Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus

Corrêa, Juliana Moço; Graciano, Luciana; Abrahão, Josielle; Loth, Eduardo Alexandre; Gandra, Rinaldo Ferreira; Kadowaki, Marina Kimiko; Henn, Caroline; Simão, Rita de Cássia Garcia

doi:10.1007/s12010-012-9931-1

Cited by 32 publications

(14 citation statements)

References 21 publications

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“…Many xylanases have been previously isolated and characterized from different strains of bacteria and fungi [ 11 – 13 ]; β-xylosidases and the co-activity of two enzymes on xylan hydrolysis however have been described to a lesser degree [ 14 – 16 ]. In this study, we have cloned two genes coding for endo-xylanase and β-xylosidase from a recently-isolated strain Bacillus subtilis M015 and strategically expressed them in a periplasmic-leaky Escherichia coli strain which resulted in successful secretion of the recombinant proteins outside of the cells at high levels.…”

Section: Introductionmentioning

confidence: 99%

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

Banka

Guralp

Gülari

2014

Appl Biochem Biotechnol

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Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-β-xylanase and β-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and β-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for β-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5–6 for xylanase and 35 °C and 7.0–7.5 for β-xylosidase using beech wood xylan and p-nitrophenyl-β-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/β-xylosidase combination as opposed to xylanase alone.

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Section: Introductionmentioning

confidence: 99%

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

Banka

Guralp

Gülari

2014

Appl Biochem Biotechnol

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“…In Caulobacter sp. two groups of polysaccharide-hydrolyzing enzymes were reported to date: the cellulases including endoglucanase, exoglucanase and β-(1 → 4)-glucosidase (Song et al 2013), and the β-xylosidases, xylosidase I and II (Graciano et al 2012; Corrêa et al 2012). In addition, the maltodextrin transport protein, MalA ( cc2287 gene product), was induced by either maltose or maltodextrins, and transported maltodextrins ranging from maltose to maltopentaose (Neugebauer et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification

et al. 2014

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The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusion protein with Staphylococcus Protein A. The fusion protein was purified using an IgG Sepharose column and was identified as the active GA. The optimum temperature and pH for the activity of this GA toward maltotriose as a substrate were approximately 40°C and 5.0, respectively, and a differential scanning fluorimetry (DSF) analysis revealed that the melting temperature (Tm) of CauloGA was 42.9°C. The kinetic analyses with maltotriose and other maltodextrins as the substrates indicated that CauloGA has higher kcat and smaller Km values at 30°C with both substrates compared with other GAs at lower substrate concentration. However, the enzyme activities toward the substrates decreased as the substrate concentrations increased at concentrations higher than approximately 10-fold the Km. The function-based identification of thermolabile Caulobacter GA contributes to the understanding of the maltodextrin-degradation system of C. crescentus as well as the bacterial GA’s function-structure relationship.

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“…The synergistic ability to hydrolyze xylan (1%, w/v) and sugarcane bagasse (1%, w/v) with enzyme purified fungal xylanase and purified bacterial β-xylosidase to measure the contribution of β-xylosidase in the process of deconstructing hemicellulose, and they showed increased production of reducing sugars with the purified enzymes as compared to the amount produced with pre-hydrolysis by xylanase. 15 In other study with recombinant proteins β-xylosidase and xylanase purified from Humicola insolens expressed in E. coli to hydrolyze xylan from birchwood and beechwood at 37 °C, which resulted in saccharification increases of 1 and 1.29 times, respectively, indicating the synergistic action of these enzymes. 16 …”

Section: Discussionmentioning

confidence: 93%

High levels of β-xylosidase in Thermomyces lanuginosus : potential use for saccharification

Corrêa¹,

Christi²,

Torre

et al. 2016

Brazilian Journal of Microbiology

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A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

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Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus

Cited by 32 publications

References 21 publications

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification

High levels of β-xylosidase in Thermomyces lanuginosus : potential use for saccharification

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