Raw materials, chemicals and microorganism strain
All chemicals including 3,5-Dinitrosalicylic acid (DNS), Bovine serum albumin (BSA), beechwood xylan, Na2CO3, avicel, (upbeta )-glucan, xylan, filter paper, locust bean gum (LBG), carboxymethylcellulose (CMC), coomassie brilliant blue, ethanol, phosphoric acid, metal ions, ethylenediaminetetra acetic acid (EDTA), urea, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), cetrimonium bromide (CTAB), Tween 20, Triton X-100, NaN3, HCl, NaOH, beta mercaptoethanol, methanol, acrylamide, bisacrylamide, temed, ammonium persulfate (APS), glycine, tris-base, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide, trichloroacetic acid, ferric chloride, Folin and gallic acid were from Sigma-Aldrich. Corn flour was purchased from local market (Karaj, Iran). The Saccharomyces cerevisiae IBRC-M 30069 was purchased from the Iranian Biological Resource Center. To obtain inoculum for SSF, the selected fermentative microorganism, Saccharomyces cerevisiae was grown in 250 mL flasks (pH 6.8) and medium containing 0.75 g yeast extract, 0.75 g malt extract, 1.25 g soy peptone, and 1.25 g glucose at 28 °C in a rotary shaker 180 rpm for 24 h until the absorbance at 600 nm reached to approximately 1.2.
Poultry feed gained from the Nican Dam Arvin. The α-amylase (PersiAmy3) was cloned, expressed, purified and characterized according to the previous study26. Luria–Bertani medium (LB broth), T4 DNA ligase (Thermo Fisher Scientific), Kanamycin (Duchfa), Isopropyl β-D-1-thiogalactopyranoside (IPTG), BamHI and SalI restriction enzyme (Thermo Fisher Scientific), Gel Extraction kit (Thermo Scientific), Ni–NTA Fast Start Kit (Qiagen, Hilden, Germany) were used for production, expression, and purification of xylanase (PersiXyn8). The phosphate buffer with appropriate pH and distilled water were used to prepar the samples.
In-silico identification, cloning, expression and purification of the novel PersiXyn8
In this study we used, raw cattle rumen Metagenomic data submitted to NCBI with Bio-project ID: PRJNA63195127. The cattle rumen metagenomic data was mined to discover a thermostable xylanase enzyme. The quality of metagenomic data was verified by FASTQC and the high-quality data was assembled using meta-velvet assembler. For targeted screening of the assembled contigs, first, the xylanase genes were predicted using MetaGeneMark. Then, TAXyl (Thermal Activity Prediction for Xylanase)28 as a machine learning assisted web-based tool which employs supervised algorithms to predict the thermal activity of GH10 and GH11 xylanases was applied on the predicted putative xylanases. TAXyl uses sequence-based and length-independent protein descriptors to predict the thermal activity of xylanases in one of the non-thermophilic, thermophilic, and hyper-thermophilic classes. Considering the application in poultry feed industry, one of thermophile GH10 xylanases predicted by TAXyl, was selected for the next steps. For further confirmation, the NCBI Conserved Domains Database (CDD)29 was employed to identify the xylanase domain in predicate xylanase, PersiXyn8. Also, the Phyre2 server30 was used to confirm the similarity of the tertiary structure of PersiXyn8 with GH 10 xylanase enzymes. Finally, PersiXyn8 sequence data was deposited in the GenBank under accession number MW349589.
To ensure that PersiXyn8 is a novel xylanase enzyme, we aligned it against NCBI non-redundant protein sequences database using multiple sequence alignments with CLUSTALW (Supplementary Fig. 1).
