
  15. Jeong MY, Kim S , Yun C W, Choi YJ, Cho SG. 2 007. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. J Biotechnol. 127: 300-309.
    Pubmed CrossRef
  16. Li YY, Zhong KX, Hu AH, Liu DN, Chen LZ, Xu SD. 2015. High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris. Protein Expr. Purif. 108: 90-96.
    Pubmed CrossRef
  17. Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu X, Rouvinen J, et al. 2001. A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism. J. Biotechnol. 88: 37-46.
    CrossRef
  18. Song L, Dumon C , Siguier B , Andre I , Eneyskaya E , Kulminskaya A, et al. 2014. Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus. J. Biotechnol. 174: 64-72.
    Pubmed CrossRef
  19. Xiong A S, Yao QH, Peng RH, Li X, Fan H Q, Cheng ZM, et al. 2004. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32: e98.
    Pubmed KoreaMed CrossRef
  20. Peng RH, Xiong AS, Yao QH. 2006. A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl. Microbiol. Biotechnol. 73:234-240.
    Pubmed CrossRef
  21. Xiong AS, Peng RH, Li X, Fan HQ, Yao QH, Guo MJ, et al. 2003. [Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris]. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 35: 154-160.
  22. Miao S, Ziser L, Aebersold R, Withers SG. 1994. Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry 33: 7027-7032.
    Pubmed CrossRef
  23. Bradford MM. 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.
    CrossRef
  24. Miller GL Jr. 1959. Measurement of methods for assay of xylanase activity. Anal. Biochem. 2: 127-132.
  25. Sun JY, Zhao D, Wang W, Liu J, Cheng J, Li Y, Jia YN. 2007. Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Food Chem. 104: 1055-1064.
    CrossRef
  26. Wakarchuk WW, Campbell RL, Sung WL, Davoodi J, Yaguchi M. 1994. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 3: 467-475.
    Pubmed KoreaMed CrossRef
  27. Bray MR, Clarke AJ. 1994. Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune. Eur. J. Biochem. 219: 821-827.
    Pubmed CrossRef
  28. Krengel U, Dijkstra BW. 1996. Three-dimensional structure of endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum. J. Mol. Biol. 263: 70-78.
    Pubmed CrossRef
  29. Qiu J , Han H , Sun B , Chen L, Yu C, Peng R, et al. 2016. Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH. Microbiol. Res. 182: 1-7.
    Pubmed CrossRef
  30. Sapre M P, Jha H , Patil M B. 2 005. P urification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn. J. Gen. Appl. Microbiol. 51:327-334.
    Pubmed CrossRef
  31. Wang K, Luo H, Tian J. 2014. Thermostability improvement of a Streptomyces xylanase by introducing proline and glutamic acid residues. Appl. Environ. Microbiol. 80: 2158-2165.
    Pubmed KoreaMed CrossRef
  32. Li H, Kankaanpää A, Xiong H. 2013. Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid on the enzymatic performance. Enzyme Microb. Technol. 53: 414-419.
    Pubmed CrossRef
  33. Yin X , Yao Y , Wu M C, 2014. A unique d isulfide b ridge of the thermophilic xylanase SyXyn11 plays a key role in its thermostability. Biochemistry (Mosc.) 79: 531-537.
    Pubmed CrossRef
  34. Facchiano AM, Colonna G, Ragone R. 1998. Helix stabilizing factors and stabilization of thermophilic proteins: an X-ray based study. Protein Eng. 11: 753-760.
    Pubmed CrossRef
  35. Davoodi J, Wakarchuk WW, Carey PR, Surewicz WK. 2007. Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds. Biophys. Chem. 125: 453-461.
    Pubmed CrossRef
  36. Betz SF. 1993. Disulfide bonds and the stability of globular proteins. Protein Sci. 2: 1551-1558.
    Pubmed KoreaMed CrossRef