Bioproduction of quercetin using recombinant thermostable glycosidases from Dictyoglomus thermophilum
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ABSTRACT:
SUBMITTER: Yu S
PROVIDER: S-EPMC10991118 | biostudies-literature | 2022 Apr
REPOSITORIES: biostudies-literature
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Project description: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 K m and V max 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.
| S-EPMC5963010 | biostudies-literature
Project description:Cellulose-based products constitute the great majority of municipal waste, and applications of cellulases in the conversion of waste biomass to biofuels will be a key technology in future biorefineries. Currently, multi-enzymatic pre-treatment of biomass is a crucial step in making carbohydrates more accessible for subsequent fermentation. Using bioinformatics analysis, endo-β-(1,4)-glucanase from Dictyoglomus thermophilum (DtCel5H) was identified as a new member of glycosyl hydrolase family 5. The gene encoding DtCel5H was cloned and the recombinant protein was overexpressed for crystallization and biophysical studies. Here, it is shown that this enzyme is active on cellulose substrates and is highly thermostable. Crystals suitable for crystallographic investigations were also obtained in different crystallization conditions. In particular, ordered crystals of DtCel5H were obtained using either ammonium sulfate or polyethylene glycol (PEG) as a precipitant agent. The crystals obtained in the presence of ammonium sulfate belonged to space group P32, with unit-cell parameters a = 73.1, b = 73.1, 73.1, c = 127.8 Å, and diffracted to 1.5 Å resolution, whereas the second crystal form belonged to the orthorhombic space group P212121, with unit-cell parameters a = 49.3, b = 67.9, c = 103.7 Å, and diffracted to 1.6 Å resolution. The crystal structure was solved in both space groups using molecular-replacement methods. Structure-activity and structure-stability studies of DtCel5H will provide insights for the design of high-performance enzymes.
| S-EPMC5947686 | biostudies-literature
Project description:Here, we present the complete genome of the extreme thermophile, Dictyoglomus thermophilum H-6-12 (phylum Dictyoglomi), which consists of 1,959,987 bp.
| S-EPMC3931368 | biostudies-literature
Project description:Dictyoglomus thermophilum strain:PYS_80_B Genome sequencing and assembly
| PRJNA562146 | ENA
Project description:Isolated from an alkaline hot spring
| PRJNA30731 | ENA
Project description:A two-step PCR protocol was used to identify and sequence a family 11 xylanase gene from Dictyoglomus thermophilum Rt46B.1. Family 11 xylanase consensus fragments (GXCFs) were amplified from Rt46B.1 genomic DNA by using different sets of consensus PCR primers that exhibited broad specificity for conserved motifs within fungal and/or bacterial family 11 xylanase genes. On the basis of the sequences of a representative sample of the GXCFs a single family 11 xylanase gene (xynB) was identified. The entire gene sequence was obtained in the second step by using genomic walking PCR to amplify Rt46B.1 genomic DNA fragments upstream and downstream of the xynB GXCF region. The putative XynB peptide (M(r), 39,800) encoded by the Rt46B.1 xynB open reading frame was a multidomain enzyme comprising an N-terminal catalytic domain (M(r), 22,000) and a possible C-terminal substrate-binding domain (M(r), 13,000) that were separated by a short serine-glycine-rich 23-amino-acid linker peptide. Seven xylanases which differed at their N and C termini were produced from different xynB expression plasmids. All seven xylanases exhibited optimum activity at pH 6.5. However, the temperature optima of the XynB xylanases varied from 70 to 85 degrees C. Pretreatment of Pinus radiata and eucalypt kraft-oxygen pulps with XynB resulted in moderate xylan solubilization and a substantial improvement in the bleachability of these pulps.
| S-EPMC106227 | biostudies-literature
Project description:Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 β-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75°C, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 β-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.
| S-EPMC9628812 | biostudies-literature
Project description:The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax. The recombinant and native enzymes share a high degree of similarity for most properties examined.
| S-EPMC94644 | biostudies-literature
Project description:Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.
| S-EPMC6429052 | biostudies-literature