The ORF coding of xylanase gene was acquired from the cattle rumen metagenome DNA with a pair of forward (5′- TGATAGGGATCCATGAATGAATGGGAAAAGGAAT-3’) and reverse (5′- TGATAGGTCGACTCAGCGTATACTACTGAATC-3’) primers which contain BamHI and SalI restriction sites, respectively. The gradient PCR protocol performed as follows: initial denaturation at 95 °C for 5 min; 10 cycles of denaturation at 95 °C for 40 s, gradient annealing at 60 °C to 50 °C for 40 s, and elongation at 72 °C for 90 s and after that 35cycles of denaturation at 95 °C for 40 s, annealing 54 °C for 40 s, and elongation at 72 °C for 90 s. The final cycle was followed by an extension at 72 °C for 10 min. For gel extraction of xylanase DNA fragments, the PCR product was run on the 1% (w/v) agarose gel and DNA fragments was cut with a razor blade and purified then using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific). Further, the extracted fragment was ligated with T/A cloning vector pTZ57/RT and transformed into competent E. coli DH5a cells. For the expression of xylanase gene, BamHI and SalI digested purified from agarose gel and was ligated to the downstream of the 6 × His tag of similarly digested pET-28a (+) vector and transformed into E. coli BL21 (DE3) by heat shock method. Transformants were selected on LB- kanamycin (50 μg/mL) agar plates and confirmed by colony PCR. For expression and purification of the recombinant xylanase, 250 mL of Luria-Bertoni (LB) broth containing 50 μg.mL−1 kanamycin was inoculated with 10 mL of overnight grown culture of E. coli BL21 (DE3) containing pET-28a-xylanas and was grown in the shaker incubator at 37 °C/180 rpm. When the optical density (OD) of the cell culture reached 0.6 at 600 nm, Isopropyl β-D-1-thiogalactoside (IPTG) (0.25 mM) was added for 18 h at 25 °C in order to xylanase protein induction. The N-terminal Histidine-tagged recombinant protein was purified by Ni–NTA Fast Start Kit (Qiagen, Hilden, Germany) with some modification. Cell suspension was harvested by centrifugation at 8000 × g for 10 min and resuspend the cells pellet in 10 mL native Lysis Buffer supplemented with PMSF and DTT and incubated on ice for 30 min after that sonication was down (60% amplitude of 5*1 min with 1 min interval), centrifuge lysate at 14,000 × g for 30 min at 4 °C to pellet the cellular debris. Apply the cell lysate supernatant to the Ni–NTA column and 2 times column washed with wash buffer. Elute bound 6xHis-tagged protein with 1 mL aliquots of Native Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole; pH 8.0). Protein concentration was measured by the method of Bradford31.
Xylanase activity assay
The activity of the xylanase was investigated by preparation of the beechwood xylan in the concentration of 1% (w/v) in 50 mM phosphate buffer, pH 6.0. The reaction mixture composed of 20 (mathrm{mu L}) of the PersiXyn8 (1 mg/mL) and 60 (mathrm{mu L}) of xylan was incubated at 50 (^circ{rm C} ) for 20 min. The amounts of released reducing sugars were measured based on the DNS assay32. In this connection, 180 (mathrm{mu L}) of DNS reagent was added to the samples and boiled for 5 min. The absorbance was recorded at 540 nm. One unit of the xylanase activity was defined as the amount of enzyme that liberates 1 μmol of reducing sugar per minute based on the xylose standard curve. The specific activity of the enzyme was defined as unit per mg of protein which was estimated according to the Bradford procedure31.
Influence of temperature, pH, metal ions and inhibitors
The optimum temperature of the xylanase activity was studied by incubating the enzyme with beechwood xylan (1% w/v) in phosphate buffer (50 mM, pH 7.0) in a temperature range of 30 to 80 (^circ{rm C} ) for 20 min. By fixing the optimum temperature for enzyme, the influence of pH on the xylanase activity was examined by the incubation of PersiXyn8 with the substrate (1% w/v) in 50 mM sodium citrate buffer (pH 4.0–5.0), 50 mM potassium phosphate buffer (pH 6.0–8.0) and 50 mM carbonate-bicarbonate buffer (pH 9.0) at 50 (^circ{rm C} ) for 20 min. The xylanase activity was investigated using DNS method, and the relative enzymatic activity was measured by expressing the maximum activity as 100%.
To analyze thermal stability, the PersiXyn8 was evaluated by incubating the enzyme for 120 min at temperatures 40 to 80 ℃ at optimum pH (pH 6.0). The storage stability of the enzyme was estimated by incubating the PersiXyn8 for 8 h at 50 °C. The enzymatic activity was measured at 30 min intervals under mentioned reaction condition.
