Cloning Cellulase Genes from Victoria Falls Rainforest Decaying Logs Metagenome

Sampling was carried out at the Victoria Falls rain forest (17°55′28″S; 25°51′19″E). Logs with visible signs of decomposition were collected by breaking, using gloved hands, into sterile sampling bags along with soil from underneath the logs. The sampling was not selective of wood type; both hard and soft woods were included. A compound sample was prepared by grinding the various logs and soils into fine particles using a sterile pestle and mortar. The logs were further grounded using a Waring^® laboratory blender 7010S (Waring, USA). A 20 g portion was suspended in 180 ml sterile distilled water and used to extract metagenomic deoxyribonucleic acid (DNA).

The Epicentre Meta-G-Nome DNA isolation kit (Epicentre Biotechnologies, USA) was used to extract metagenomic DNA from 100 ml of the compound sample in distilled water, following the manufacturer’s instructions. Gel electrophoresis on a 1% agarose gel was used to validate the quality of the isolated DNA by comparison to the Fosmid Control DNA (40 kb; 100 ng/μl) provided in the kit. Universal degenerate cellulase primers: primer 1 5’-CATATGGCAAGTCCGTCCGTGACGAA-3’ and primer 2 5’-CTCCAGTTTCTGATACCCCATTCACC-3’ (Liu et al. 2011) were used in PCR reactions with the metagenomic DNA as the template. The PCR reactions were carried out in 25 μl volume, which comprised of 0.2 mM dNTPs, 10 X Dream Taq buffer, 1 μM primer 1, 1 μM primer 2, 1 μg DNA sample, and 1.25 U Dream Taq polymerase enzyme. A titration of MgCl₂ of concentrations 2 mM, 3 mM, and 4 mM was carried out, and the reaction made up to 25 μl accordingly with nuclease-free water. The PCR reactions were carried out in the steps of primary denaturation at 95°C for 2.5 minutes, followed by 30 amplification cycles of denaturation at 95°C for 0.5 minutes, annealing at 60°C for 30 seconds, extension at 72°C for 1.5 minutes, and a final extension step of 10 minutes at 72°C.

The amplicons from the cellulase PCR reaction using primer 1 and primer 2 were cleaned and concentrated using the Zymoresearch Clean and Concentrate Kit according to the manufacturer’s instructions (Zymo Research, USA). The purified amplicons of sizes 400 base pairs to 1,200 base pairs were treated with the blunting enzyme mix from the Thermo Scientific pJet cloning kit (Thermo Fischer Scientific, USA) and subsequently ligated using T4 ligase into pUC19 that had been cut with SmaI. The ligation reaction mixture was kept at –20°C and used directly for transformation. For transformation, 3 μl of the ligation reaction mixture was added to 100 μl of Z competent Escherichia coli DH5α cells thawed on ice. The transformation reaction mixture was gently mixed while on ice, 50 μl of the reaction was spread on a pre-warmed (37°C) LB agar plate (yeast extract 10 g/l; tryptone 10 g/l; NaCl 10 g/l; Agar 15 g/l) plate supplemented with 50 mg/ml ampicillin, 0.1 M IPTG and 50 mg/ml X-Gal (LB-AMP-IPTG-X-Gal) and incubated at 37°C for 18 to 24 hours according to the method by Maniatis et al. (1982). White colonies were picked and enriched in LB broth with ampicillin for 24 hours at 37°C with vigorous shaking. The clone cultures were stored in 15% glycerol at –20°C for further analysis.

The alkaline lysis method (Birnboim and Doly 1979) was used to extract plasmids from the selected white colonies. The presence and quality of plasmid DNA were validated by gel electrophoresis on a 1% agarose gel.

