Strains, vectors, and culture media
E. coli BL21(DE3) and pET-28a(+) (Novagen, Madison, WI, USA) were used for the construction and screening of a site-saturation mutagenesis library, while E. coli DH5α and pPIC9K (Invitrogen, San Diego, CA, USA) for the construction of recombinant expression vectors. The vectors pET-28a-Aoxyn11A and pPIC9K-Aoxyn11A, and transformants E. coli/Aoxyn11A and P. pastoris/Aoxyn11A were constructed in our lab (Li et al. 2013). The genes, Aoxyn11A and its variants, were expressed in P. pastoris GS115. E. coli BL21 and DH5α were grown in the LB medium (10 g L−1 tryptone, 5 g L−1 yeast extract and 10 g L−1 NaCl, pH 7.2). P. pastoris and its transformants were cultured and induced in the YPD, MD, geneticin G418-containing YPD, BMGY and BMMY media, which were prepared as described in the manual of Multi-Copy Pichia Expression Kit (Invitrogen, USA).
Multiple templates-based homology modeling
It was demonstrated that the validity of multiple templates-based modeling mainly relies on the primary structure identities of a target protein with template ones, and on the multiple alignment accuracy among crystal structures of templates (Madhusudhan et al. 2009). Compared to the single template-based modeling method, the multiple templates-based one greatly increased the facticity and accuracy of the modeled 3-D structure of a target protein (Sokkar et al. 2011). Therefore, using AoXyn11A primary structure as the template, homology sequences were searched among GHF11 xylanases at NCBI website (http://www.ncbi.nlm.nih.gov/) towards Protein Data Bank (PDB). The three known crystal structure xylanases separately from Penicillium funiculosum (PDB code: 1TE1) (Payan et al. 2004), Talaromyces cellulolyticus (3WP3) (Kataoka et al. 2014) and E. coli (2VUL) (Dumon et al. 2008), possessing the highest primary structure identities with AoXyn11A, were selected as homology modeling templates. The 3-D structures of AoXyn11A and its variants were modeled based on the three templates using the SALIGN program (http://salilab.org/DBAli/) and MODELLER 9.11 program (http://salilab.org/modeller/), and analyzed using the PyMol software (http://pymol.org/).
Prediction of the three variants of AoXyn11A
The 3-D structure of a mesophilic protein/enzyme was more flexible than that of the corresponding thermophilic analog (Jaenicke and Böhm 1998). Therefore, to quantify the protein/enzyme flexibility, the notion of B-factor values was introduced to reflect the smearing of atomic electron densities as a result of thermal motion and positional disorder. B-factor values, namely atomic displacement parameters, of amino acids, were generated by molecular dynamics (MD) simulation on the crystal or modeled 3-D structure of protein using the GROMACS 4.5 package (http://www.gromacs.org/) and analyzed using the B-FITTER program (Reetz and Carballeira 2007). In this work, the modeled 3-D structure of AoXyn11A was subjected to MD simulation at 300 K for 15 ns, followed by calculating the B-factor values of residues. Gly21 in AoXyn11A with the maximum B-factor value was confirmed, and then randomly substituted by site-saturation mutagenesis.
AoXyn11AY13F or AoXyn11AG21I–Y13F was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe, based on the multiple alignment of AoXyn11A with seven representative thermophilic GHF11 xylanases using the ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Root mean square deviation (RMSD) value, which was defined as the Cα-atomic displacement range of a protein from its original configuration to changed one at a high temperature and a certain time, was negatively correlative to its thermostability (Badieyan et al. 2012). Thereby, to predict the thermostabilities of AoXyn11AY13F and AoXyn11AG21I–Y13F, the modeled 3-D structures of AoXyn11A, AoXyn11AY13F and AoXyn11AG21I–Y13F were subjected to MD simulations, respectively, at 500 K for 7 ns using the GROMACS 4.5 package, followed by calculating their RMSD values using a g_rms software in the GROMACS 4.5.
