Cloning, Expression, and Characterization of a Family 52 β-Xylosidase Gene (xysB) of a Multiple-xylanase-producing Bacterium, Aeromonas caviae ME-1

Suzuki, Tomohiko; Kitagawa, Emiko; Sakakibara, Fukumitsu; Ibata, Keiji; Usui, Kengo; Kawai, Kazuhiro

doi:10.1271/bbb.65.487

Cited by 27 publications

(8 citation statements)

References 14 publications

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“…As with other β-D-xylosidases, XynB2 also hydrolyzes 4-nitrophenyl R-L-arabinofuranoside (k cat ) 0.11 s -1 ) although with very low catalytic efficiency (measured by relative k cat /K m values). Another family 52 β-xylosidase from A. caVie ME-1 also exhibited very low R-L-arabinofuranosidase activity (28). Interestingly, XynB2 also hydrolyzes 4-nitrophenyl R-L-arabinopyranoside.…”

Section: Resultsmentioning

confidence: 99%

Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A β-Xylosidase from Geobacillus stearothermophilus

et al. 2003

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Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Brønsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.

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Section: Resultsmentioning

confidence: 99%

Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A β-Xylosidase from Geobacillus stearothermophilus

et al. 2003

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“…Before the discovery of the RUM630-AG enzyme, all reported a-glucuronidase genes had been isolated from individually cultured microorganisms using a variety of methods. Some were discovered because the genes were on the same genomic fragments as other hemicellulase genes that were being targeted [28,32].…”

Section: Discussionmentioning

confidence: 99%

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

et al. 2012

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α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.

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“…The α-glucuronidase genes that are currently known were isolated by several methods. Genes were discovered serendipitously because they were located on the same genomic DNA fragment as another hemicelluloytic gene that was the original target [29,30]. Other α-glucuronidase genes have been tentatively identified based on homology to data from microbial genome sequencing databases.…”

Section: Discussionmentioning

confidence: 99%

An α-Glucuronidase Enzyme Activity Assay Adaptable for Solid Phase Screening

Lee

Wagschal

Kibblewhite-Accinelli

et al. 2008

Appl Biochem Biotechnol

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Glucuronic acid is a common chemical moiety that decorates the xylan polymer of hemicellulose. This chemical substituent impairs both enzymatic and acidic hydrolysis of xylosidic bonds. The alpha-glucuronidase enzyme hydrolyzes the 1,2-linked glucuronic acid from the terminal, non-reducing xylose of xylo-oligosaccharides. There are relatively few alpha-glucuronidase genes in the public databases. We have developed an assay with commercially available reagents that can be used to search DNA libraries for alpha-glucuronidase genes in a high-throughput, solid phase activity screen.

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Cloning, Expression, and Characterization of a Family 52 β-Xylosidase Gene (xysB) of a Multiple-xylanase-producing Bacterium, Aeromonas caviae ME-1

Cited by 27 publications

References 14 publications

Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A β-Xylosidase from Geobacillus stearothermophilus

Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A β-Xylosidase from Geobacillus stearothermophilus

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

An α-Glucuronidase Enzyme Activity Assay Adaptable for Solid Phase Screening

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