Insight into the functional roles of Glu175 in the hyperthermostable xylanase XYL10C-ΔN through structural analysis and site-saturation mutagenesis

Abstract

BackgroundImproving the hydrolytic performance of hemicellulases to degrade lignocellulosic biomass is of considerable importance for second-generation biorefinery. Xylanase, as the crucial hemicellulase, must be thermostable and have high activity for its potential use in the bioethanol industry. To obtain excellent xylanase candidates, it is necessary to understand the structure–function relationships to provide a meaningful reference to improve the enzyme properties. This study aimed to investigate the catalytic mechanism of a highly active hyperthermophilic xylanase variant, XYL10C-ΔN, for hemicellulose degradation.ResultsBy removing the N-terminal 66 amino acids, the variant XYL10C-ΔN showed a 1.8-fold improvement in catalytic efficiency and could hydrolyze corn stover more efficiently in hydrolysis of corn stover; however, it retained similar thermostability to the wild-type XYL10C. Based on the crystal structures of XYL10C-ΔN and its complex with xylobiose, Glu175 located on loop 3 was found to be specific to GH10 xylanases and probably accounts for the excellent enzyme properties by interacting with Lys135 and Met137 on loop 2. Site-saturation mutagenesis confirmed that XYL10C-ΔN with glutamate acid at position 175 had the highest catalytic efficiency, specific activity, and the broadest pH-activity profile. The functional roles of Glu175 were also verified in the mutants of another two GH10 xylanases, XylE and XynE2, which showed increased catalytic efficiencies and wider pH-activity profiles.ConclusionsXYL10C-ΔN, with excellent thermostability, high catalytic efficiency, and great lignocellulose-degrading capability, is a valuable candidate xylanase for the biofuel industry. The mechanism underlying improved activity of XYN10C-ΔN was thus investigated through structural analysis and functional verification, and Glu175 was identified to play the key role in the improved catalytic efficiency. This study revealed the importance of a key residue (Glu175) in XYN10C-ΔN and provides a reference to modify GH10 xylanases for improved catalytic performance.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1150-8) contains supplementary material, which is available to authorized users.

Extremophilic Prokaryotic Endoxylanases: Diversity, Applicability, and Molecular Insights

“…The GH-11 xylanases are therefore preferred that are xylan specific and release the sugars in a controlled manner (Henrissat and Davies, 1997;Qing et al, 2010). GH-11 xylanase (XYL10C-N) supports this hypothesis that showed a synergistic effect with cellulose and generates the sugars by reducing the sugar-producing rate to reduce the xylan-induced obstacles (You et al, 2018). Partial utilization of LCB-generated sugars by native microorganisms is one of the limiting factors that could be overcome by developing recombinant strains that can transform the available sugar in an efficient manner (Aftab et al, 2018).…”

Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase

“…The catalytic domains of XylE and thermophilic XYL10C share 53% sequence identity (Additional file 1: Figure S1), and their crystal structures showed a common (α/β) 8 -barrel fold of GH10 xylanases [22, 23]. Two standards were used to determine the demarcation point of the sequences: (1) Each sequence retains the local secondary structure and (2) except for the N- and C-terminal sequences, each sequence has a length of less than 20 amino acid residues.…”

A novel acid‐tolerant β‐xylanase fromScytalidium candidum3C for the synthesis ofo‐nitrophenyl xylooligosaccharides

“…To gain insights into the structural features responsible for the functional properties of ScXynA, we built a homology model. It should be noted that structures for eukaryotic GH10 xylanases are available in complex with xylobiose or xylotriose only [44,68], while bacterial xylanases are represented by inactive mutants complexed with oligosaccharides of DP up to 7, and provide a better overview of a binding cleft. A brief phylogenetic analysis of xylanase GH10 sequences available in PDB showed that ScXynA is closely related to some bacterial xylanases ( Figure S3).…”