Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil

Background Cellulose is the most prevalent biomass and renewable energy source in nature. The hydrolysis of cellulosic biomass to glucose units is essential for the economic exploitation of this natural resource. Cellulase enzyme, which is largely generated by bacteria and fungus, is commonly used to degrade cellulose. Cellulases are used in a variety of industries, including bioethanol manufacturing, textiles, detergents, drugs, food, and paper. As part of our quest to find an efficient biocatalyst for the hydrolysis of cellulosic biomass, we describe the amplification, cloning, and sequencing of cellulase (cel9z) from Bacillus licheniformis strain Z9, as well as the characterization of the resulting enzyme. Results Cellulase was partially purified from B. licheniformis strain Z9 using (NH4)2SO4 precipitation and Sephadex G-100 gel column chromatography with 356.5 U/mg specific activity, 2.1-purification fold, and 3.07 % yield. The nucleotide sequence of the cellulase gene was deposited to the GenBank, B. licheniformis strain Z9 cellulase (cel9z) gene, under accession number MK814929. This corresponds to 1453 nucleotides gene and encodes for a protein composed of 484 amino acids. Comparison of deduced amino acids sequence to other related cellulases showed that the enzyme cel9z can be classified as a glycoside hydrolase family 9. SDS-PAGE analysis of the purified enzyme revealed that the molecular mass was 54.5 kDa. The optimal enzyme activity was observed at pH 7.4 and 30 °C. The enzyme was found to be strongly inhibited by Mg2+ and Na+, whereas strongly activated by Fe3+, Cu2+, and Ca2+. Conclusions B. licheniformis strain Z9 and its cellulase gene can be further utilized for recombinant production of cellulases for industrial application. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-022-00317-4.

enzymes [5]. Endoglucanase works by cleaving intermolecular β-1,4-glycosidic bonds inside the cellulose chain to liberate oligosaccharides for exoglucanase and β-glucosidase to further hydrolyze [6]. Endoglucanase and exoglucanase create cello-oligosaccharides and cellobiose from cellulose, which are then converted to glucose by β-glucosidase [7]. Cellulases derived from fungi, bacteria, and yeasts have been studied extensively [3]. Different bacterial species as Bacillus [8], Clostridium, and Ruminococcus [9] have all been reported to produce cellulases. Cellulases are used in juice extraction processes, pulp and paper, textile industry, secondary metabolites, animal feed, extraction of vegetable dyes, and the production of fermentable sugars for biofuels [10]. Thus, the demand for this enzyme is increasing exponentially [11].
Cellulases were isolated and identified first only from culturable bacteria using a fermentation technique, and the whole cellulase potential of the site was not completely investigated. Due to the high substrate cost necessary for cellulase induction and the problems of maintaining the appropriate conditions for cellulase production, fermentation approaches have limitations [12]. As a result, recent breakthroughs in molecular approaches, such as the production of metagenomic libraries, will expand the pool of cellulolytic enzymes suitable for biofuel research, potentially solving these challenges. This new method will allow the extraction of cellulases and related enzymes from bacteria that are otherwise unculturable and may create novel enzymes with specialized applications [13]. Uncultured microorganisms make up a large part of the natural world's biodiversity. Only 0.1-1% of the natural environment is made up of microorganisms that can be cultivated using conventional laboratory techniques [14]. Genes derived from metagenomic techniques have proven to be useful in identifying novel genes with specialized functions [15]. A new strategy for discovering novel enzymes is to clone and express the cellulase gene in an efficient host cell like Escherichia coli [16].
Our earlier studies based on isolation and identification of cellulolytic B. licheniformis strain Z9 from soils having the highest cellulolytic activity [17]. The present study is concerned with the amplification, sequencing, and cloning of the gene encoding cellulase. Also, biochemical characterization was investigated to determine the optimum enzyme activity. This research, combined with ongoing expression research, could lead to a low-cost system based on genetically recombinant Escherichia coli with industrial applications.

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′ATG GCT TAT TCT GCC GCA ATC CTG TCA-3′) and reverse primer Zr1 (5′ GGC CAT GTC GCT CTG CAC GTA GTG G-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  [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:// Unipr otKB. 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.

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.

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 nontreated 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 + , Cu 2+ , Co 2+ , Ca 2+ , Fe 3+ , Mg 2+ , 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. graph pad. com.

Molecular identification of cellulase gene
A total volume of 50 μL of PCR reaction was used for amplification of cellulase cel9z from B. licheniformis strain Z9. The length of the fragments was about 1500 bp. The PCR products of strain Z9 were sequenced to obtain an open reading frame (ORF) of 1453-bp gene sequence, which was consistent with the result of electrophoresis. This band was then excised, eluted, purified, and then subjected to cloning. The phylogenetic analysis of the cel9z and its related hits was carried out. Analysis revealed that the isolated B. licheniformis strain Z9 cellulase (cel9z) gene formed a distinct clade, containing endoglucanase and cellulase of B. licheniformis (Fig. 1A).