The enzymatic activity was measured at the presence of 5 mM MgCl2, CaCl2, NaCl, MnCl2, CuSO4, FeSO4, ZnCl2, EDTA, Urea, PMSF and NaN3 and 1% SDS, CTAB, Tween 20 and Triton X-100. The enzyme was preincubated with each denaturant for 30 min at room temperature. The substrate (1% w/v) was added to the mixtures and incubated in optimum condition for 20 min. The DNS method was used for determining the amounts of generated reducing sugars, and the relative xylanase activities were investigated. The activity found in the absence of chemicals was taken as control (100%).
Determination of kinetic parameters
To measur the kinetic parameters of the enzyme, it was incubated in various concentrations of the beechwood xylan from 0.001 mM to 0.04 mM in potassium phosphate buffer (pH 6.0, 50 mM) at 50 °C. The enzymatic activity was determined according to the DNS method and Km, Kcat and Kcat/Km of the xylanase were calculated based on the Lineweaver–Burk plot.
Substrate spectrum of the PersiXyn8
Using different substrates, including avicel, (upbeta )-glucan, beechwood xylan, filter paper, LBG and CMC, the substrate specificity of the PersiXyn8 was analyzed. Toward that end, the substrates were prepared at the concentration of 1% in phosphate buffer (50 mM, pH 6.0) and incubated with the enzyme (1 mg/mL) at 50 (^circ{rm C} ) for 20 min. The amounts of reducing sugars were examined according to the DNS method33. One unit of the xylanase activity was defined as the amounts of enzyme that liberated 1 μmol of reducing sugar per minute and the specific activity was expressed as unit per mg of protein.
For thin layer chromatography (TLC) analysis, the PersiXyn8 was incubated with 10 mL of beechwood xylan (0.5 g in 50 mM phosphate buffer pH 9.0) followed by incubation at 50 °C for 12 h. The hydrolyzed products were separated and detected by TLC and D-xylose was used as the reference standard. The mobile phase was a mixture of chloroform/acetic acid/water (6:7:1 by volume). To detect sugar spots, TLC plate was sprayed by solution of 5% H2SO4 and 95% ethanol and dried the plate at 105 °C for 10 min34.
Developing the carbohydrate-hydrolyzing enzyme cocktail capable of degrading the poultry feed
To investigate the synergistic relationship between the PersiXyn8 (322 U/mg protein) and the reported α-amylase, PersiAmy3 (65 U/mg protein), 20 μL of enzyme mixture (PersiXyn8:PersiAmy3) in various ratios of 100:0, 80:20, 60:40, 40:60, 20:80, 0:100 was added to the 60 μL of poultry feed (20 mg/mL) in 50 mM phosphate buffer (pH 6.0) and incubated at 50 (^circ{rm C} ) for 20 min. The DNS method was used for enzymatic activity estimation and the relative activities were measured by taking the maximum activity as 100%.
To study the ability of the enzyme cocktail to hydrolyze the poultry feed, 20 μL of enzyme cocktail in an optimum ratio of 20:80 (PersiXyn8:PersiAmy3, 10 U/g) was added to the 60 μL of poultry feed (20 mg/mL) in 50 mM phosphate buffer (pH 6.0) and incubated at 50 (^circ{rm C} ) for 72 h. Samples were taken in 24 h intervals followed by the reducing sugars estimation via DNS method. The sample without enzyme addition was used as a control.
Solid-state fermentation of corn and enzyme cocktail treatment
Ingredients and nutrients of poultry feed for this study are given in supplementary Table.1. Due to the high percentage of corn in this substrate, it was selected for SSF in the presence of a thermostable enzyme cocktail. Corn flour was sieved through a 50 mesh and sterilized by autoclaving at 110 °C for 10 min. The SSF was processed in the presence of 10 g corn flour, 50 mL medium, active yeast (5%) and enzyme cocktail at the optimum ratio of 20:80 (PersiXyn8:PersiAmy3, 10 U/g) under 80% humidity for 7 days at 28 (^circ{rm C} ). The sample without enzyme cocktail and inoculation was used as a control. The fermented materials were lyophilized for 24 h and stored at 4 °C for further analysis.
Scanning electron microscopy (SEM) analysis
The morphological changes of the corn during SSF and enzyme cocktail treatment was investigated by scanning electron microscopy (SEM). The fermented and control (without enzyme cocktail and yeast) samples were lyophilized for 24 h and their structural changes were analyzed using a scanning electron microscope (FEI Quanta 200, USA) at 15 kV.
