Research Progress Concerning Fungal and Bacterial ß-Xylosidases.

In the present review, we briefly summarize the biotechnological applications of microbial ß-xylosidases in the processing of agro-industrial residues into fuels and chemicals and report the importance of using immobilization techniques to study the enzyme. The advantages of utilizing genes that encode ß-xylosidases are readily apparent in the bioconversion of abundant, inexpensive, and renewable resources into economically important products, such as xylitol and bioethanol. We highlight recent research characterizing fungal and bacterial ß-xylosidases, including the use of classical biochemical methods such as purification, heterologous recombinant protein expression, and metagenomic approaches to discovery ß-xylosidases, with focus on enzyme molecular and kinetic properties. In addition, we discuss the relevance of using experimental design optimization methodologies to increase the efficacy of these enzymes for use with residual biomass. Finally, we emphasize more extensively the advances in the regulatory mechanisms governing ß-xylosidase gene expression and xylose metabolism in gram-negative and gram-positive bacteria and fungi. Unlike previous reviews, this revision covers recent research concerning the various features of bacterial and fungal ß-xylosidases with a greater emphasis on their biochemical characteristics and how the genes that encode these enzymes can be better exploited to obtain products of biotechnological interest via the application of different technical approaches.

Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus.

Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for ß-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, ß-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 µM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

Expression and characterization of a GH39 ß-xylosidase II from Caulobacter crescentus.

In the present work, the gene xynB2, encoding a ß-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ß-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K (M) and V (Max) values were 9.3 ± 0.45 mM and 402 ± 19 µmol min(-1) for ρ-nitrophenyl-ß-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ß-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus ß-Xylosidase I.

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved ß-Xylosidase and α-L-Arabinofuranosidase (ß-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant ß-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant ß-Xyl I-α-L-Ara exhibited a specific ß-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its ß-Xylosidase I activity, and the enzymatic characterization was focused on it. The ß-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, ß-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 µM min(-1) to oNPX, respectively. The modeled structure of ß-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.