Nucleotide sequence analysis
The obtained sequence was analyzed and compared with a sequence in the nucleotide database (NCBI) using the BLAST algorithm. The 1453 bp fragment of cel9z was fully sequenced at Macrogen Company, Seoul, South Korea, encoding a protein of 484 amino acids, with a calculated molecular mass of about 54.4 kDa as shown in Supplementary data 1.

Analysis and alignment of cel9z protein sequences
The deduced amino acid sequence encoding cel9z was conducted from the sequence of nucleotides data. The amino acid sequence was aligned with the amino acid sequences of other organisms using UniProtKB program. The results Conserved domain analysis of cellulase cel9z protein revealed the presence of glycosyl hydrolase 9 domain of glycoside hydrolase 9 superfamily in the protein. The domain analysis found a specific hit for the with e value at 2.12e−165. Consequently, there was high confidence in the association between the protein query sequence and a conserved domain, resulting in a high confidence level for the inferred function of the protein query sequence. Further, hydropathy plots indicated that the protein was hydrophilic with a hydropathy score of − 3.

Enzyme purification
The purification profile of cellulase (cel9z) of B. licheniformis were listed in Table 1. Ammonium sulfate precipitation gave a purification fold of about 1.96 with a specific activity of 333.4 U/mg and 51.1% yield, while purification fold of about 2.1 and specific activity of 356.5 U/mg with 3.07% yield was achieved for gel filtration chromatography ( Fig. 2A).

SDS-PAGE and zymogram analysis
Enzyme activity is concentrated and subjected to SDS-PAGE The enzyme cel9z showed a single band on  Table 1 Overall purification profile of cellulase from Bacillus licheniformis strain-Z9 a , b values represent the mean of the values from three independent experiments, with a standard deviation of < ± 5% Specific activity = total activity/total protein c Purification fold = specific activity of particular purification step/specific activity of the crude enzyme d Yield (%) = (total activity of the particular purification step/total activity of the crude enzyme) × 100 SDS-PAGE with a molecular weight of approximately 54.4 kDa for both crude extract and partially purified enzyme. The enzymatic activity of partially purified cel9z is confirmed by CMC zymographic analysis. It is shown as a yellow halo against a red background (Fig. 2B). Enzyme activity was concentrated and subjected to SDS-PAGE. The enzyme cel9z showed a single band on SDS-PAGE with a molecular weight of approximately 54.4 kDa for the partially purified enzyme. The enzymatic activity of purified cel9z was confirmed by CMC zymographic analysis. It was shown as a yellow halo against a red background (Fig. 2B).

Screening of optimal temperature
The effect of temperature on the enzyme activity of cel9z from B. licheniformis was determined at various temperatures ranging from 10 to 60 °C as shown in (Fig. 3A).
Results demonstrated that the optimum temperature of the enzyme was around 30 °C.

Screening of optimal pH
The effect of pH on the enzyme activity of cel9z from B. licheniformis was also examined at various pH levels ranging from pH 4.3 to pH 10.5. Cel9z exhibited the highest activity at pH 5.5-8, with an optimum pH of the enzyme activity at pH 7.4 (Fig. 3B).

Screening of the effect of metal ions and chemical reagents
The effect of metal ions and some chemical reagents at a final concentration of 1 mM (Ag + , Na + , Cu 2+ , Co 2+ , Ca 2+ , Fe 3+ , Mg 2+ , SDS, and EDTA) on cel9z enzyme activity from B. licheniformis strain Z9 was studied. The enzyme activity was strongly inhibited by Mg 2+ and Na + between 31 and 53.8% whereas Fe 3+ , Ca 2+ , and Cu 2+ significantly activated the enzyme activity between 98.4 and 83.2% (Fig. 3C). Enzyme activity demonstrated a