Extraction and determination of total polyphenolic composition
The phenolic compounds of the fermented samples were extracted according to the described method with some modifications35. In brief, 2 g of each lyophilized sample was extracted in 40 mL solution of methanol 95% and HCl 1 N (85:15, v/v) for 2 h at room temperature. Later, the solutions were centrifuged at 2500 g for 10 min and the supernatants were obtained. Next, the residues from the centrifugation were re-extracted using the methanol 95% and HCl 1 N (85:15, v/v) after 2 h of stirred condition at room temperature followed by the second centrifugation at 2500 g for 10 min. The final supernatants were mixed, concentrated under a vacuum at 45 (^circ{rm C} ) and stored in the freezer until use. The Folin-Ciocalteu colorimetric method was used to examine the total phenol content of samples36. Therefore, 500 μL of Folin reagent 10% was added to 100 μL of diluted sample and mixed with 400 μL of Na2CO3 7.5%. The reaction mixture was equilibrated at room temperature for 30 min and the absorbance was recorded at 750 nm. Different concentrations of gallic acid (5–85 μg/mL) were used to plot the standard curve and the phenolic concentration of the sample expressed as the mg of gallic acid equivalents per gram of sample dry weight (mg GAE/g sample).
Determination of the antioxidant capacity
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ABTS radical scavenging activity
The ability of the fermented samples (1 mg/mL) to scavenge the radical ABTS was performed based on the previous method37. At first, the stock solution of 7.4 mM ABTS was mixed with 2.6 mM potassium persulfate solution at the ratio of 1:1 in 50 mM phosphate buffer saline (PBS, pH7.4) and kept in a dark place at room temperature for 12–16 h. Afterwards, the ABTS+ solution was diluted with distilled water to reach an absorbance of 0.70 ± 0.02 at 734 nm. Next, 1 mL of ABTS+ solution was added to 100 μL of diluted sample or distilled water (control) and mixed by vortexing. The reaction mixture was proceeded at 25 °C for 6 min and the absorbance was read at 734 nm using spectrophotometer. Radical scavenging activity was calculated using the following equation:
$$mathrm{%ABTS scavenging}=frac{{mathrm{Abs}}_{mathrm{control}}-{mathrm{Abs}}_{mathrm{sample}}}{{mathrm{Abs}}_{mathrm{control}}}times 100$$
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DPPH radical scavenging activity
The ability of the samples (1 mg/mL) to eliminate the DPPH free radicals was assessed according to the procedure described38. Briefly, 1 mL of a freshly prepared ethanolic solution of DPPH (0.1 mM) mixed with 100 μL of diluted sample and shook vigorously. The distilled water was used as a control, after incubating the solution stand in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against a methanol blank. DPPH radical scavenging activity was calculated using the following equation:
$$mathrm{%DPPH scavenging}=frac{{mathrm{Abs}}_{mathrm{control}}-{mathrm{Abs}}_{mathrm{sample}}}{{mathrm{Abs}}_{mathrm{control}}}times 100$$
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Reducing power assay
The reducing power was measured according to the method described before with some modification39. For analysis, 0.5 mL of diluted sample (1 mg/mL) was mixed with 1.25 mL phosphate buffer (200 mM/L, pH 6.6) and 1.25 mL potassium ferricyanide (1% w/v). After incubation for 20 min in a water bath at 50 °C, 1.25 mL trichloroacetic acid (10% w/v) was added to the mixture. The resulting solutions were centrifuged for 10 min at 1500 g. Then 1.25 mL of the supernatants were mixed with 1.25 mL distilled water and 0.25 mL ferric chloride (0.1% w/v). The absorbance was measured at 700 nm after 10 min incubating at room temperature using a UV visible spectrophotometer.
Determination of protein, lipid and ash contents
Total crude protein was evaluated using the Kjeldahl procedure40. Lipid content was measured by Soxhlet extraction method using n-hexane as a solvent41. Ash content was determined based on the AOAC standard methods42.
Statistical analysis
Three replications were performed for each experiment and the results were analyzed with SPSS software (SPSS, Inc., IBM, Somers, NY, USA, Version 22.0), (https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-22). The statistical analysis of data was performed using one-way analysis of variance (ANOVA) and differences were analyzed by Duncan post-hoc test considering the significance at p (le ) 0.05.
