Bacterial strain
B. licheniformis strain Z9 (KT693282) was isolated from farm soil at Menoufia governorate, Egypt (30°35′50.09′′ North, 30°59′15.48′′ East) [17] and tested for its high performance for cellulase activity. The bacterial strain was cultured in nutrient broth and stored on nutrient agar at 4 °C and as 50% glycerol stocks at − 80 °C.
Enzyme assay
The cellulase activity was observed by the 3, 5 dinitrosalicylic acid (DNS) method as described by Miller [18]. CMCase activity was determined by incubating 500 μl of 1% CMC in 50 mM sodium phosphate buffer (pH 7.2) with 500 μl cell free extract for 30 min at 50 °C. The reaction was stopped by adding 1 mL of 3, 5 dinitrosalicylic acid (DNS) reagent and incubated in a water bath for 10 min at 50 °C. After cooling at room temperature, the amount of glucose released was investigated with a spectrophotometer at 540 nm against a blank containing all the reagents minus the crude enzyme. A calibration curve for glucose was constructed to determine the CMCase activity. One unit (U) of cellulase activity was defined as the amount of enzyme that released 1 μmol of glucose per minute under the standard assay conditions. All assays were performed in triplicate.
Protein assay
Protein concentration was estimated by the method of Bradford [19] using bovine serum albumin (BSA) as a standard against a blank was set with only distilled water.
Molecular identification and DNA sequence analysis
To amplify the cellulase gene from the B. licheniformis strain Z9, degenerated gene-specific primers were designed complementary to the B. licheniformis strain SRCM100027 (CP021677) cellulase [ARW53264] gene sequence retrieved from the NCBI nucleotide database. This includes Forward primer Zf1:(5′ATGGCTTATTCTGCCGCAATCCTGTCA-3′) and reverse primer Zr1 (5′ GGCCATGTCGCTCTGCACGTAGTGG-3′). The PCR amplification reaction was performed in a total volume of 50 μl containing 2 μL of template DNA (50 ng/μL), 25 μL of 2X Taq PCR Master Mix (contains Taq DNA polymerase (0.05 U/μL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP) provided by Thermo Fisher Scientific, USA, 2 μL of forward primer, 2 μL of reverse primer and 19 μL of Nuclease-free water. The following PCR conditions were used for amplification of cellulase gene: initial denaturation at 94 °C, 5 min, and 35 cycles of the following steps: denaturation at 94 °C, 30 s; annealing at 55 °C, 30 s; extension at 72 °C, 1 min; and final extension at 72 °C, 5 min. The amplified PCR products were checked on 1% agarose gel stained with ethidium bromide and visualized on a UV transilluminator. The Purified PCR products were cloned into pSC-A-amp/kan PCR Cloning Vector as recommended by the manufacturer (Stratagene, Agilent Technologies, USA). StrataClone SoloPack competent cells were used for the transformation and recovery of high-quality recombinant DNA. The purified PCR products were Sanger-sequenced with the BigDye terminator v3.1 sequencing kit and ABI PRISM® 3730xl Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Nucleotide sequences were determined on both strands of PCR amplification products at Macrogen Company, Seoul, South Korea. The nucleotide sequence data was assembled, analyzed with GENETYX computer software (Software Development Co. Ltd., Tokyo, Japan). The consensus sequence obtained was compared with other sequences available in the GenBank/NCBI database using the BLAST tool [20] and aligned using CLUSTAL O [21]. The sequence was deposited with the GenBank Data Library under accession number MK814929. The deduced amino acids sequence was analyzed with UniprotKB database Release 2020, Washington, USA (http:// UniprotKB.org/). The phylogenetic tree was drawn with MEGA11 software [22], using Kimura’s two-parameter model of sequence evolution. The robustness of the phylogenetic tree was estimated via bootstrap analysis using 1000 resampling.
Conserved domain analysis and hydropathy plots of predicted cellulase cel9z
The protein sequences of cellulase cel9z were subjected to conserved domain analysis using the Conserved Domain Database tool of NCBI [23] (http://www.ncbi.nlm.nih.gov/Structure/cdd/). A hydropathy plot was generated using Expasy-Protscale (https://web.expasy.org/protscale/).
Preparation of cell extracts
Cellulase enzyme was produced under submerged fermentation from isolated B. licheniformis strain Z9. Conical flasks containing 50 mL of a carboxymethylcellulose medium [24] were supplemented with 0.5% (w/v) of CMC at pH 7.2. The flasks were autoclaved at 121 °C for 20 min. These sterilized flasks were inoculated with 10% of an inoculum culture of B. licheniformis strain Z9 and incubated under agitation at 150 pm, for 72 h at 30 °C according to the method of MARCO [25]. Then, the culture was centrifuged at 10,000×g for 15 min at 4 °C to separate the cells. The clear cell-free supernatant (crude extract) was collected and concentrated by ultrafiltration using a MILTEX-HV ultrafiltration cell (Millipore, Ireland). The crude extract was stored at 4 °C and used for further analyses [26].
