Improvements in xylose stability and thermalstability of GH39 β-xylosidase from Dictyoglomus thermophilum by site-directed mutagenesis and insights into its xylose tolerance mechanism

https://doi.org/10.1016/j.enzmictec.2021.109921Get rights and content

Highlights

  • β-Xylosidase Xln-DT mutant showed high xylose tolerance, with the Ki value of 4602 mM, which is the highest in the reported literature so far.

  • The Xln-DT mutant would be more advantageous in the degradation of hemicellulose and the biotransformation of natural active substances.

  • This study supplys new insights into general mechanism of xylose effect on the activity of GH 39 β-xylosidases.

  • This study provides a reference for the related glycoside hydrolases that modulate their activity via feedback control mechanism.

Abstract

β-Xylosidases are often inhibited by its reaction product xylose or inactivated by high temperature environment, which limited its application in hemicellulosic biomass conversion to fuel and food processing. Remarkably, some β-xylosidases from GH39 family are tolerant to xylose. Therefore, it is of great significance to elucidate the effect mechanism of xylose on GH39 β-xylosidases to improve their application. In this paper, based on the homologous model and prediction of protein active pocket constructed by I-TASSA and PyMOL, two putative xylose tolerance relevant sites (283 and 284) were mutated at the bottom of the protein active pocket, where xylose sensitivity and thermostability of Dictyoglomus thermophilum β-xylosidase Xln-DT were improved by site-directed mutagenesis. The Xln-DT mutant Xln-DT-284ASP and Xln-DT-284ALA showed high xylose tolerance, with the Ki values of 4602 mM and 3708 mM, respectively, which increased by 9–35% compared with the wildtype Xln-DT. The thermostability of mutant Xln-DT-284ASP was significantly improved at 75 and 85 °C, while the activity of the wild enzyme Xln-DT decreased to 40–20%, the activity of the mutant enzyme still remained 100%. The mutant Xln-DT-284ALA showed excellent stability at pH 4.0–7.0, but Xln-DT-284ASP showed slightly decreased activity. Furthermore, in order to explore the key sites and mechanism of xylose’s effect on β-xylosidase activity, the interaction between xylose and enzyme was simulated by molecular docking. Besides binding to the active sites at the bottom of the substrate channel, xylose can also bind to sites in the middle or entrance of the channel with different affinities, which may determine the xylose inhibition of β-xylosidase. In conclusion, the improved xylose tolerance of mutant enzyme could be more advantageous in the degradation of hemicellulose and the biotransformation of other natural active substances containing xylose. This study supplies new insights into general mechanism of xylose effect on the activity of GH 39 β-xylosidases as well as related enzymes that modulate their activity via feedback control mechanism.

Introduction

β-Xylosidase (EC 3.2.1.37), as the major exoglycoside hydrolase to degrade the terminal, non-reducing β-D-xylosyl residues to release xylose, has great potential applications in many biotechnological industries [[1], [2], [3]]. In the biomass industry, xylan in plant fiber raw materials can be converted into xylose by endo-β-1,4-xylanase and β-xylosidase, and then into ethanol, furfural and other valuable fuels or chemicals [[4], [5], [6]]. By degrading xylobiose and xylooligosaccharides into several molecules of xylose, β-xylosidases relieve the inhibition of endo-xylanases and accelerate the overall rate of hemicellulose conversion [7]. In the pulp and paper industry, the synergistic effect of β-xylosidase and endo-xylanase can effectively improve the bleaching performance [8]. In recent years, β-xylosidases have been widely used in the pharmaceutical industry to hydrolyze the outer xylose residues at the glycosyl end of some natural compounds (glucoside bond formed by glucosides such as steroids and terpenes), so as to transform them into products with important application value [[9], [10], [11]]. However, when the content of xylose in the hydrolysate is too high, the activity of β-xylosidase will be production inhibition, which limits its practical application. In addition, in most food and pharmaceutical industries, where high temperature was needed to improve the utilization and solubility of substrates, a thermostable β-xylosidase could improve the conversion efficiency of enzyme and reduce the possibility of microbial contamination [12].

Most β-xylosidases, especially the β-xylosidases belong to GH 3, are sensitive to xylose and even inhibited by xylose. The Ki values were in range of 1.857–29 mM reported for β-xylosidases from Aspergillus niger, Humicola insolens Y1, Dictyoglomus turgidum and Thermotoga petrophila [[13], [14], [15], [16]]. This caused by the hydrolyzed xylose, which will compete with the substrate for the active binding sites on the enzyme, resulting in the decrease of enzyme activity. Therefore, on the one hand, during the last decade it has become a research hotspot to search and explore β-xylosidases that are tolerant to xylose and xylose stimulation. On the other hand, it is vital important to elucidate the effect mechanism of xylose on β-xylosidases for improving the application potential of enzyme by enzyme engineering. Although there are some papers have been reported the glucose tolerance mechanism of β-glucosidase, the molecular detail of xylose tolerance of β-xylosidase is still unclear so far. Through the research for β-glucosidases, there are two points of view in β-glucosidase sugar tolerance. The first one is that facilitates transglycosylation of substrate is the reason for avoiding the phenomenon of substrate competitive inhibition [17]. The other one is that glucose tolerance has been caused by the deep and narrow active sites of GH1 β-glucosidases [18,19]. Yang et al. [19] studied two GH 1 family β-glucosidases with similar sequence but different sugar dependence. The results showed that some sites of the enzyme at the entrance and middle of the substrate channel regulated the effects of glucose. Based on the analysis of glucose tolerance mechanism of GH1 β-glucosidase, it can provide a reference for the mechanism of xylose feedback inhibition of β-xylosidase catalytic activity.

