Expression, purification, crystallization and preliminary crystallographic analysis of an endo-1,5-α-<scp>L</scp>-arabinanase from hyperthermophilic<i>Thermotoga petrophila</i>

Squina, Fábio M.; Prade, Rolf A.; Wang, Hongliang; Murakami, Mário Tyago

doi:10.1107/s1744309109029844

Cited by 11 publications

(9 citation statements)

References 19 publications

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“…However, it revealed a close relationship with T. thermarum and T. petrophila , indicating that they share the similar properties [1,17]. Other endo-arabinanases from genus Thermotoga including T. thermarum endo-arabinanase have not yet been studied.…”

Section: Discussionmentioning

confidence: 99%

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Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum

et al. 2014

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BackgroundArabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated.ResultsThe recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent Km and Vmax values were determined to be 12.3 ± 0.15 mg ml−1 and 1,052.1 ± 12.7 μmol ml−1 min−1, respectively (at pH 6.5, 75°C), and the calculated kcat value was 349.3 ± 4.2 s−1.ConclusionsThis work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.

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Section: Discussionmentioning

confidence: 99%

“…Only a few endo-arabinanases especially that from hyperthermophile have been reported to possess the function in generating arabino-oligosaccharides [1,17]. Although it was insensitive to the branched arabinan (eg.…”

Section: Discussionmentioning

confidence: 99%

Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum

et al. 2014

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“…Cloning, expression and purification of the TpBGL1 (GenBank: ABQ46970.1) and TpBGL3 (GenBank: ABQ46916.1) were carried out as described previously (Cota et al 2015;Squina et al 2009). The purity of the final products, TpBGL1 and TpBGL3, were checked by Coomassie-stained 15 % SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

et al. 2015

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The β-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable β-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the β-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two β-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable β-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

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“…Thus, to shed light on the structural determinants for the distinct molecular mechanisms governing the catalysis of this important family of glycosidases, we have investigated three GH43 ABNs: two endo-acting ABNs, TpABN and ARN2, which are two-domain enzymes, from Thermotoga petrophila RKU-1 (23)(24) and from a bovine rumen metagenomic library (22), respectively; and, a single-domain exo-ABN (ARN3) recovered from the same metagenomic DNA library (18). Biochemical and biophysical studies with TpABN contributed to elucidate the structural basis for calcium dependence of some GH43 ABNs and the role of the ancestral ancillary module for function and stability of two-domain ABNs.…”

Section: Endo-15-␣-l-arabinanases (Abns)mentioning

confidence: 99%

Mechanistic Strategies for Catalysis Adopted by Evolutionary Distinct Family 43 Arabinanases

Santos

Polo

Costa

et al. 2014

Journal of Biological Chemistry

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Background: Arabinanases are key enzymes involved in hemicellulose degradation. Results: Crystallographic, mutational, and biochemical assays of three arabinanases reveal the molecular mechanisms governing their catalysis and activation. Conclusion: Accessory domain and metal ion are essential for catalysis. Structural adaptations in the catalytic interface confer unique action modes to ruminal arabinanases. Significance: This work provides new molecular strategies for arabinan hydrolysis.

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Expression, purification, crystallization and preliminary crystallographic analysis of an endo-1,5-α-L-arabinanase from hyperthermophilicThermotoga petrophila

Cited by 11 publications

References 19 publications

Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum

Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

Mechanistic Strategies for Catalysis Adopted by Evolutionary Distinct Family 43 Arabinanases

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