Purification of cellulase
All steps were performed at 4 °C unless otherwise noted. The crude extract was saturated with (20–80%) ammonium sulphate with continuous stirring at 4 °C followed by centrifugation at 10,000×g for 15 min. Ammonium sulfate fraction (the developed pellet) was dialyzed against 50 mM sodium phosphate buffer (pH 7.2) for 6 h at 4 °C in a dialysis bag (20,000 kDa) and immersed in the same buffer at 4 °C overnight. Changing buffer at every 1 h intervals is important to achieve proper purification [27]. Fractions with high activity of cellulase were pooled together, dialyzed towards the above buffer, and concentrated by lyophilization (− 50 °C) for the next purification step. The dialysate was loaded onto a Sephadex G-100 column (2.5 × 40 cm) equilibrated with 50 mM sodium phosphate buffer (pH 7.2), and eluted in a gradient of NaCl (0–1 mol L−1) [25]. The cellulase was eluted from the column at a flow rate of 5 ml/min using the same buffer. Thirty-five fractions (5 ml each) were collected, dialyzed against the same buffer and the protein content was measured with a spectrophotometer at 280 nm. Fractions were checked for their purity by applying sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE)
To estimate the molecular weight of the partially purified enzyme, SDS-PAGE was done as reported by Laemmli [28]. Briefly, the SDS-PAGE gel slabs were prepared with upper 4% stacking gel and lower 10% resolving gel using a Bio-Rad electrophoresis system (Bio-Rad, CA, USA). The protein samples were mixed with sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 0.01% bromophenol blue, 2% SDS, 10% b-mercaptoethanol, and then heated for 3 min before loading to the gel. The electrophoresis was carried out in running buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3) and the gel was then stained by a solution of 0.15% Coomassie Brilliant Blue (CBB) R-250 in 50% ethanol and 10% glacial acetic acid. The samples were dissolved with sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenol blue) and then applied to the wells, resolved by applying a constant current (100 V) across the gel. After the run, the resolved bands were visualized by Coomassie brilliant blue R-250 staining method. The molecular weights were estimated by comparing with standard broad range protein marker (iNtRON Biotechnology, Gangnam-STAIN™ Prestained Protein Ladder ranging from 10 to 245 kDa).
Zymographic analysis
According to the method of Schwarz [29], zymograms were conducted to detect the proteins of cellulolytic activity from B. licheniformis strain Z9, with minor modifications as described below. A 0.2% CMC was added before polymerization to the resolving portion of 12% resolving gels except for no SDS and reducing agent were presented (native PAGE). For preventing aggregation, CMC was added slowly to the gel mixture while stirring. Gel polymerization was induced after all CMC was dissolved. Gels were allowed to polymerize overnight at room temperature, then kept at 4 °C until used (< 2 weeks). Solubilized protein samples were mixed with native sample buffer and then heated at 70 °C for 20 min to partially denature enzymes and reduce smearing of activity due to continuous enzymatic activity during electrophoresis. Following heating, the sample was briefly centrifuged to collect evaporated solution and loaded on gels for detection of cellulase activity. Electrophoresis was carried out at 4 °C at constant voltage (100 V) for approximately 4 h. For cellulolytic activity staining, gels were washed five times (6 min each) in 50 mL of washing buffer (50 mM phosphate buffer pH 7.2). The gel was incubated for 30 min at 37 °C in washing buffer without DTT to develop cellulase activity. Gels were then stained with 0.1% Congo Red for 15 min at room temperature and washed with 1 M NaCl until the clear cellulase band was visible. Gels were then immersed in 5% (v/v) acetic acid and photographed. The position of the cellulase enzyme on the gels was detected with a standard broad range protein marker (iNtRON Biotechnology, Korea).
Characterization of the purified cellulase
Optimum temperature
To assess the effect of temperature on the enzyme activity, a reaction mixture of substrate and the partially purified enzyme was incubated at various temperatures. 100 μL of the appropriate concentration of enzyme was added to 100 μL of 1% CMC and completed to 1 mL with 800 μL of 20 mM glycine-NaOH buffer (pH 7.4), and incubated at 10 °C, 20 °C, 30 °C, 37 °C, 45 °C, and 60 °C temperature for 30 min. The non-incubated enzyme was used as the control (100%). The activity was then measured according to the method of Miller [18].
Optimum pH
This experiment was performed to investigate the effect of different buffers at different pH values on the partially purified cel9z protein. One hundred microliters of the appropriate concentration of enzyme was added to 100 μL of 1% CMC and completed to 1 mL with 800 μL of various buffers. The buffers were citrate phosphate (pH 4.2–7), Tris (Hydroxymethyl) aminomethane (Tris) (pH 7.2–9.0), and glycine-NaOH (9.5-10.6), and the activity was measured as described by Miller [18]. The non-treated enzyme activity was regarded as control (100%).
Effect of metal ions and chemical reagents
For activators and/or inhibitors sensitivity studies, the partially purified enzyme was pre-incubated with a final concentration of 1mM of various metal ions and chemicals (Ag+, Na+, Cu2+, Co2+, Ca2+, Fe3+, Mg2+, SDS, and EDTA) dissolved in 50 mM Tris (Hydroxymethyl) aminomethane (pH 7.2) at 37 °C for 30 min. The activity assay in the absence of any chemical reagent or metal ions was recorded as a control (100%). The residual activity was measured by using the standard assay [18].
Statistical analysis
Results were expressed as mean ± standard deviation and the data was analyzed using one-way ANOVA using GraphPad Prism for windows, www.graphpad.com.