| Title |
Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design
|
|---|---|
| Published in |
AMB Express, May 2017
|
| DOI | 10.1186/s13568-017-0399-9 |
| Pubmed ID | |
| Authors |
Xue-Qing Li, Qin Wu, Die Hu, Rui Wang, Yan Liu, Min-Chen Wu, Jian-Fang Li |
| Abstract |
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E. coli/Aoxyn11A (G21X) (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A(Y13F) or AoXyn11A(G21I-Y13F), was designed by replacing Tyr(13) in AoXyn11A or AoXyn11A(G21I) with Phe. Finally, three variant-encoding genes, Aoxyn11A (G21I), Aoxyn11A (Y13F) and Aoxyn11A (G21I-Y13F), were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T opt) of recombinant (re) AoXyn11A(G21I-Y13F) was 60 °C, being 5 °C higher than that of reAoXyn11A(G21I) or reAoXyn11A(Y13F), and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2) of reAoXyn11A(G21I-Y13F) at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11A(G21I) and reAoXyn11A(Y13F). The melting temperature (T m) values of reAoXyn11A, reAoXyn11A(G21I), reAoXyn11A(Y13F) and reAoXyn11A(G21I-Y13F) were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k cat/K m) of reAoXyn11A(G21I-Y13F) was 473.1 mL mg(-1) s(-1), which was 1.65-fold higher than that of reAoXyn11A. |
X Demographics
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Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 23 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 23 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Master | 6 | 26% |
| Student > Ph. D. Student | 5 | 22% |
| Researcher | 3 | 13% |
| Student > Doctoral Student | 2 | 9% |
| Student > Bachelor | 1 | 4% |
| Other | 0 | 0% |
| Unknown | 6 | 26% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 6 | 26% |
| Biochemistry, Genetics and Molecular Biology | 3 | 13% |
| Chemistry | 2 | 9% |
| Social Sciences | 1 | 4% |
| Neuroscience | 1 | 4% |
| Other | 1 | 4% |
| Unknown | 9 | 39% |
Attention Score in Context
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#15,459,013
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Outputs from AMB Express
#445
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#194,845
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Outputs of similar age from AMB Express
#38
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