Abstract
Backgroundβ-D-xylosidase is a vital exoglycosidase with the ability to hydrolyze xylooligosaccharides to xylose and to biotransform some saponins by cleaving outer β-xylose. β-D-xylosidase is widely used as one of the xylanolytic enzymes in a diverse range of applications, such as fuel, food and the pharmaceutical industry; therefore, more and more studies have focused on the thermostable and xylose-tolerant β-D-xylosidases.ResultsA thermostable β-xylosidase gene (xln-DT) of 1509 bp was cloned from Dictyoglomus thermophilum and expressed in E.coli BL21. According to the amino acid and phylogeny analyses, the β-xylosidase Xln-DT is a novel β-xylosidase of the GH family 39. The recombinant β-xylosidase was purified, showing unique bands on SDS-PAGE, and had a protein molecular weight of 58.7 kDa. The β-xylosidase Xln-DT showed an optimal activity at pH 6.0 and 75 °C, with p-nitrophenyl-β-D-xylopyranoside (pNPX) as a substrate. Xln-DT displayed stability over a pH range of 4.0-7.5 for 24 h and displayed thermotolerance below 85 °C. The values of the kinetic parameters Km and Vmax for pNPX were 1.66 mM and 78.46 U/mg, respectively. In particular, Xln-DT displayed high tolerance to xylose, with 60% activity in the presence of 3 M xylose. Xln-DT showed significant effects on the hydrolyzation of xylobiose. After 3 h, all the xylobiose tested was degraded into xylose. Moreover, β-xylosidase Xln-DT had a high selectivity for cleaving the outer xylose moieties of natural saponins, such as notoginsenoside R1 and astragaloside IV, which produced the ginsenoside Rg1 with stronger anti-fatigue activity and produced cycloastragenol with stronger anti-aging activity, respectively.ConclusionThis study provides a novel GH 39 β-xylosidase displaying extraordinary properties of highly catalytic activity at temperatures above 75 °C, remarkable hydrolyzing activity of xylooligosaccharides and rare saponins producing ability in the pharmaceutical and commercial industries.
Highlights
More and more studies have focused on cellulose and hemicellulose hydrolysis in an effort to use lignocellulosic residues as feedstock for producing bioethanol [1, 2]
Cloning and sequencing of the β-xylosidase gene xln-DT According to the analysis of the complete genome sequence of Dictyoglomus thermophilum DSM 3960, a protein with possible β-xylosidase activity (GenBank accession No KX449145) encoded 502 amino acids with a full-length of 1509 bp and belonged to Glycosyl hydrolase (GH) 39
Among the β-xylosidases from Clade II (GH 39), the members of the thermophilic genus Dictyoglomus had a distant relationship with Thermoanaerobacterium
Summary
More and more studies have focused on cellulose and hemicellulose hydrolysis in an effort to use lignocellulosic residues as feedstock for producing bioethanol [1, 2]. Ginsenoside Rg1 (Rg1) and cycloastragenol (CA) are the major bioactive constituents in the plants Panax notoginseng and Astragali radix, respectively [18, 19], which have been considered to be remarkable ingredients with pharmacological activities [20, 21]. Conversion from ASI to CA could increase the content of CA efficiently Methods such as acid hydrolysis, heating treatment, steaming treatment, and microbial and enzymatic transformation, have been used to convert these saponins [24,25,26]. Among these methods, enzymatic transformation with β-xylosidase has exhibited potential because of its mild reaction condition and high specificity
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