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β-Xylosidases (EC 188.8.131.52) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.
Xylan is the most abundant hemicellulosic polysaccharide found in plant cell walls and consists of a main chain of β-1,4-linked xylosyl residues, which can be further substituted at the 2-OH and 3-OH positions with a variety of side groups such as arabinose, acetic acid, d-glucuronic acid, p-coumaric acid and ferulic acid (Rennie & Scheller, 2014 ).
The depolymerization of xylan to free xylose has a great impact in the production of biofuels by pentose-fermenting microorganisms (Dodd & Cann, 2009 ) and in a number of other industrial processes such as the paper, animal-feed and food industries (Zhao et al., 2013 ; Driss et al., 2014 ; Valenzuela et al., 2014 ). The complete enzymatic hydrolysis of xylan requires the synergistic action of several enzymes to cope with the distinct chemical linkages and side groups. For the xylan backbone in particular, endo-β-1,4-xylanases (E.C. 184.108.40.206) are required to cleave internal β-1,4-glycosidic bonds, generating xylooligosaccharides, and β-xylosidases are required to catalyze the breakdown of short xylooligosaccharides into xylose.
These enzymes also complement the activity of cellulases, improving the lignocellulose hydrolysis efficiency (Hu et al., 2011 ; Gao et al., 2011 ) and consequently contributing to C6-fermenting technologies. β-Xylosidases, in particular, have an important role in alleviating the inhibition of cellulases by xylooligosaccharides during biomass degradation. On the other hand, analogously to β-glucosidases, β-xylosidases are also competitively inhibited by high concentrations of free xylose in the course of enzymatic hydrolysis. Therefore, for industrial applications it is of great interest to have β-xylosidases with a high tolerance to xylose (Yan et al., 2008 ; Shi et al., 2013 ) and with a stability compatible with the harsh conditions typically found in biotechnological processes involving biomass deconstruction (Bhalla et al., 2013 ).
These enzymes have been identified, purified and characterized from a number of microorganisms, including actinomycetes, eubacteria and fungi (Holtz et al., 1991 ; Collins et al., 2005 ; Polizeli et al., 2005 ), and they have been found in GH families 1, 3, 30, 31, 39, 43, 51, 52, 54, 116 and 120 (Cantarel et al., 2009 ). Despite the large expansion of characterized enzymes with this activity, the structural requirements for xylooligosaccharide specificity among the different GH families are still not fully understood and neither is the molecular basis for xylose tolerance. In this study, we report the cloning, overexpression, purification, crystallization and preliminary X-ray analysis of a β-xylosidase belonging to the GH43 family from the bacterium Bacillus licheniformis (BlXylA). This enzyme shares approximately 45% sequence similarity with other GH43 β-xylosidases; however, the loops that are considered to be important for substrate binding are not conserved in BlXylA, indicating a distinct functional behaviour, which is being investigated along with this structural characterization.
The open reading frame (ORF) encoding a β-xylosidase belonging to the GH43 family (accession code WP_011197672; Table 1 ) was amplified from the genomic DNA of B. licheniformis by touchdown PCR using Phusion DNA polymerase (Thermo Scientific). The amplified ORF was cloned into the pGEM-T Easy vector (Promega) and further subcloned into the pET-28a bacterial expression vector between the NheI and EcoRI restriction sites. The resulting construct consisted of the full-length BsXylA sequence with an N-terminal hexahistidine tag.
The recombinant plasmid BlXylA-pET-28a was transformed into Escherichia coli strain BL21(DE3) and the cells were grown in selective Luria broth (LB) medium at 310 K and 250 rev min−1 to an A 600 of ~0.8. Expression was then induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 310 K for 4 h. The cells were harvested by centrifugation for 30 min at 8000g and resuspended in lysis buffer [50 mM phosphate pH 7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mg ml−1 lysozyme]. The cell suspension was lysed by sonication and the supernatant was separated by centrifugation for 30 min at 277 K and 12 000g.
The clarified supernatant was subjected to metal-affinity chromatography using a 5 ml HisTrap colunm (GE Healthcare) pre-equilibrated with 50 mM phosphate pH 7.4, 150 mM NaCl, 20 mM imidazole. The bound proteins were eluted using a 0–0.5 M imidazole gradient. Fractions containing the target protein were pooled and concentrated to a final volume of 2 ml for subsequent size-exclusion chromatography on a Superdex 200 column which had been equilibrated with 50 mM phosphate pH 7.4, 150 mM NaCl.
