Plasmid construction
The chromosomal region 3168595–3169587 from E. coli K12 including the signal sequence and the protein coding sequence of the xylF gene encoding the xylose binding protein (XBP) (Gene ID: 948090, 993 nt) was cloned into the vector pT7T3GFP to construct the plasmid pT7T3GFP_XBP as described previously [17]. The protein coding sequence without the signal sequence from the xynA gene from B. subtilis (GeneID: 939861, 558 nt) was cloned into the plasmid pT7T3 18U (GeneID: U13869.1; GE Healthcare, Fairfield, Connecticut, USA) using the restriction sites HindIII and BamHI, to generate the construct pT7T3/XynA.
Library creation by random insertion of XynA into XBP
A random insertion library of xylanase in XBP was created using the protocol developed by Guntas and Ostermeier [14, 15] with some modifications. Fifty micrograms of the pT7T3GFP_XBP plasmid was mixed with 50 mM Tris HCl (pH 7.5), 1 mM MnCl2 and 50 µg/mL BSA, and the volume was completed to 95 µL with DNase-free water. Five µL of DNase I (Promega) containing 5 mU of the enzyme was then added. The mixture was incubated for 8 minutes at 22 °C, and the reaction was stopped by the addition of 2.4 µL of 0.5 M EDTA and incubation at 75 °C for 10 minutes. The DNA from this reaction was purified and eluted in 200 µL of water, and a sample of 100 ng of DNA was run on an agarose gel to estimate the percentage of linear DNA. The repair step was then performed in which T4 DNA ligase and T4 DNA polymerase (both enzymes from New England Biolabs, Ipswich, MA, USA) were added in a ratio of 160:1 respectively, per 1 µg of linear DNA. The mixture was incubated at 12 °C for 20 minutes with T4 ligase buffer and 200 μM dNTPs. The reaction was stopped by the addition of 10 mM EDTA and heating to 75 °C for 15 minutes. The linear DNA (5041 bp) was then purified using agarose gel. Approximately 1.5 µg of DNA was dephosphorylated using Antarctic phosphatase 12.5 U (New England Biolabs, Ipswich, MA, USA) in Antarctic phosphatase buffer for 30 minutes at 37 °C.
To create the random insertion library, the xynA gene without the stop codon was amplified by PCR from the pT7T3/XynA vector using phosphorylated primers and ligated to the gel purified pT7T3GFP_XBP plasmid prepared as described in the previous paragraph. The product of the ligation reaction was purified, concentrated and used to transform electrocompetent kanamycin resistant JW3538-1 E. coli cells that lack the XBP gene [the xylF749(del)::kan strain from the Coli Genetic Stock Center, Yale University, USA, hereafter referred to as ΔxylF]. After regeneration, the cells were plated on LB-agar containing 34 µg/mL kanamycin and 100 µg/mL ampicillin, on bioassay plates (245 × 245 cm). After growth, all the bacterial colonies present on the plates were harvested in storage media [LB + 10 % glycerol (v/v)] and stored at −80 °C.
Screening for the binding activity of XBP
A 500 µL aliquot of cells from the libraries stored at −80 °C was used to inoculate 50 mL of tryptone broth (TB) (10 g tryptone and 5 g NaCl per liter) containing 34 µg/mL kanamycin, 100 µg/mL ampicillin and 10 mM xylose. The culture was grown in an orbital shaker at 250 rpm and 37 °C for 10 h, and the culture was centrifuged. The cell pellet was resuspended in phosphate buffered saline (PBS) to a concentration of ~106 cells mL−1, after which the cells were kept on ice. Flow cytometry analysis fluorescent assisted cell sorting (FACS) was performed on a FACSAria cytometer (Becton, Dickinson and Company, East Rutherford, NJ, USA) equipped with a 405 nm excitation laser and a 530/30 nm bandpass emission filter. For each sample, 104 events were collected at a rate of 500–1000 events per second, where data collection and analysis used the FACSDiva software (Version 6.1.1., BD Biosciences, San Jose, CA, USA). Cells transformed with the pT7T3XBP vector were used as the negative control for correction of the auto-fluorescence, and cells transformed with the pT7T3GFP_XBP vector were used as the positive control. Clones that produced higher fluorescence than cells transformed with pT7T3GFP were collected and were denominated as XBP+ clones.