Discussion
The increased demand for finding new sources of biofuels and renewable energy as an alternative to fossil fuels is one of the most common interests of researches worldwide. The enzymatic hydrolysis of cellulosic biomass by cellulases has been increased in several studies for their employment in many industrial applications. Different Bacilli species presented relevant results related to cellulase production [30]. In this study, gene encoding cellulase (cel9z) was derived from B. licheniformis strain-Z9 and was successfully amplified, purified, and sequenced. Cloning of powerful cellulase genes might be very important for the successful production and consequently industrial application of the enzyme. Furthermore, researchers have concentrated on producing recombinant cellulase. As a host, E. coli does not require any special media and grows rapidly. The key benefit of recombinant cellulase is that it may be scaled up to commercial levels without the usage of expensive substrates [31]. Cel9z from B. licheniformis strain Z9 has been cloned into the pSC-A-amp/kan PCR Cloning Vector, and further research into cel9z in a recombinant expression system is underway.
To provide pure and homogeneous cellulase for industrial applications, a comprehensive purification process is required. Therefore, the purification of crude enzyme extracts from B. licheniformis strain-Z9 was achieved through the combination of ammonium sulphate precipitation, 40-80% saturation followed by gel filtration on Sephadex G-100 with a total yield of 3.07% and 2.1-fold purification. Enhancement of specific activity is observed in each of the purification steps. In this respect, Azadian et al. [24] reported overall purification fold of the enzyme about 8.85 with the specific activity of 412.32 U/ mg of cellulase from Bacillus sp. The purified enzyme was emerged as only one protein band with a molecular mass of 54.4 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming its purity.
The molecular weight 54.4 kDa of the partially purified enzyme is close to that reported by Assareh et al. [32]. Our results are also similar to those obtained by Rawat and Tewari [8] where cellulases of Bacillus species reveal a variable molecular mass ranging from 24.4 to 185 kDa.
CMCase production over a broad pH range is considered a characteristic of cellulases secreted by Bacillus sp. [33]. Similar results were obtained by Aygan et al. [34] who observed endoglucanases activity for the genus Bacillus at pH values from 8.5 to 10.0. In contrast, other studies have reported that the optimum pH for purified cellulase from B. circulans was 4.5 [35] while cellulase was produced by Bacillus sp. C14 was 11 [36]. The pH of the growth medium influences many enzymatic reactions by affecting the transport of chemical products and enzymes across the cell membrane [37]. A wide pH range is required for the application of enzymes in numerous conditions [3]. In the present study, the optimum pH was 7.4 and it was active over the broad pH region of 5.5-8. Obtained results are in good agreement with Pokhrel et al. [36] who reported that the optimum pH for the enzyme of Bacillus subtilis ranged between 6.5 and 7.5. Also, the optimum pH for B.licheniformis NLRI-X33 was 7.5 [38]. Other similar findings by Ekwealor et al. [39] showed very good activity at pH range 6 to 9.
The optimum temperature of the partially purified cellulase was found to be around 30 °C. The results are in close agreement with the study of B. licheniformis isolated from compost by Nallusamy et al. [40] at 37 °C, cellulase activity retained 87% activity. Also, the maximum endo (1,4) β-d-glucanase production from B. licheniformis KIBGE-IB2 was observed at 37 °C [41]. The same maximum cellulase activity at 37 °C of B. pumilus EWBCM1 isolated from the gut of earthworm was determined by Shankar and Isaiarasu [42]. There is a remarkable decrease of enzyme activity over 45 °C and retained only 42% activity at 60 °C. In this respect, Maurya et al. [43] reported that over a certain temperature, enzyme activity decreases with an increase in temperature because of enzyme denaturation. During the saccharification and fermentation processes, a lowtemperature adaptation can save energy and money [3]. As a result, it can be used in a variety of industries, including food, feedstuffs, textiles, and pharmaceuticals [44]. According to current research, cel9z optimal activity occurs at a low temperature, which may be helpful for its prospective application.
The partially purified cellulase was also screened to determine its enzymatic activity in the presence of different metal ions Ag + , Na + , Cu 2+ , Co 2+ , Ca 2+ , Fe 3+ , Mg 2+ , SDS, and EDTA. The cellulase activity was found to be enhanced in the presence of Fe 3+ , Cu 2+ , and Ca 2+ . However, the enzyme was inhibited by the presence of metal ions Mg 2+ , and Na + . The opposite result was reported by Azadian et al. [24], where the CMCase activity was enhanced in the presence of Mg2+ (110%). The inhibition by the same divalent cations was also reported in cellulase enzymes from B. licheniformis strain MK7 and Bacillus amyoliquefaciens DL-3 [45]. The inhibitory effect of Mg 2+ in this study is contrary to the work of Ekwealor et al. [39] who reported the stimulatory effect of MgSO 4 on the activity of CMCase. Reports also revealed that metal ion in the form of a salt such as CaCl 2 ·6H 2 O provides protection to some enzymes against thermal denaturation and plays an important role to stabilize the native forms at high temperatures [32]. The inhibition of cellulase by Mg 2+ and Na + ions may be linked to the competition between the exogenous cations and the protein-associated cations, resulting in a decreased metalloenzyme activity. EDTA was found to be inhibitory to the activity of cellulase used in this study. Low concentration of low valent metal ions had almost no inhibition effects on enzyme activity. Therefore, employing cel9z in the industry is promising.
Currently, some developed studies were applied to produce industrial cellulase by Bacilli strains due to the high bacterial growth, compared to fungi, and their ability to adapt to low-cost carbon sources, such as sugar cane bagasse. In addition, bacterial cellulase is considered a potent enzyme for the application in second-generation ethanol produced from sugarcane biomass in Brazil [46]. Further studies of the cellulase gene (cel9z) will provide insights into the function of the protein and its biochemical properties.

Conclusion
In the current study, successful cloning of the geneproducing cellulase appears to be a viable technique that will lead to the creation of a low-cost effective strategy for achieving considerable lignocellulosic waste bioconversions. Furthermore, the purified cellulase (cel9z) from B. licheniformis Z9 showed a wide pH and temperature range. Further expression of the cloned gene will reveal information regarding the function of the produced protein, as well as its biochemical properties and prospective industrial applications.