Construction and screening of the site-saturation mutagenesis library
The site-saturation mutagenesis of a Gly21-encoding codon in Aoxyn11A into any amino acid-encoding codon was performed using the two-stage whole-plasmid PCR technique (Sanchis et al. 2008). The PCR primers used for gene mutagenesis were listed in Additional file 1: Table S1. The recombinant vectors, pET-28a-Aoxyn11A
G21X, were amplified from pET-28a-Aoxyn11A. In brief, the first-stage PCR amplification was carried out using a pair of PCR primers G21X-F (forward) and X11-R (reverse) under the following conditions: an initial denaturation at 98 °C for 3 min, followed by 30 cycles of at 98 °C for 10 s, 53 °C for 30 s and 72 °C for 45 s. Then, the second-stage PCR was performed using the first-stage PCR products as the primers: 30 cycles of at 98 °C for 10 s, 55 °C for 30 s and 72 °C for 5 min. The target PCR products (pET-28a-Aoxyn11A
G21X) were digested by DpnI, and transformed into E. coli BL21, thereby constructing a site-saturation mutagenesis library (E. coli/Aoxyn11A
G21X).
Single colonies (E. coli/Aoxyn11A
G21X strains) were separately inoculated into 500 μL LB medium containing 50 μg mL−1 kanamycin in a 96-well plate, and cultured at 37 °C overnight as the seed culture. Then, the same medium was inoculated with 2% seed culture, and grown until OD600 reached 0.6. Expression of Aoxyn11A
G21X was induced by 1 mM IPTG at 28 °C for 8 h. The cells were collected, suspended in 100 μL Na2HPO4–citric acid buffer (50 mM, pH 5.5), and disrupted by ultrasonic (650 W, 180 cycles of work for 2 s and rest for 8 s). The resulting supernatant was divided into two aliquots and added into two 96-well plates. To screen the mutagenesis library, one plate was treated at 60 °C for 20 min, while the other plate used as the control. Then, aliquots of 5 μL treated or untreated supernatant were correspondingly added into two new plates with 95 μL 5.0 mg mL−1 xylan in each well, incubated at 50 °C for 10 min and stopped by adding 50 μL DNS reagent. Thus, the positive strains were selected, by which the expressed recombinant xylanases retained over 80% of their original activities, and in which the mutant genes were DNA-sequenced. Furthermore, one strain expressing reAoXyn11AG21I with the highest thermostability, E. coli/Aoxyn11A
G21I, was obtained by extending treating time to 30 min at 60 °C.
Site-directed mutagenesis of Aoxyn11A and Aoxyn11A
G21I
The recombinant vector, pET-28a-Aoxyn11A
G21I, was extracted from the obtained strain E. coli/Aoxyn11A
G21I, and digested using EcoRI and NotI to release Aoxyn11A
G21I. Then, Aoxyn11A
G21I was inserted into pPIC9K vector digested using the same enzymes, followed by transforming it into E. coli DH5α. The recombinant vector, pPIC9K-Aoxyn11A
G21I, was confirmed by DNA sequencing. Using pPIC9K-Aoxyn11A or -Aoxyn11A
G21I as the template, the site-directed mutagenesis of Tyr13-encoding codon in Aoxyn11A or Aoxyn11A
G21I into Phe-encoding codon was performed by two-stage whole-plasmid PCR using a pair of PCR primers Y13F-F (forward) and X11-R (reverse). The amplified target PCR products, that is, the recombinant vectors, pPIC9K-Aoxyn11A
Y13F and -Aoxyn11A
G21I–Y13F, were digested using DpnI, transformed into E. coli DH5α and confirmed by DNA sequencing.
Transformation of recombinant vectors and screening of P. pastoris transformants
Three recombinant vectors, pPIC9K-Aoxyn11A
G21I, -Aoxyn11A
Y13F and -Aoxyn11A
G21I–Y13F, were linearized with SalI, and transformed into P. pastoris GS115, respectively, by electroporation using the Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA). All P. pastoris transformants were primarily screened based on their abilities to grow on the MD plate, and then successively inoculated on the YPD plates containing G418 at concentrations of 1.0, 2.0 and 4.0 mg mL−1 for the screening of multiple copies of the integrated target gene. The P. pastoris transformant resisting high geneticin G418 concentration may have multiple copies of a gene, which can lead to the high expression of recombinant protein/enzyme (Invitrogen, USA). However, the expression level was not directly proportional to G418 concentration (Zhang et al. 2014). Thereby, a total of 60 P. pastoris transformants resistant to G418 of 4.0 mg mL−1, separately containing Aoxyn11A
G21I, Aoxyn11A
Y13F and Aoxyn11A
G21I–Y13F, were picked out for expression tests.