Screening for xylanolytic activity
The XBP+ clones separated by FACS were plated on selective LB-agar media. After incubation at 37 °C for 12 h, individual colonies were transferred to 384-well microplates containing 60 µL selective TB media using an automated colony picker (model K6, Kbiosystems, Basildon, Essex, UK). The plates were incubated at 37 °C for 24 h, and replicated onto 245 × 245 mm bioassay plates containing TB-agar media, supplemented with 0.6 % (m/v) xylan, 1 % (m/v) xylose; 34 µg/mL kanamycin; 100 µg/mL ampicillin. After incubation of the plates at 37 °C for 24 h, the clones expressing xylanase activity were identified by the formation of halos after staining with Congo red [34], and were denominated as XBP+/XynA+ clones.
Measurement of xylose stimulated catalytic activity
The XBP+/XynA+ clones were grown in TB supplemented with 34 µg/mL kanamycin, 100 µg/mL ampicillin for 48 h in 96-well plates (deep well). The supernatants were analyzed for hydrolysis of Remazol Brilliant Blue Xylan (RBB-xylan, Sigma-Aldrich, St. Louis, Missouri, USA), using a modification of a previously described protocol [35]. Fifty µL of culture supernatant was mixed with 50 µL of a solution containing RBB-xylan (4 mg/mL) in 100 mM acetate buffer (pH 5.5), in the presence or absence of 1 % (m/v) d-xylose (Sigma-Aldrich, St. Louis, Missouri, USA), and incubated at 37 °C for 12 h. After incubation, the reaction was stopped by the addition of 2 volumes (200 µL) of 96 % (v/v) ethanol. The insoluble material was removed by centrifugation (2000g/2 minutes), and the increase in the absorbance of the supernatant was measured at 595 nm. A single clone that showed activity ratio (with xylose)/(without xylose) greater than 1.3 was selected, and nucleotide sequencing indicated that the XynA domain was inserted within the XBP at position 271, and this chimeric enzyme was denominated as XynA–XBP271.
Expression and purification of the recombinant enzymes
The parental XynA, parental XBP and the chimeric XynA–XBP271 enzyme were expressed in E. coli [Rosetta™ (DE3)] transformed with pET28a (+) (Novagen, Billerica, MA, USA) carrying XynA, XBP or XynA–XBP271 with a N-terminal His6-tag and grown in HDM medium containing (per liter) 25 g of yeast extract, 15 g of tryptone, 1.2 g of MgSO4, supplemented with 34 µg/mL kanamycin and 40 µg/mL chloramphenicol. The cells were grown at 30 °C/120 rpm to an OD600 of 0.6. In all the cases, protein expression was induced with 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) for 5 h at 20 °C/120 rpm. Cells were harvested by centrifugation (8000g, 4 °C, 10 minutes). Whole-cell extracts were prepared from cell pellets by ultrasonication in 4 % (v/v) of the original culture volume of lysis buffer (100 mM HEPES, 300 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 % (v/v) Triton X-100, and 20 mM imidazole, pH 7.5). The cell extracts were cooled on ice and cleared of cell debris by centrifugation (10,000g, 4 °C, 30 minutes). The supernatants were loaded on an immobilized metal affinity column Ni–NTA (GE Healthcare, Fairfield, Connecticut, USA) pre-equilibrated with a buffer containing 100 mM HEPES, 300 mM NaCl, and 20 mM imidazole (pH 7.5). The column was washed with buffer containing 100 mM HEPES (pH 7.5), 300 mM NaCl, and 40 mM imidazole until no further reduction in the A280 was observed. Protein was eluted with 300 mM imidazole, and protein samples were dialyzed against 20 mM Tris–HCl (pH 8.0) and 200 mM NaCl and stored at 4 °C for further use. The protein concentrations were determined by measurement of the A280.
