Dataset Information

Biochemical Characterization of the α-l-Rhamnosidase DtRha from Dictyoglomus thermophilum: Application to the Selective Derhamnosylation of Natural Flavonoids.

ABSTRACT: α-l-Rhamnosidases are catalysts of industrial tremendous interest, but their uses are still somewhat limited by their poor thermal stabilities and selectivities. The thermophilic DtRha from Dictyoglomus thermophilum was cloned, and the recombinant protein was easily purified to homogeneity to afford 4.5 mg/L culture of biocatalyst. Michaelis-Menten parameters demonstrated it to be fully specific for α-l-rhamnose. Most significantly, DtRha demonstrated to have a stronger preference for α(1 → 2) linkage rather than α(1 → 6) linkage when removing rhamnosyl moiety from natural flavonoids. This selectivity was fully explained by the difference of binding of the corresponding substrates in the active site of the protein.

SUBMITTER: Guillotin L

PROVIDER: S-EPMC6649072 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Biochemical Characterization of the α-l-Rhamnosidase DtRha from Dictyoglomus thermophilum: Application to the Selective Derhamnosylation of Natural Flavonoids.

Guillotin Laure L Kim Hyuna H Traore Yasmina Y Moreau Philippe P Lafite Pierre P Coquoin Véronique V Nuccio Sylvie S de Vaumas René R Daniellou Richard R

ACS omega 20190124 1

α-l-Rhamnosidases are catalysts of industrial tremendous interest, but their uses are still somewhat limited by their poor thermal stabilities and selectivities. The thermophilic DtRha from Dictyoglomus thermophilum was cloned, and the recombinant protein was easily purified to homogeneity to afford 4.5 mg/L culture of biocatalyst. Michaelis-Menten parameters demonstrated it to be fully specific for α-l-rhamnose. Most significantly, DtRha demonstrated to have a stronger pref ...[more]

PMID: 31459445

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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.

Dataset Information

Biochemical Characterization of the α-l-Rhamnosidase DtRha from Dictyoglomus thermophilum: Application to the Selective Derhamnosylation of Natural Flavonoids.

Publications

Biochemical Characterization of the α-l-Rhamnosidase <i>Dt</i>Rha from <i>Dictyoglomus thermophilum</i>: Application to the Selective Derhamnosylation of Natural Flavonoids.

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