Sample collection and preparation
The soil samples (2) were collected from Alexandria (2018), Egypt’s Wadi El Natrun (Latitude & Longitude (WGS84): 30° 27′ 20′′ North, 30° 10′ 20′′ East), secondly were transferred to the laboratory in ice box, thirdly were placed in a deep freezer (− 20 °C), and then were inspected.
Chromosomal DNA preparation
DNA isolation from soil sample was carried out using soil-isolation DNA kit (QIAGEN). In brief, the soil sample (500 mg) was suspended in 10 ml of 0.1 M sodium phosphate buffer (pH 8) before being added to 0.5 ml of 20% sodium dodecyl sulfate (SDS) and was mixed numerous times by inversion. Afterwards, 20 μl lysozyme (10 mg/ ml) was added and was mixed well (30 s); then the protocol of DNA isolation was completed according to the manufacturer’s recommendation. Afterwards, the purity and the concentration of the isolated DNA were detected by using NANO DROP (Thermo Scientific™ NanoDrop 2000).
DNA restriction digestion
The isolated soil- DNA was digested in 20 μl reaction volume; 2 μl of enzyme buffer and 1–2 U restriction enzyme were added. DNA digestions with restriction enzymes were performed under the reaction conditions specific for each enzyme, as being suggested by the manufacturers (Fermentas). Different restriction enzymes were used (BamHI, EcoRI, HindIII, and SalI) for the DNA digestion and the cloning vector (pUC19), as well.
DNA ligation
A ligation of the digested DNA-vector (ratio 4:1) was made into 20 μl reaction volume using T4 DNA ligase (1U) (Fermentas) and 2 μl buffer as the mixture reaction was kept at 16 °C overnight. PUC 19 (plasmid cloning vectors) was used as cloning and expression vector which allowed blue/white screening for the recombinant clones through Lacz promoter in the presence of the lactose analogue (IPTG, 1M) and the chromogenic agent 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal, 200 mg/ ml).
Competent cells preparation and transformation
Escherichia coli DH5α was prepared according to Sambrook et al. [24]; 100 ml Luria-Bertani (LBlow salt) liquid medium of the following composition (g/l): yeast extract, 10; peptone, 5; NaCl, 5; they were inoculated with 100 μl overnight culture of E. coli DH5α and were kept warm at 37 °C with shaking (170 rpm) till optical density (OD 600) 0.6–0.7. The flask was ice-chilled and was aliquoted into 50 ml portions. Cells were collected with centrifugation at 4000 rpm and re-suspended in one ml of transformation storage solution (TSS). This solution composed of (w/v %) polyethylene glycol 6000 (PEG); 10, MgCl2.6H2O; 1 and 5 ml dimethyl sulfoxide (DMSO), complete to 100 ml final volume with LB broth then adjust the pH at 6.5 as described by Chung and Miller [25].
Cell suspension was dispensed into sterile Eppendorf tube (200 μl aliquots), which was frozen immediately at − 80 °C. Frozen aliquots of the competent cells were allowed to thaw on ice. DNA (ligation mixture) was added to the tube, and then incubated for 20 min on ice. The tube was heat shocked at 42 °C for 60 s then 800 μ1of LB medium was added and then incubated at 37 °C for 1 h with shaking. Aliquots (200 μl) were spread on selective LB plates containing ampicillin (amp), X-Gal, and IPTG with a final concentration % 10 mg, 2 mg, and 1 mM, respectively. A master plate for white clones was prepared to test individually clone ability to degrade the cellulose macromolecule.
Screening soil metagenomic clones for expressing cellulase gene
To differentiate the positive cellulase producing clones among soil metagenomic clones (white clones), qualitative detection (plate assay) was carried out. The tested clones were screened for cellulase activity in Nutrient agar (NAamp& IPTG) supplemented with 0.5% of carboxymethylcellulose (CMC). The positive clones characterize by formation of a yellowish halo around the colonies after addition of indicator (0.2% w/v Congo red) followed by washing with 1 M sodium chloride [26].