Enzyme activity assays
The effect of pH on xylan hydrolysis by the purified enzymes was determined at 40 °C in 50 mM with 0.2 % (w/v) RBB-xylan substrate (Sigma-Aldrich, St. Louis, Missouri, USA) buffered with one of the following buffer systems: acetic acid/acetate (pH 4.5-5.5), potassium phosphate (pH 5.5–6.5), MOPS-NaOH (pH 6.5–7.5) and Arginine-NaOH (pH 9.0). The effect of temperature on xylanase activity was conducted at temperatures between 30 and 55 °C in 50 mM acetate, pH 5.5. Thermostability was assessed by incubation of the purified enzymes at 55 °C and residual activity was measured in aliquots collected at increasing times. The kinetic parameters for xylanase were determined using the RBB-xylan substrate at concentrations ranging from 0.5 to 10 mg/ml, with and without 1 % (w/v) d-xylose (Sigma-Aldrich, St. Louis, Missouri, USA). The reactions were initiated by the addition of 50 nM of the purified enzyme to MOPS buffer (pH 6.5) at 45 °C. After 15 minutes, the enzyme was inactivated by incubation at 80 °C for 10 minutes, followed by incubation at 4 °C for 5 minutes. One hundred microliters of ethanol were then added and the mixture was incubated at 25 °C for 15 minutes. The samples were centrifuged at 2000g for 2 minutes and 90 µL of each sample and transferred to a 96-well plate. The absorbance values were measured at 595 nm and converted to μmols of released dye using a RBB-xylan substrate standard curve generated under the same conditions. All enzymatic activities were determined in triplicate and the maximum velocity (V
max), apparent dissociation constant (K
RBB-Xylan), and catalytic constant (k
cat) were calculated by nonlinear regression fitting of the data to the semi-logarithmic form of the Hill equation using the SigrafW software [36].
Enzyme assays using sorghum stover
The activity of enzymes on a natural substrate was evaluated using ground sorghum stover, previously washed with 100 mM MOPS buffer (pH 6.5) to remove residual soluble sugars. A 1 % (w/v) suspension of the washed substrate was prepared in the same buffer and mixed with either 30 nmol of the purified chimera, 30 nmol of individual purified xylanase or with an equimolar mixture comprised of 30 nmol of xylanase and 30 nmol of XBP, in a final reaction volume of 5 mL. The reaction was incubated at 40 °C for 15 h in a temperature controlled orbital shaker at 250 rpm to avoid substrate precipitation, and the total reducing sugar release was measured using the 3,5-dinitrosalicylic acid (DNS) assay [37]. All samples were assayed in triplicate and the mean of the three values was used for subsequent comparisons.
Molecular dynamics simulations and molecular modelling
Molecular dynamics simulations (MDS) and analyses were performed with the XynA–XBP271 chimera, both with and without bound xylose using the GROMACS 5.0.2 software package [38–40] with the GROMOS-96(53A6) force field [41]. The starting atomic coordinates of the chimeras were obtained by comparative protein modeling with program MODELLER 9.13 [42] to merge the XynA (pdb code:1XXN) and the XBP in the open (PDB code: 3M9 W, xylose-free) and closed conformations (PDB code: 3MA0, xylose-bound), respectively. These initial structures were validated using Procheck software [43] with a subsequent energy minimization step using the steepest descent method. The resulting structures were solvated by SPC water molecules at a concentration of approximately 54 mol/L in dodecahedron simulation boxes. Na+ ions were added to ensure the electroneutrality of the systems. Position restrained dynamics were performed for 400 ps at a reference temperature of 300 K to improve the equilibration phase. All systems were carried out in the NVT ensemble at neutral pH and 300 K, with a total time simulation of 120 ns. Temperature was controlled by a V-rescale thermostat [44] and covalent bonds involving hydrogen atoms in the protein and water molecules were restrained by LINCS [45] and SETTLE [46] algorithms, respectively. Newton’s equations of motion were solved using the Leap-Frog integration method [47] with dt = 2.0 fs. The Maxwell–Boltzmann distribution at a reference temperature was employed to generate the initial atomic velocities. The particle-mesh Ewald sum (PME) [48] was used to treat the long-range interactions with a 1.2 nm cutoff distance. The interaction potential energy (IPE) can be defined as the total interaction energy between protein A and protein B, and was computed according to the equation:
$$IPE = \sum_{i}^{NA}\sum_{j}^{NB}E_{i,j}$$
where \(E_{ij}\) is the interaction energy between an atom \((i)\) from protein A and an atom \((j)\) from protein B, and NA and NB are the total number of protein A and B atoms, respectively. Computational Alanine Scanning (CAS) was performed using the ROBETTA program [49] to identify “hotspot” residues at the protein–protein interface.