Browse > Article
http://dx.doi.org/10.4014/jmb.1705.05026

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

Tian, Yong-Sheng (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Xu, Jing (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Chen, Lei (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Fu, Xiao-Yan (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Peng, Ri-He (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Yao, Quan-Hong (Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences)
Publication Information
Journal of Microbiology and Biotechnology / v.27, no.10, 2017 , pp. 1783-1789 More about this Journal
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 $65^{\circ}C$ increased from 10 to 50 min after a disulfide bridge was introduced between the ${\alpha}$-helix and its adjacent ${\beta}$-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this ${\alpha}$-helix enhanced the half-life of xylanase for 60 min at $65^{\circ}C$. Therefore, the mutant may be utilized for industrial applications.
Keywords
Xylanase thermostability; Thermobacillus composti; disulfide bridge; structure;
Citations & Related Records
연도 인용수 순위
  • Reference
1 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.   DOI
2 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.   DOI
3 Sapre MP, Jha H , Patil MB. 2005. Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn. J. Gen. Appl. Microbiol. 51: 327-334.   DOI
4 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.   DOI
5 Li H, Kankaanpaa 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.   DOI
6 Yin X , Yao Y , Wu MC, 2014. A uniqued isulfide bridge of the thermophilic xylanase SyXyn11 plays a key role in its thermostability. Biochemistry (Mosc.) 79: 531-537.   DOI
7 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.   DOI
8 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.   DOI
9 Betz SF. 1993. Disulfide bonds and the stability of globular proteins. Protein Sci. 2: 1551-1558.   DOI
10 Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23.   DOI
11 Shi H, Zhang Y , Zhong H , Huang Y , Li X, Wang F. 2014. Cloning, over-expression and characterization of a thermotolerant xylanase from Thermotoga thermarum. Biotechnol. Lett. 36: 587-593.   DOI
12 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.   DOI
13 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.   DOI
14 Davies G, Henrissat B. 1995. Structures and mechanisms of glycosyl hydrolases. Structure 3: 853-859.   DOI
15 Jeffries TW. 1996. Biochemistry and genetics of microbial xylanases. Curr. Opin. Biotechnol. 7: 337-342.   DOI
16 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.   DOI
17 Fukunaga N , Iwasaki Y , Kono S, Kita Y , Izumi Y. 1998. Thermostable xylanase. US Patent 5, 916,795.
18 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.   DOI
19 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.
20 Paloheimo M, Mantyla A, Vehmaanpera 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.
21 Samain E, Debeire P, Debeire-Gosselin M, Touzel JP. 1991. Xylanase, souches de Bacillus productrices de xylanase et leurs ytilisation. Patent FR-9101191.
22 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.   DOI
23 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.   DOI
24 Jeong MY, Kim S , Yun CW, Choi YJ, Cho SG. 2007. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. J Biotechnol. 127: 300-309.   DOI
25 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.   DOI
26 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.   DOI
27 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.
28 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.   DOI
29 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.   DOI
30 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.   DOI
31 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.   DOI
32 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.
33 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.   DOI
34 Miller GL Jr. 1959. Measurement of methods for assay of xylanase activity. Anal. Biochem. 2: 127-132.
35 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.   DOI
36 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.   DOI