Enzyme colorimetric assay
The cellulase activity was determined by monitoring the released reducing sugar (measured as glucose) upon enzymatic hydrolysis of CMC substrate, by applying the dinitrosalicylic acid (DNS) method and a standard curve was generated using crystalline glucose powder [27]. Cell pellets of positive clones were sonicated in 2 ml phosphate buffer pH 7. A 0.5 ml of tested pellet lysate was mixed with 0.5 ml of the CMC-substrate (0.5% w/v) in tris-HCl buffer (50 mM, pH 7.5); the reaction mixture was incubated for 15 min at 50 °C. Afterwards, the reaction was stopped by adding 1 ml of DNS and boiling for 10 min. The released reducing sugar producing color was measured at 540 nm by spectrophotometer (UV, SHIMADZU); the absorbance was measured against control (without active enzyme). Under standard test conditions, enzyme activity (U) was defined as the amount of enzyme that produced 1 μg of reducing sugar equivalent to glucose per minute. All assays measurements were calculated after subtracting from the individual control which composed of mixture of the enzyme and substrate boiled before being used as a control sample.
Preparation of plasmid-DNA from E. coli recombinant cells harboring insert
Mini-plasmid extraction was carried out via alkali lysis method as defined by Sambrook et al. [24]. Cells (1.5 ml) from overnight culture of the selected clone were collected by centrifugation at 7000 rpm and were sequentially suspended in three solutions (I, II, III) by equal volume; then, using mild shaking after the addition of solution I& II and standing 15 min after solution III. The first (Tris-HCl-pH 7.5 and 100 mM EDTA) was for solubilizing the cell pellet , the second was for lysis (1 M NaOH and 5.3% (w/v) SDS), and the third (60 ml of 5 M K-acetate and 11.5 ml acetic acid w/v%) was for the precipitation of the protein and the high molecular weight DNA. The plasmid containing supernatant was separated after applying centrifugation for 10 min at 13,000 rpm. Finally, the extracted plasmids were precipitated by isopropanol, were washed with 70% ethanol, and then were suspended in 30 μl water. Polymerase chain reaction (PCR) was applied to amplify the cellulase gene harboured-plasmid by the most active clone(s) using M13 flanking primer (M13F: 5′AGGCCCTGCACCTGAAG3′ and M13R: 5′ TCAGCGCCTGGTACC3′ [28]. The PCR was carried out for 30 cycles at 94 °C (denaturation) for 1 min, 55 °C (annealing) for 1 min and 72 °C (extension) for 2 min, followed by 10 min at 72 °C (final extension). After completion, a fraction of the PCR product was examined by using the agarose gel electrophoresis [29], and the remnant mixture was purified by using the QIAquick PCR purification reagent (QIAGEN) according to the manufacturer’s recommendation. Afterwards, PCR product was subjected to automated DNA sequencing using the ABI PRISM model 3730 [30].
Sequence similarity
Basic Local Alignment Tool (BLAST) (www.ncbi.nlm.gov/blast) is an algorithm and is an applied program for comparing the primary biological sequence information such as the nucleotides of DNA and/or ribosome-ribonucleic acid (RNA) sequences and amino-acid sequences of proteins. Thus, it was used to determine the similarity of the received assembled sequence of PCR product-insert (upon using the universal primer of pUC19) with the already submitted sequence in the data base. In addition, multiple sequence alignments were performed by using sequence retrieval system (SwissProt http://hcuge.ch/srs5/). SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation (such as protein-function description, domain structure, post translational modification … etc.), a minimal level of redundancy, and a high level of integration with other databases.