Expression and purification of the recombinant xylanases
Expression of Aoxyn11A
G21I, Aoxyn11A
Y13F or Aoxyn11A
G21I–Y13F in P. pastoris GS115 was performed according to the instruction of Multi-Copy Pichia Expression Kit (Invitrogen, USA) with slight modification. In brief, each single colony of 60 pastoris transformants was inoculated into 30 mL BMGY medium and cultured at 30 °C with 220 rpm until OD600 reached 2–4. Then, the cells were harvested by centrifugation, suspended in 30 mL BMMY medium and induced for the expression of recombinant xylanase by adding methanol to a final concentration of 1.0% at 24 h intervals at 30 °C for 72 h.
After expression, 10 mL supernatant containing the recombinant xylanase with a 6-His-tag at its C-terminus was loaded onto a nickel–nitrilotriacetic acid (Ni–NTA) column (Tiandz, Beijing, China; 1 × 6 cm) equilibrated with buffer A (20 mM Tris–HCl, 500 mM NaCl and 20 mM imidazole, pH 7.9), followed by elution at a flow rate of 0.3 mL min−1 with buffer B as same as buffer A except for 250 mM imidazole. Aliquots of 1 mL eluent containing the target xylanase were pooled, dialyzed against 50 mM Na2HPO4–citric acid buffer (pH 5.5), and concentrated by ultrafiltration.
Enzyme activity and protein assays
Xylanase activity was assayed by measuring the amount of reducing sugars from birchwood xylan (Sigma, St. Louis, MO, USA) using the 3,5-dinitrosalicylic acid (DNS) method (Gao et al. 2013). One unit (U) was defined as the amount of xylanase liberating 1 μmol of reducing sugar equivalent per min under the assay conditions (at pH 5.5 and 50 °C for 10 min). The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970). The separated proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Sigma, USA) and apparent molecular weights of xylanases were estimated by comparison with the standard proteins using the Quantity One software. The protein concentration was measured with a BCA-200 Protein Assay Kit (Pierce, Rockford, IL).
pH characteristics of the recombinant xylanases
The pH optimum of purified recombinant xylanase was determined under the standard assay conditions, except 5.0 mg mL−1 birchwood xylan in 50 mM Na2HPO4–citric acid buffer at a pH range of 3.0–7.5. Aliquots of xylanase were incubated at 40 °C and varied pH values (Na2HPO4–citric acid buffer: pH 3.0–7.5; Tris–HCl buffer: pH 8.0–9.0) for 60 min. The pH stability in this work was defined as a pH range, over which the residual xylanase activity retained over 85% of its original one.
Temperature characteristics of the recombinant xylanases
The temperature optimum (T
opt) of recombinant xylanase was measured, at pH optimum, at temperatures ranging from 40 to 70 °C. The inactivation half-life (t
1/2) of enzyme, a parameter for estimating its thermostability, was defined as a time, when the residual enzyme activity was 50%. In this work, to measure the t
501/2
of recombinant xylanase, aliquots of xylanase were incubated at 50 °C for different times.
The melting temperature (T
m) was defined as a temperature, at which a protein configuration is half unfolded. The protein/enzyme with high T
m meant it has a high thermostability (Jang et al. 2010). The T
m of xylanase here was measured by protein thermal shift (PTS) method (Dong et al. 2016), using a PTS Kit (Applied Biosystems, Carlsbad, CA, USA) and a LightCycler 480II 96 Real-Time PCR system (Roche, Basel, Switzerland). Three replicates were conducted independently. The T
m was a temperature which corresponds to the peak value of a derivative melting curve plotted using the ‘T
m calling’ method.
Kinetic parameters of the recombinant xylanases
The reaction rate (U/mg) of birchwood xylan by xylanase was measured under the standard assay conditions, but the substrate concentrations from 1.0 to 10 mg mL−1. The reaction rate versus xylan concentration was plotted to verify whether the reaction mode conformed to the Michaelis–Menten equation. The kinetic parameters, K
m and k
cat, were determined by non-linear regression analysis using an Origin 9.0 software (http://www.originlab.com/). All data were expressed as the mean ± standard deviation (SD) from three independent replicates.