Strains, plasmids, and reagents
The E. coli strains Trans I-T1 and BL21(DE3) from Transgen (Beijing, China) were used for plasmid amplification and expression, respectively. The white-rot fungus Trametes sp. is a strain isolated from Shennongjia Nature Reserve (Hubei province, China) (Yang et al. 2011, 2012). The plasmid pET-28a (+) (Invitrogen, Carlsbad, CA) was used for gene expression in E. coli. The LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). The T4 DNA ligase was obtained from New England Biolabs (Hitchin, UK). The Pfu DNA polymerase was purchased from Tiangen (Beijing, China). The substrates d-glucose, d-xylose, l-sorbose, d-mannose, and d-galactose were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were of analytical grade and commercially available.
Gene cloning
Trametes sp. was grown at 28 °C in 100 mL of medium containing 20 g/L of glucose, 5 g/L of yeast extract, 5 g/L of peptone, 1 g/L of MgSO4 and 2 mg/L CuSO4 with constant agitation at 160 rpm. The mycelia were collected on the 5th day for RNA extraction when the Trametes sp. culture produced maximum POx activity. The total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The first strand cDNA was synthesized using the First Strand cDNA Synthesis kit (Fermentas, Ontario, Canada). The cDNA of TsPox was amplified using the primers TsPOxf and TsPOxr (Additional file 1: Table S1) and inserted into the expression vector pET-28a(+) between the EcoRI and NotI restriction sites to obtain the expression plasmid pET-28a-TsPox.
Sequence analysis
Sequence comparisons with known sequences were conducted with BlastP at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A multiple amino acid sequence alignment was carried out using the ClustalW program (http://www.ebi.ac.uk/clustalW). The molecular mass of the mature peptide was calculated using Vector NTI 10.0 software (Invitrogen, Carlsbad, CA). No signal peptide was predicted for POx by using the SignalP server (http://www.cbs.dtu.dk/services/SignalP/).
Construction of mutants
Two single mutants (E539K and K312E) and two double mutants (T166A/E539K and K312E/E539K) were generated by site-directed mutagenesis using pET-28a-TsPox as the template, which was carried out by using the Fast Mutagenesis System (Transgen, Beijing, China) according to the instructions of the manufacturer. Specific primers (Additional file 1: Table S1) were used to introduce residue substitution, which was confirmed by DNA sequencing.
Expression and purification of the POx wild-type and mutants
To express active POx and its mutants, all recombinant plasmids constructed above were individually transformed into the chemically competent cells of E. coli. Positive transformants were cultivated in 1 L-flasks at 37 °C and shaken at 200 rpm. When the OD600 values reached 0.8‒1.0, IPTG at a final concentration of 1 mM was added to induce the protein expression. After cultivation at 16 °C for an additional 16 h, the cells were harvested by centrifugation at 12,000g for 10 min and then re-suspended in a binding buffer (20 mM Tris–HCl, 500 mM NaCl, 10% glycerol, pH 7.6). The cell wall was disrupted by sonication followed by centrifugation at 10,000g for 15 min. The supernatant was separated and loaded into a Nickle-NTA (nitrilotriacetic acid) chelating column (GE Healthcare, Uppsala, Sweden). The bound proteins were eluted from the resin with a linear imidazole gradient (40–500 mM) in the binding buffer. Fractions with POx activity were analyzed by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 12% separation gel and 5% spacer gel) and the pure fractions were collected and combined. The protein concentration was determined using a Protein Assay Kit (Bio-Rad, Hercules, CA).
Biochemical characterization
The POx activity was determined spectrophotometrically at 420 nm and 30 °C by monitoring the formation of H2O2 for 3 min with a peroxidase-coupled assay using 2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid) (ABTS) as the chromogen. The standard assay mixture (total volume, 1 mL) contained 1 mM of ABTS in 50 mM potassium phosphate buffer (pH 6.5), 2 U of horseradish peroxidase, 100 mM of d-glucose, and properly diluted POx sample. One unit (U) of POx activity was defined as the amount of enzyme to oxidize 2 μmol of ABTS per min under the conditions described above.
The pH optimum for the POx activity was estimated at 30 °C in the 200 mM Tris–HCl (pH 2.0–5.0), McIlvaine buffer (200 mM sodium phosphate, 100 mM sodium citrate, pH 6.0–8.0), and 200 mM glycine–NaOH (pH 9.0–12.0). To determine the optimal temperature for activity, the assays were performed at temperatures from 20 to 70 °C at the pH optimum as determined above. For analysis of pH stability, the enzymes were pre-incubated in the 200 mM Tris–HCl (pH 4.0–5.0), McIlvaine buffer (pH 6.0–8.0), or 200 mM glycine–NaOH (pH 9.0) without substrate at 30°C for 1 h, and residual enzyme activities were measured under the standard condition (pH 6.0, 30 °C and 10 min). The thermal stability of the enzymes was determined by measuring the residual activities after incubation at 60 and 65 °C for various durations. The influence of metal ions and chemical reagents on the TsPOx activity was tested at concentrations of 5 and 10 mM, respectively.
Determination of substrate specificity and kinetic parameters
The substrate specificities of wild-type TsPOx and its mutants were determined by measuring the enzyme activities at pH 6.5 and 30 °C in the McIlvaine buffer containing 100 mM of d-glucose, d-xylose, d-mannose, l-sorbose or d-galactose as the substrate. Their kinetic parameters were determined in the McIlvaine buffer containing 1‒50 mM of d-glucose under pH 6.5 and 30 °C for 5 min. The K
m
and kcat values were estimated by fitting the data to the Michaelis–Menten equation using the software GraphPad Prism 5.01 (GraphPad Softwares, La Jolla, CA). The experiments were carried out three times, and each experiment included triplicates.
Determination of the wheat gluten content
The wheat gluten was checked for residual starch by iodine solution (0.1 g of iodide and 1 g of potassium iodide in 250 mL water). The wheat gluten content was determined according to the State Standard of the People’s Republic of China GB/T 14608-93 (“Method for determination of wet gluten in flour”). The dough was prepared by mixing 100 g of wheat flour and 50 mL of water, followed by extensive washing with water until no gluten was present. The wet gluten was incubated at 105 °C for 12 h to eliminate the bound water. To investigate the effect of POx on gluten agglomeration, 0.25, 0.5, 0.75, and 1.0 nkat/g flour of wild-type TsPOx or its mutants was supplemented. The wet and dry gluten were then recorded.
Application potentials of TsPOx and its mutants in breadmaking industry
The dough was prepared by mixing 100 g of flour, 3.2 g of yeast, 3.0 g of salt, 12.0 g of sugar, 8.0 g of milk powder, 6.0 g of butter, and 120 g of water. The wild-type TsPOx or its mutants was supplemented at the dosage of 0.25, 0.50, 0.75 or 1.00 nkat/g flour. Dough mixing was performed at room temperature for 3 min. Mixed dough was then transferred to a lightly greased beaker and incubated at 37 °C in a fermentation cabinet. The fermentation in the breadmaking process continued for 90 min. After fermentation, the dough was punched, transferred to a greased metal baking plate, and incubated at room temperature for a final proof of 45 min. The proofed dough was subsequently baked at 180 °C for 30 min. The loaf volume and weight were measured after cooling down to room temperature for 60 min.
Nucleotide sequence accession number
The nucleotide sequence of the TsPOx gene was deposited in the GenBank database with the accession numbers of MG344741.