The purified BlXylA was concentrated to 15 mg ml−1 using Amicon centrifugal filter units (Millipore) for crystallization experiments. The protein concentration was determined by absorption (A 280 nm) using a Nanodrop spectrophotometer (Thermo Scientific) and a protein molar extinction coefficient of 119 220 M −1 cm−1.
Sample monodispersity was checked by dynamic light scattering using a Zetasizer Nano Series (Malvern Instruments Ltd) device. 20 measurements were taken at 291 K and the hydrodynamic parameters were determined using the Zetasizer Software v.6.32 (Malvern Instruments Ltd). The hydrodynamic radius (R h) was extrapolated from the translational diffusion coefficient (D t) using the Stokes–Einstein equation.
Initial screening was performed with approximately 600 different crystallization conditions based on the commercial kits Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard I and II (Emerald Bio), JCSG+ (Emerald Bio), Precipitant Synergy (Emerald Bio), PACT (Qiagen) and SaltRx (Hampton Research). The sitting-drop experiments were prepared with a Cartesian Honeybee 963 robot (Genomic Solutions) and were systematically analyzed with the assistance of a Rock Imager 1000 automated imaging system (Formulatrix). A single condition [40%(v/v) 2-methyl-2,4-pentanediol, 5%(w/v) polyethylene glycol 8000, 0.1 M sodium cacodylate pH 6.5] yielded small crystals after 10 d. Crystals with adequate dimensions for X-ray diffraction analysis on the MX2 beamline at LNLS, Campinas, Brazil were obtained by reducing the precipitant to 36% in steps of 0.5%(v/v) (Table 2 ).
For X-ray diffraction data collection, a BlXylA crystal was soaked in a cryoprotectant solution consisting of the reservoir solution supplemented with 20%(v/v) glycerol and was further flash-cooled to 100 K in a nitrogen-gas stream. Shutterless data collection was performed with a single-photon-counting hybrid pixel detector using the fine ϕ-slicing strategy to optimal data quality. The data were indexed, integrated and scaled using the XDS package (Kabsch, 2010a ,b ) taking into account the CC1/2 criterion (Karplus & Diederichs, 2012 ) for high-resolution data cutoff. Additional data-collection information and processing statistics are summarized in Table 3 .
The recombinant full-length BlXylA with an N-terminal His6 tag was purified to homogeneity by two chromatographic steps (Fig. 1 ), yielding approximately 5 mg target protein per litre of expression medium. The few contaminants that remained from Ni2+-chelating affinity chromatography (Fig. 1 a) were successfully removed by a gel-filtration purification step (Fig. 1 b), which improved the reproducibility of BLXylA crystallization. The purified protein remained monodisperse in the crystallization concentration (15 mg ml−1) as assessed by dynamic light scattering (R h = 4.3 nm and polydispersity of 25%) and only crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol. Despite the imperfection of the crystal surface as can be observed in the microphotograph (Fig. 2 ), the crystal diffracted to 2.49 Å resolution and yielded an interpretable diffraction pattern (Fig. 3 ). The crystal belonged to the base-centred monoclinic space group C2 characterized by β = 91.72°. The calculated Matthews coefficient of 1.90 Å3 Da−1 corresponds to a solvent content of 35.41% (Matthews, 1968 ), assuming the molecular weight of the recombinant BlXylA to be 60 387 Da. This analysis statistically confirms the presence of one protomer in the asymmetric unit. BlXylA shares a maximum sequence identity of 45% with homologous β-xylosidases belonging to the GH43 family, and molecular-replacement calculations resulted in an initial model with an R factor of 0.40 and an R free of 0.46, which were further improved by isotropic restrained refinement to an R factor of 0.34 and an R free of 0.39, indicating it to be a correct solution. The structural characterization of this enzyme combined with site-directed mutagenesis and functional studies will shed light on the molecular requirements for xylooligosaccharide specificity within the enigmatic GH43 family, which have great biotechnological relevance in the conversion of lignocellulosic materials into biofuels and other chemicals.
This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (MTM; 2014/07135-1 and 2013/13309-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (RR; 481666/2012-5). JAD is the recipient of a FAPESP fellowship (2013/10443-7).