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 2017-05, Vol.7 (1), p.97-11, Article 97
• Main Authors: Li, Xue-Qing, Wu, Qin, Hu, Die, Wang, Rui, Liu, Yan, Wu, Min-Chen, Li, Jian-Fang
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Biomedical and Life Sciences; Biotechnology; Catalysis; Catalytic efficiency; Deactivation; E coli; Efficiency; Glycoside hydrolase; Hydrolase; In silico design; Iterative mutagenesis; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular dynamics; Mutagenesis; Original; Original Article; Pichia pastoris; Saturation mutagenesis; Site-saturation mutagenesis; Temperature characteristics; Temperature effects; Thermal stability; Xylanase; Yeast
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-017-0399-9

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.