β-d-Xylosidase From Selenomonas ruminantium of Glycoside Hydrolase Family 43

Jordan, Douglas B.; Li, Xinliang; Dunlap, Christopher A.; Whitehead, Terence R.; Cotta, Michael A.

doi:10.1007/978-1-60327-181-3_9

Cited by 4 publications

(7 citation statements)

References 23 publications

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“…The GH43 xylosidases of Enterobacter sp. [33], Lactobacillus brevis [34], Lactobacillus rossiae DSM 15814 T [35], and Penicillium purpurogenum [36] have an optimum pH and temperature of approximately pH 6 and 40 °C, respectively, while the Selenomonas ruminantium enzyme has an optimal temperature of 40 °C but a lower optimum pH of 5.3 [37].…”

Section: General Characterization Of Rswu43amentioning

confidence: 99%

Identification and characterization of the first β-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10

Phuengmaung

Fujiwara

Sukhumsirichart

et al. 2018

Enzyme and Microbial Technology

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In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a β-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40°C. The enzyme was stable up to 40°C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe and Mn and inhibited by various metals (Ag, Cd, Co, Cu, Hg, Ni, and Zn), d-xylose, and l-arabinose. rSWU43A showed activity on p-nitrophenyl-β-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside substrates, with specific activities of 0.09 and 0.06U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded β-1,3-xylooligosaccharides to produce xylose but showed little activity towards β-1,4-xylobiose, with specific activities of 1.33 and 0.003U/mg, respectively. These results demonstrate that SWU43A is a β-1,3-d-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first β-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from β-1,3-xylan in seaweed cell walls.

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Section: General Characterization Of Rswu43amentioning

confidence: 99%

Identification and characterization of the first β-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10

Phuengmaung

Fujiwara

Sukhumsirichart

et al. 2018

Enzyme and Microbial Technology

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“…Size-exclusion chromatography of the two proteins (Fig. 3d) determined Stokes radii of 54.8 ± 0.8 Å (W145P) and 56.0 ± 0.8 Å (wild-type SXA), expected values for the SXA tetramer [15], thus rejecting the hypothesis that a perturbed quaternary structure provides an explanation for the cooperative binding kinetics of W145P.…”

Section: Resultsmentioning

confidence: 85%

“…Substrates, buffers, and other reagents [7,17,21,22], circular dichroism spectroscopy [17], HPLC with pulsed amperometric detection [19], isothermal calorimetry [21], SDS-PAGE analysis [15], UV-V is spectroscopy [17], and molecular modeling [7] have been described. D-Xylobiose (X2) was obtained from Wako Chemicals USA (Richmond, VA).…”

Section: Materials and General Methodsmentioning

confidence: 99%

“…Size-exclusion chromatography was used to determine the Stokes radii of the SXA-W145P variant and the wild-type SXA by using similar methods and the same chromatography column described previously [15]. Briefly, the column (2.6 9 61 cm) was packed with Toyo-Pearl 55F resin equilibrated with 10 mM sodium phosphate, 75 mM NaCl, pH 7.0 at 24-26°C with a flow rate of 1.0 ml/min.…”

Section: Stokes Radius Determinationsmentioning

confidence: 99%

“…Its k cat and k cat /K m are at least tenfold greater than those reported for other b-xylosidases [16,19,23] when acting on oligoxylosides of increasing degrees of polymerization from xylobiose (DP2) to xylohexaose (DP6). Its strength of catalysis [16,19,23], good stability versus moderately low pH (stable above pH 4.0) and temperature (stable below 50°C) [15], and ease of protein production in Escherichia coli (30% of soluble cell protein is SXA with greater than 5 g SXA produced per liter of culture under nonoptimized conditions) [14] recommend employment of SXA in industrial processes where it would serve in the hydrolysis of herbaceous biomass (xylan fraction) to simple sugars for fermentation to fuel ethanol and other bioproducts [11,33]. SXA belongs to glycoside hydrolase family 43 (GH43) and structural clan F of the CAZy database (Carbohydrate Active Enzymes database, http://www.cazy.org/) [4,12].…”

Section: Introductionmentioning

confidence: 99%

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Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

Jordan

Wagschal

Fan

et al. 2011

J Ind Microbiol Biotechnol

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β-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-β-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (K (i) ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K(i)(D-glucose) and K(i)(D-xylose) twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k(cat)(D-xylobiose) and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K (i) values that are 1.5-2.7-fold (D-glucose) and 1.9-4.6-fold (D-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k(cat)(D-xylobiose) than that of the wild type. At 0.1 M D-xylobiose and 0.1 M D-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.

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β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis

Terrasan

Aragon

Masui

et al. 2016

Biocatalysis and Biotransformation

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The tetrameric b-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehydedextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50 C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0-6.0 and at 55 C. The catalyst retained its side activity against p-nitrophenyl a-Larabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl b-D-xylopyranoside as substrate were V max 0.20 mmol.min À1 mg prot. À1 , K m 0.45 mM, K cat 0.82 s À1 , and K cat /K m 1.82 s À1 mM À1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chainlength. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.

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β-d-Xylosidase From Selenomonas ruminantium of Glycoside Hydrolase Family 43

Cited by 4 publications

References 23 publications

Identification and characterization of the first β-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10

Identification and characterization of the first β-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10

Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis

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