A Chromogenic Substrate for a β-Xylosidase-Coupled Assay of α-Glucuronidase

Biely, Peter; Hirsch, Ján; Grange, Daniël C. la; Zyl, Willem H. van; Prior, Bernard A.

doi:10.1006/abio.2000.4810

Cited by 43 publications

(31 citation statements)

References 31 publications

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“…strain JDR-2 have been identified in several other bacteria (G. stearothermophilus, T. maritima, and Bacillus clausii). The enzymatic activity of AguA with MeGAX 3 as the substrate shares properties with the activities of other GH67 ␣-glucuronidases (4,7,21,27,29) and supports the unique role proposed for extracellular GH10 endoxylanases, in which MeGAX 3 is generated along with xylooligosaccharides, all of which are assimilated and metabolized (3,24).…”

Section: Discussionsupporting

confidence: 66%

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism

Nong

Rice

Chow

et al. 2009

Appl Environ Microbiol

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Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 ␣-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/␤-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX 1 ), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX 3 ), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX n ). The average rates of utilization of MeGAX n , MeGAX 1 , and MeGAX 3 were 149.8, 59.4, and 54.3 g xylose equivalents ⅐ ml ؊1 ⅐ h ؊1 , respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX 1 and MeGAX 3 , releasing 4-O-methyl-D-glucuronate ␣-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX 3 , to xylose and xylobiose. The ability to utilize MeGAX 1 provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX n than that of MeGAX 3 indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

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Section: Discussionsupporting

confidence: 66%

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism

Nong

Rice

Chow

et al. 2009

Appl Environ Microbiol

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“…␣-Glucuronidase activity was measured following the method of Biely et al (11). Briefly 4-nitrophenyl-2-O-(4-O-methyl-␣-D-glucuronosyl)-␤-D-xylopyranoside, at concentrations ranging from 20 M to 15 mM was incubated with an appropriate concentration of native or mutant GlcA67A in 50 mM sodium phosphate buffer, pH 7.0, containing 1 mg/ml bovine serum albumen and 60 g of the Thermoanaerobacterium saccharolyticum GH39 ␤-xylosidase, at 37°C in a total volume of 500 l. GlcA67A activity was determined by measuring the rate of release of 4-nitrophenol spectrophotometrically at 400 nm, using the molar extinction coefficient for 4-nitrophenol of 10,300 at pH 7.0 (12).…”

Section: Methodsmentioning

confidence: 99%

The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

Nagy

Nurizzo

Davies

et al. 2003

Journal of Biological Chemistry

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“…Xylanolytic bacteria secrete endoxylanases of the glycohydrolase families GH5, GH10, and GH11 that catalyze the depolymerization of the xylan backbone with the generation of different products (4,18). The GH10 endoxylanases generate xylobiose, xylotriose, and the aldotetrauronate, methylglucuronoxylotriose (MeGAX 3 ), in which ␤-1,4-linked D-xylotriose is substituted at the nonreducing terminus with ␣-1,2-linked 4-O-methyl-D-glucuronate.…”

mentioning

confidence: 99%

Structure, Function, and Regulation of the Aldouronate Utilization Gene Cluster from Paenibacillus sp. Strain JDR-2

2007

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Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX n ), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX n and rapidly assimilates the principal products, ␤-1,4-xylobiose, ␤-1,4-xylotriose, and MeGAX 3 , the aldotetrauronate 4-O-methylglucuronosyl-␣-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 ␣-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 ␤-xylosidase/␣-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.

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A Chromogenic Substrate for a β-Xylosidase-Coupled Assay of α-Glucuronidase

Cited by 43 publications

References 31 publications

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism

The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

Structure, Function, and Regulation of the Aldouronate Utilization Gene Cluster from Paenibacillus sp. Strain JDR-2

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