Expression of recombinant cellulase gene by most active clone under lacz promoter
This was done by inoculating 50 ml of production LB broth medium (amp & IPTG) dispensed in 250 ml Erlenmeyer flask with 1 ml suspension from freshly prepared overnight pre-culture of the most active clone. A starting pre-culture was grown in 250 ml Erlenmeyer flask containing 50 ml of LBamp medium under shaking (200 rpm) at 37 °C. After inoculation of the production medium, flasks were incubated at 37 °C under shaking (200 rpm); growth and cellulase activity were detected along 48 h. Growth was monitored by measuring the optical density (OD600 nm), while the cellulase activity was followed by measuring the color intensity (DNS assay) under the inducing conditions (IPTG, 1 mM). An additional flask was prepared and was run under the same conditions without IPTG to be used as control (non-induced) in the characterization of recombinant cellSNSY through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and activity staining in the next experiment.
Characterization of the recombinant protein cellSNSY
Protein estimation
In order to estimate the total soluble protein concentration, Lowry method was performed based on pre-prepared standard curve of bovine serum albumin [31].
SDS-PAGE and the activity staining (Zymography)
The recombinant protein samples (induced and non-induced at conc.10–20 μg) were treated with SDS-loading buffer, inactivated for 5 min at 95 °C, suspended on ice, and loaded onto the SDS-PAGE (12 %) according to Laemmli [32]. The gel run for 15 min at 20 mV in the electrode buffer, and then the voltage increased to 60 mV for approximately 3 h. After racing, the gel was stripped from the glass plates, divided into two sections; the first was subjected to stain by Coomassie then de-stained for protein visualization. The second section was incubated for 4 h at 4 °C in the renaturation solution (100 mM Tris–HCl buffer, pH 7.5 containing 0.5% Triton X-100) followed by 2 h at 50 °C in the substrate solution (50 mM Tris–HCl buffer, pH 7.5 containing 0.5% CMC) for cellulase activity detection. Next, the gel was placed in a Petri dish containing Congo-red solution (0.2% w/v) for 10 min at room temperature, and then, it was washed with 1 M sodium chloride. The existence of a faint yellowish halo band indicated that the CMC substrate had been hydrolyzed.
Determination of the optimum temperature
The optimum temperature of the crude recombinant cellSNSY was estimated over different temperatures (ranged 35–80 °C). The reaction was carried out under the initial assay conditions (pH 7.5 and shaking for 15 min).
Determination of the optimum pH
The optimum pH of the crude recombinant cellSNSY was considered upon testing a wide range of pHs (3–10). The experiment was designed by using citrate buffer (pH 3–5); phosphate buffer (6.0–7.0) and tris-HCl (pH 7.5–10) at 50 mM. The reaction assay mixtures were allowed to stand for 15 min under shaking at 45 °C incubation temperature.
Temperature stability
The thermal strength of the recombinant cellSNSY was measured at different temperatures ranging from 25 to 80 °C for (1–2 h), and then the temperature stability of the recombinant cellSNSY protein was checked at − 20 °C for up to 9 months without thawing. Furthermore, the stability was tested upon gradual weakly thawing of the tested recombinant protein which was kept in buffer (50 mM tris-HCl buffer pH 7.5) over 12 days at varied period breaks and the residual activity was determined. The reaction assay was carried out under the resulted optimal conditions and was expressed as % compared to untreated enzyme sample.
pH stability
The pH stability of the recombinant cellSNSY enzyme was checked at pH 7, 7.5, 8, and 8.5 for up to 9 months. The reaction assay was carried out under the resulted optimal conditions (45 °C, pH 8.5) and was expressed as % compared to untreated enzyme.
Effect of certain metals, solvents, surfactant, and EDTA on the enzyme activity
The crude recombinant protein cellSNSY was tested against different cations (MgCl2, CaCl2, ZnCl2, NaCl, FeSO4, MnSO4, and CuSO4), chelating agent (EDTA) at concentrations 1 mM, solvent (DMSO, methanol, ethanol, isopropanol, glycerol), and surfactant (SDS) at concentrations 1% (w/v). The tested enzyme was independently incubated with the tested agent for 15 min; the residual activity was calculated then subtracted from the activity of untreated sample to know the inhibition or activation % of the tested compound.
Statistical analysis
All assays were performed in triplex reactions. The results were expressed as means ± standard deviation which was determined by using Microsoft Office Excel 2013.