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

다국어 초록 (Multilingual 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.

참고문헌 (Reference)

1 Peng RH, "direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis" 73 : 234-240, 2006

2 Collins T, "Xylanases, xylanase families and extremophilic xylanases" 29 : 3-23, 2005

3 Samain E, "Xylanase, souches de Bacillus productrices de xylanase et leurs ytilisation"

4 Krengel U, "Three-dimensional structure of endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum" 263 : 70-78, 1996

5 Fukunaga N, "Thermostable xylanase"

6 Li H, "Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid on the enzymatic performance" 53 : 414-419, 2013

7 Wang K, "Thermostability improvement of a Streptomyces xylanase by introducing proline and glutamic acid residues" 80 : 2158-2165, 2014

8 Kumar PR, "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" 295 : 581-593, 2000

9 Davies G, "Structures and mechanisms of glycosyl hydrolases" 3 : 853-859, 1995

10 Qiu J, "Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH" 182 : 1-7, 2016

11 Sapre MP, "Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn" 51 : 327-334, 2005

12 Wakarchuk WW, "Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase" 3 : 467-475, 1994

13 Davoodi J, "Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds" 125 : 453-461, 2007

14 Miller GL Jr., "Measurement of methods for assay of xylanase activity" 2 : 127-132, 1959

15 Xiong AS, "Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris" 35 : 154-160, 2003

16 Wang Y, "Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge" 112 : 275-279, 2012

17 Song L, "Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus" 174 : 64-72, 2014

18 Miao S, "Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry" 33 : 7027-7032, 1994

19 Bray MR, "Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune" 219 : 821-827, 1994

20 Li YY, "High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris" 108 : 90-96, 2015

21 Facchiano AM, "Helix stabilizing factors and stabilization of thermophilic proteins: an X-ray based study" 11 : 753-760, 1998

22 Sun JY, "Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it" 104 : 1055-1064, 2007

23 Jeong MY, "Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236" 127 : 300-309, 2007

24 Betz SF., "Disulfide bonds and the stability of globular proteins" 2 : 1551-1558, 1993

25 Shi H, "Cloning, over-expression and characterization of a thermotolerant xylanase from Thermotoga thermarum" 36 : 587-593, 2014

26 Morris DD, "Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp" 64 : 1759-1765, 1998

27 Zhou C, "Cloning of a xylanase gene 44 from Aspergillus usamii and its expression in Escherichia coli" 99 : 831-838, 2008

28 Schlacher A, "Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus" 49 : 211-218, 1996

29 Han HJ, "Characterization and high expression of recombinant Ustilago maydis xylanase in Pichia pastoris" 37 : 697-703, 2015

30 Paloheimo M, "Carbohydrate from Trichoderma reesei and Other Microorganisms" Royal Society of Chemistry 255-264, 1998

31 Jeffries TW., "Biochemistry and genetics of microbial xylanases" 7 : 337-342, 1996

32 Yin X, "A unique d isulfide b ridge of the thermophilic xylanase SyXyn11 plays a key role in its thermostability" 79 : 531-537, 2014

33 Xiong A S, "A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences" 32 : v-, 2004

34 Bradford MM., "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976

35 Fenel F, "A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II" 108 : 137-143, 2004

36 Turunen O, "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" 88 : 37-46, 2001