Along with more and more β-xylosidases of various sources are cloned, purified and characterized, β-xylosidases especially β-xylosidases from GH39 family with xylose tolerance are gradually recognized [[20], [21], [22]]. Bhalla et al. [23] cloned and expressed a GH39 β-xylosidase from Geobacillus sp. strain, which retained 70% of relative activity at 210 mM xylose concentration. In addition, our laboratory has previously characterized a thermostable β-xylosidase Xln-DT of the GH family 39 from D.thermophilum, which displayed high tolerance to xylose. At the concentration of 3000 mM xylose, over 60% of the relative enzyme activity could be retained, which the Ki value was 3394 mM [24]. Thus, the β-xylosidase Xln-DT can be used as excellent model to investigate the xylose dependence of GH39 β-xylosidases.

With the continuous innovation of modern biotechnology, in vitro directed evolution of enzyme molecules has become a proven tool for molecular transformation and an effective strategy for altering or optimizing enzymatic properties to meet specific needs, which has been used to modified cellulases, β-xylanases, β-glucosidases and β-xylosidases, where temperature optimum, thermal stability, pH optimum, pH stability and glucose tolerance are most modified and improved [[25], [26], [27]]. Therefore, by homologous modeling and site directed mutation, the xylose tolerance of β-xylosidases can be targeted for improvement.

In this paper, three dimensional structure simulation, homology comparison and molecular docking of D-xylose and p-nitrophenyl-β-D-xylopyranoside (pNPX) with β-xylosidase Xln-DT from D.thermophilum DSM 3960 were carried out. Through the comparison of Coulomb force, Van der Waals force and energy value, we identified several key xylose tolerance sites, and then confirmed the key xylose tolerance sites combined with site-directed mutagenesis and determination of xylose tolerance coefficient, so as to analyze a potential mechanism of β-xylosidase Xln-DT. This mechanism may offer a point of departure for the rational design of β-xylosidases aimed at optimizing the commercial applications of these vital enzymes by improving their xylose tolerance or stimulation effects and thermal stability.

Section snippets

Plasmids, bacterial strains and materials

The recombinant plasmid pET-28a-xln-DT was constructed and preserved by Microbial Technology Research Laboratory (College of Chemical Engineering, Nanjing Forestry University, China). The recombinant plasmid pET-28a-xln-DT was used as a template in inverse-PCR reactions [24]. Escherichia coli Top10 F’ was the cloning host and E.coli BL21 (DE3) was the expression host, which were preserved in Microbial Technology Research Laboratory. The bacterial strains were grown overnight at 37 °C in

Structural modeling and molecular docking of xylose and Xln-DT

Based on the functional screening, we cloned and heterologous expressed a xylose-tolerant GH 39 β-xylosidase Xln-DT (GenBank accession No. WP_012547134.1) from D.thermophilum DSM 3960 [24] and a xylose-inhibition GH 3 β-xylosidase Dt-xyl3 (GenBank accession No. ACK42995.1) from D.turgidum DSM 6724 [16] in our previous study, with the Ki values 3394 mM and 20 mM, respectively. Till now, there is no crystal structure for the wildtype GH39 β-xylosidase Xln-DT to evaluate the relationship between

Author contributions

The author contributions were as follow: Qi Li and Linguo Zhao conceived and designed the experiments; Qi Li performed all the experiments and analyzed the data; Xinyi Tong and Yunpeng Jiang helped to perform site-directed mutagenesis experiments; Dongdong Li helped to computer aided design and bioinformatics analysis; Qi Li wrote the paper. All authors have read and approved the manuscript.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Key Research Development Program of China (2017YFD0601001) and Practice and Innovation Training Program for College Students of Nanjing Forestry University (2020NFUSPITP0080).

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    • Transxylosylation of stevioside by a novel GH39 β-xylosidase, and simultaneous valorization of agroindustrial byproducts

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      RcXyl39A showed satisfactory thermostability, since over 70 % remained after 24 h in 50 oC, although over 60 oC the stability of the enzyme is significantly reduced after 4 h (Fig. 4). The thermostability profile of RcXyl39A is not consistent with most polymeric thermostable GH39 β-xylosidases from thermophilic microorganisms, such as CoxylA from Caldicellulosiruptor owensensis (Mi et al., 2014), WSUCF1 from Geobacillus sp. (Bhalla et al., 2014) and Xln-DT from D. thermophilum (Li et al., 2021), which retain their activity over 70 oC for up to several days. However, the thermostability of RcXyl39A resembles the profile shown by XylC from T. saccharolyticum, which drops quickly in temperatures over 65 oC (Shao et al., 2011).

    1

    Qi Li and Xinyi Tong contributed equally to this work.

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