The clones were examined for cellulase activity by drop inoculating 5 μl of overnight cultures in (LB-AMP-IPTG-X-Gal) broth on Mandel’s agar plates (CMC 10 g/l; NH₄SO₄ 1.4 g/l; KH₂PO₄ 2 g/l; Urea 0.3 g/l; CaCl₂ 0.4 g/l; MgSO₄ 0.6 g/l and trace elements MnSO₄ 1 mg; ZnSO₄ 1.4 mg; CoCl₂ 3.7 mg) (Acharya et al. 2008) enriched with ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG), and X-Gal. After incubation at 37°C for 24 hours, the plates were flooded with Gram’s iodine, according to Kasana et al. (2008). The clones showing cellulase activity were noted by the presence of clearance zones and were selected for further analysis.

The selected clones were pre-cultured overnight in LB broth formulated following Maniatis et al. (1982), enriched with IPTG, X-Gal, and ampicillin, and incubated at 37°C with shaking at 160 rpm (Liang et al. 2014). After incubation, 2 ml of the culture was inoculated into 50 ml of Mandel’s broth enriched with IPTG, X-Gal, ampicillin, and 1% CMC as the sole carbon source in 250 ml conical flasks. Incubation was done at 37°C for 72 hours with shaking at 160 rpm. The cultures were then centrifuged at 5,000 × g for 15 minutes at 4°C, and the supernatants (crude enzyme) were collected for the detection of cellulases (Sethi et al. 2013).

Cellulase activity was measured by using 3,5-dinitrosalicyclic acid (DNS) (Miller 1959) by determining the reducing sugars liberated from substrates in 50 mM citrate buffer at pH 5. The method was scaled down to micro-titre volumes, which were 25 μl substrate, 25 μl of buffer, and 25 μl of crude enzyme, according to King et al. (2009). For the endoglucanase assay, the mixture containing carboxymethylcellulose as substrate was incubated at 50°C for 30 minutes. For the exoglucanase assay, the mixture had Avicel as substrate, and incubation time was 1 hour at the same temperature as for the endoglucanase detection. The micro-titre-based filter paper assay was carried out for total cellulases by incubating a 7 mm disc of Whatman No. 1 filter paper in crude enzyme and 40 μl of 50 mM citrate buffer pH 5 for 1 hour at 50°C (Xiao et al. 2004). The reactions were stopped by the addition of DNS reagent and boiling for 5 minutes at 100°C for color development. The absorbance of the reaction mixtures was read at 540 nm. A calibration curve of glucose standard solutions was used to determine the concentration of glucose produced. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of glucose per minute.

The selected clones were cultured in Mandel’s media broth enriched with IPTG, X-Gal, and ampicillin at 37°C with agitation at 160 rpm for five days. At 24-hour intervals, 2 ml of broth was aseptically withdrawn from the cultures and centrifuged at 3,300 × g for 5 minutes. The obtained supernatants were assayed for cellulase activity over time, as previously described.

The optimum temperature for enzyme activity was determined by incubating the crude enzyme in 1% CMC, 1% Avicel, or 7 mm filter paper discs substrates at temperatures 45, 50, 55, 60, and 70°C for 5 minutes (Quiroz-Castañeda et al. 2009). Production of reducing sugars was then determined using DNS as previously described for cellulase assessment.

To determine the optimum pH for cellulase activity, 1% CMC, 1% Avicel or a 7 mm filter paper disc were prepared in 10 mM buffer solutions in the 4–9 pH range (acetate buffer for pH 4 and 5, phosphate buffer for pH 6, and sodium for 7 and TRIS-HCl for pH 8 and 9). The substrates were then incubated with 0.1 ml of the crude enzymes at their optimum temperatures. Production of reducing sugar was determined using the DNS as previously described for cellulase.

Crude enzymes sampled at optimum production time for each of the clones were used to determine the diversity of cellulases produced by the bacterial isolates. Enzyme assays were carried out at optimum temperatures and pH for the isolate, as predetermined in the sections above. Production of reducing sugars from the exoglucanase, endoglucanase, and total cellulases were determined using the DNS, as previously described.