High-level expression of a novel multifunctional GH3 family β-xylosidase/α-arabinosidase/β-glucosidase from Dictyoglomus turgidum in Escherichia coli

https://doi.org/10.1016/j.bioorg.2021.104906Get rights and content

Highlights

  • β-xylosidase was produced to highest yield by recombinant E. coli in shake flask.

  • A novel multifunctional glucoside hydrolase was cloned and over-expressed in E. coli.

  • Dt-2286 shows great thermostable/haloduric/organic solvent-tolerance.

  • Dt-2286 showed significant synergistic effects on the degradation of xylans.

  • Dt-2286 also has a good application in the hydrolysis of natural products.

Abstract

A novel β-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of β-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities, and Kcat/Km was 5245.316 mM−1 s−1, 2077.353 mM−1 s−1, 1626.454 mM−1 s−1, and 470.432 mM−1 s−1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.

Introduction

In recent years, with the continuous exploitation of fossil fuels and the global warming of the climate, people have become increasingly interested in using renewable energy sources, such as solar, hydro, wind and biomass energy [1]. Among them, the production of biofuels from lignocellulose has aroused great interest [2]. Lignocellulose is an organic flocculent fibrous material obtained from natural and renewable wood by chemical and mechanical processing and it is mainly comprised of cellulose, hemicellulose and lignin, which are arranged in a complex three-dimensional matrix [3]. Xylan is the main component of hemicellulose, which is mainly heteropolysaccharide and it can be modified by the attachment of l-arabinofuranose, 4-O-methyl-d-glucuronic acid, p-cumaric acid and ferrulic acid residues in nature [4].

Due to the structural complexity of xylan, complete degradation of xylan to achieve biotransformation requires a complex hemicellulase system, including endoxylanases, β-d-xylosidases, α-l-arabinosidases and β-d-glucosidases [5]. Among them, endoxylanase is used to hydrolyze the main chain and randomly split the main chain into xylo-oligosaccharides [6]; α-l-arabinosidases and β-d-glucosidases can remove the side chains of the main chain and β-d-xylosidase hydrolyzes xylooligosaccharide into monosaccharides [6]. Therefore, it is of great value to search for glycoside hydrolases with simultaneous activities of arabinase, xylosidase and glucosidase. Glycoside hydrolase family 3 (GH3) is the main GH family of exoacting β-d-glucosidases, α-l-arabinofuranosidases, and β-d-xylosidases (www.cazy.org).

Because of the complex three-dimensional structure of lignocellulose, different pretreatment processes are usually required to improve the hydrolysis efficiency of hydrolases and nonhydrolases [6]. However, after pretreatment, some organic reagents and salts would remain or accumulate in the biomass, which would lead to inhibition of subsequent enzymatic hydrolysis [7]. Therefore, the glycoside hydrolases used should have high tolerance to organic solvents and salts.

In addition, in recent years, the application of glucosidolytic enzymes has not only been limited to the hydrolysis of xylan, but has also been applied to the conversion of bioactive natural products with glycoside bonds [8], [9]. Compared with traditional chemical catalysts, biocatalysts are more specific and environmentally friendly [8], [9], [10], [11]. However, glucoside hydrolase produced by microorganisms is often unable to be put into industrial production due to its low expression level [12].

Baohuoside Ⅰ and sagittatoside B are flavonoids from epimedium [13]. Baohuoside I is a potential antitumor drug that can be used to treat a variety of malignancies and osteoporosis and has been approved for clinical use [14], [15], [16]. However, due to its low content in plants and low extraction and separation rate, it is too expensive to be routinely used in clinical practice [17]. Sagittatoside B is also a rare and active substance produced by epimedium. Because of its low content and high price, few people have studied the activity of this substance. Fortunately, epimedin B is a major flavonoid compound from epimedium, which has a low separation cost and is the precursor of baohuoside Ⅰ and sagittatoside B [17]. One glucose is removed from epimedium B to obtain sagittatoside B, and another xylose is removed to obtain baohuoside Ⅰ. If an enzyme could be found to hydrolyze xylose and glucose on epimedium B, these useful compounds could be produced in abundance.

In this study, we cloned and characterized a novel thermostable/haloduric/ organic solvent-tolerance multifunctional GH3 β-xylosidase from Dictyoglomus turgidum. This enzyme was highly expressed in E. coli, showed high activity in the hydrolysis of poplar xylan, and displayed highly selective hydrolysis performance in removing xylose and glucose groups from epimedium B.

Section snippets

Reagents, strains and medium

All artificial substrates used for enzyme characterization were purchased from Sigma-Aldrich (St Louis, MO, USA), including p-nitrophenyl-α-l-rhamnopyranoside (pNPR), p-nitrophenyl-β-d-glucopyranoside (pNPGlu), pNP-α-l-arabinofuranoside (pNPArf), pNP-α-l-arabinopyranoside (pNPArp) , pNP-β-d-xylopyranoside (pNPX) and p-nitrophenol (pNP).

Genomic DNA from Dictyoglomus turgidum DSM6724, which was grown as described (Takahata et al., 2001), was purchased from DSMZ (www.dsmz.de). Escherichia coli

Sequence analysis and phylogenic analysis

According to the analysis of the whole genome sequence of D. turgidum DSM 6724, it was speculated that it might have β-xylosidase/α-arabinose/β-glucosidase activity (GenBank accession No. ACK42133.1). The Dt-2286 gene fragment was amplified from the genomic DNA of D. turgidum DSM 6724. The full-length gene Dt-2286 encodes a polypeptide of 762 amino acid residues with a predicted molecular weight of 85.1 kDa.

Dt-2286 and other β-xylosidase from organisms isolated from other environments and

Conclusion

In this paper, a rare GH family 3 multifunctional β-xylosidase gene (Dt-2286) from D. turgidum was cloned and expressed in E. coli BL21 (DE3). The expression of recombinant Dt-2286 in E. coli BL21 was 270 U/mL in TB medium. To the best of our knowledge, this level of β-xylosidase production in E. coli in shake flask is the highest ever reported. The thermostable/haloduric/organic solvent-tolerance characteristics of Dt-2286 make it possible to be used in practical industry. The hydrolysis of

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key Research and Development Program of China National Key R&D Program of China (2017YFD0601001).

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    Xinyi Tong and Zhipeng Qi contributed equally to this work.

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