Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

Xue-Qing Li,Min-Chen Wu,Qin Wu,Jian-Fang Li,Die Hu,Rui Wang,Yan Liu

doi:10.1186/s13568-017-0399-9

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, Gly21 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, AoXyn11AG21I, was selected from the mutagenesis library, E. coli/Aoxyn11AG21X (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11AY13F or AoXyn11AG21I–Y13F, was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe. Finally, three variant-encoding genes, Aoxyn11AG21I, Aoxyn11AY13F and Aoxyn11AG21I–Y13F, were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (Topt) of recombinant (re) AoXyn11AG21I–Y13F was 60 °C, being 5 °C higher than that of reAoXyn11AG21I or reAoXyn11AY13F, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) of reAoXyn11AG21I–Y13F at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11AG21I and reAoXyn11AY13F. The melting temperature (Tm) values of reAoXyn11A, reAoXyn11AG21I, reAoXyn11AY13F and reAoXyn11AG21I–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 (kcat/Km) of reAoXyn11AG21I–Y13F was 473.1 mL mg−1 s−1, which was 1.65-fold higher than that of reAoXyn11A.

Highlights

IntroductionXylanase (endo-β-1,4-d-xylanase, EC 3.2.1.8) exclusively catalyzes the hydrolysis of internal β-1,4-d-xylosidic linkages in the xylan backbone, producing xylooligosaccharidesLi et al AMB Expr (2017) 7:97 et al 2012)
Xylanase exclusively catalyzes the hydrolysis of internal β-1,4-d-xylosidic linkages in the xylan backbone, producing xylooligosaccharidesLi et al AMB Expr (2017) 7:97 et al 2012)
Homology modeling of the xylanase 3‐D structures The primary structure identities of AoXyn11A with the three known crystal structure glycoside hydrolase family 11 (GHF11) xylanases from P. funiculosum (PDB code: 1TE1), T. cellulolyticus (3WP3) and E. coli (2VUL) were 72, 70 and 68%, respectively, suggesting that the crystal structures were suitable as templates

Summary

Introduction

Xylanase (endo-β-1,4-d-xylanase, EC 3.2.1.8) exclusively catalyzes the hydrolysis of internal β-1,4-d-xylosidic linkages in the xylan backbone, producing xylooligosaccharidesLi et al AMB Expr (2017) 7:97 et al 2012). The majority of wild-type xylanases displayed low thermostability, preventing them from being applied in bioprocesses where the high temperature was required and encountered, exemplified by enzyme-added feeds, baking and pulp bleaching (Kumar et al 2016). A handful of thermophilic xylanases have been produced by thermophiles, but their specific activities and other enzymatic properties were very poor, making them unable to be applied effectively (Zhang et al 2014). To meet the increasing demands for thermophilic xylanases, some modifications in the primary and/or 3-D structures of mesophilic counterparts with superior enzymatic properties were conducted by peptide segment substitution, site-directed mutagenesis, DNA shuffling and error-prone PCR (Song et al 2014; Stephens et al 2014; Watanabe et al 2016; Zheng et al 2014). The site-saturation mutagenesis of Glu434 on the surface of RGI lyase was carried out to improve its thermostability. For the site-directed or site-saturation mutagenesis, the selection of the amino acid positions or the substituted residues was a pivotal step, which could be predicted based on in silico design or computer-aided design (Yin et al 2015)

Methods

Results

Conclusion