The rumen fluid was obtained from the rumen-fistulated Holstein steers fed alfalfa and commercial concentrates and kept anaerobic condition using stainless steel vacuum insulated bottle and directly transferred to the laboratory. The rumen fluid was filtered through 8 layers of cheesecloth and homogenized using a mixer for 1 min. To maintain anaerobic condition, flushing the oxygen-free CO₂ gas into the bottle was done continuously. Then, the prepared rumen fluid was inoculated into sterilized Dehority’s artificial medium [12] in the Hungate tubes and serially diluted 10⁴ to 10⁶ fold with same medium. The dilutions from each tubes were spread (100 μL) on the plate having chromogenic carboxymethylcellulose (Azo-CMC, Megazyme Inc., Dublin, Ireland) and incubated at 39°C for 48 h in anaerobic chamber filled with 95% of CO₂ and 5% of H₂.

After incubation, the cellulolytic microorganisms were screened through the presence of yellow halos around the colonies (Figure 1). A total of 18 colonies were screened and inoculated into the fresh Dehority liquid medium, and incubated for 3 days. The grown microorganisms were subcultured twice as described above. For identification of the selected bacterial strain, 16S ribosomal RNA gene sequence was amplified by polymerase chain reaction (PCR). Bacterial cell pellet was obtained from each isolated culture by centrifugation (10,000 g×1 min) prior to DNA extraction, which was performed with aid of DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The universal primers 27f (5′-AG AGTTTGATCMTGGCTCAG-3′) and 1492r (5′-ACGGCTACC TTGTTACGACTT-3′) were used to amplify the 16S rRNA gene fragments using PCR [13]. The amplification was carried out using HotStar Taq Master Mix Kit (Qiagen, Germany) with the following cycling parameter: 95°C for 15 min, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 90 s at 72°C with final extension for 7 min. The amplified PCR product was loaded on agarose gel. The 16S rRNA band was purified using a Gel DNA extraction kit (Qiagen, Germany). A sequence similarity search of the purified PCR product was done using BLAST in National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).

Escherichia coli (E. coli) BL21 (DE3) (Novagen pET Systems, Merck Millipore Corp., Darmstadt, Germany) were used for the constructed vector’s host. The E. coli strain was grown in 37°C in Luria-Bertani (LB) Broth containing 30 μg/mL kanamycin with shaking (180 rpm) and the isolated bacteria (E. cellulosolvens sp.) were incubated anaerobically in 39°C in Dehority’s artificial medium containing 1% of carboxymethylcellulose (CMC) without agitation.

Genomic DNA and plasmid DNA were purified using G spin TM Genomic DNA extraction kit and plasmid DNA extraction kit (iNtRON Inc., Seoul, Korea) respectively. The plasmid vector, pET41b (Novagen’s pET Systems, Merck Millipore Corp., Germany) is used for cloning and expression of Cel5A. For amplification of the Cel5A gene’s open reading frame (ORF) from genomic DNA of E. cellulosolvens, AccuPower PCR PreMix (Bioneer Inc., Daejeon, Korea) and primer set EC-f (5′-AG ATATACATATGAAAGGAAACTGGTTG-3′ with NdeI site) and EC-r (5′-AGATATACTCGAGCGGGATCAGGTTGC-3′ with XhoI site) were used and PCR was performed with the following cycling temperature and time: 95°C for 15 min, followed by 40 cycles of 30 s at 94°C, 1 min at 60°C, and 3 min 30 s at 72°C with final elongation for 10 min. The DNA fragment of Cel5A and pET-41b was digested with the same restriction enzymes (NdeI and XhoI). The enzyme treated target DNA and vector was purified from the Agarose gel and ligated, then transformed into E. coli T10F′ for checking its sequence using NCBI sequence database. The recombinant plasmid was purified from E. coli T10F′ and then transformed into E. coli BL21 (DE3) for protein expression. The transformant was identified by colony PCR [14] using same primer set and PCR condition. The transformants were incubated overnight in LB media containing kanamycin (30 μg/mL) and the culture was used as the DNA templates for the colony PCR. The PCR products were electrophoresed with 0.8% agarose gel. The isolates which had the clear 3.5 kb band at the gel, were selected for the further experiment.

A total of 150 mL of LB broth containing 30 μg/mL kanamycin was prepared and inoculated with transformed E. coli with pET41b-Cel5A then grown at 37°C for 3 h in a shaking incubator set at 180 rpm. After the optical density in 600 nm (OD₆₀₀) value of the culture reached around 1.0, Isopropyl β-D-1-thiogalactopyranoside (IPTG) 1 mM was added then shook overnight in 16°C. The cell pellet was collected after centrifugation at 10,000 g for 10 min and then lysed by Bugbuster reagent (Novagen’s pET Systems, Merck Millipore Corp., Germany). The cell lysate was harvested by centrifugation at 12,000 g for 20 min and the His-tagged Cel5A was purified using Ni-NTA His-Bind column (Novagen’s pET Systems, Merck Millipore Corp., Germany). A gradient of 5 to 250 mM imidazole in Tris-HCl buffer containing 500 mM NaCl was employed to purify the fusion protein. After elution of the target protein from the resin, the product was used for further characterization of its properties.

Endoglucanase activity was determined using 3, 5-dinitrosalicylic acid (DNS) reagent [15]. The purified enzyme with the addition of 1% (w/v) of CMC prepared in 0.05 M sodium citrate buffer (pH 4.0) was incubated in a water bath for 1 h at 37°C. After the reaction was ended by addition of DNS reagent, the mixture was boiled for 5 min in water for color development and then cooled rapidly. The reducing sugar (liberated) was measured at 540 nm in a UV-VIS spectrophotometer (UV-1601 PC, Shimadzu, Japan). Glucose (Sigma, St. Louis, MO, USA) was used for the standard curve of endoglucanase and one unit of enzyme activity (U/mL) was defined as the amount of enzyme releasing 1 μM of reducing sugar per a minute. All the assays were done in triplicate.

The optimum pH of the purified enzyme was measured in appropriate buffers (0.05 M KCl-HCl, pH 1.0 to 2.0; 0.05 M sodium citrate, pH 3.0 to 5.0; 0.05 M sodium phosphate, pH 6.0 to 8.0; 0.05 M glycine-NaOH buffer, pH 9.0 to 10.0) at 50°C. Enzyme stability at each different pH was determined by pre-incubating the enzyme in the pH buffer solutions as described above at 4°C for 24 h. To determine the optimum temperature for endoglucanase, the purified enzyme preparation was incubated at 20°C to 80°C in 0.05 M sodium citrate buffer (pH 4.0). The effect of temperature on enzyme stability was measured by pre-incubating the enzyme for 30 or 60 min at selected temperatures (20°C to 50°C) then mixed with buffer and CMC solution for 30 min at 50°C. The relative activity of the purified enzyme at various pH or temperature was indicated as the percentage of the maximum enzyme activity at optimal condition. While, the relative activity in stability studies was calculated in contrast to the observed maximum activity at individual pH or temperatures. The influence of chemicals on the enzyme activity was measured after pre-incubating the purified enzyme with 1 mM final concentration of various metal ions (Ca²⁺, Co²⁺, K⁺, Mn²⁺, Ni²⁺, Mg²⁺, Fe²⁺, Cu²⁺, and Zn²⁺) and nonionic reagents (Triton, Tween 20, sodium dodecyl sulfate [SDS], and ethylene diamine tetra acetic acid [EDTA]) in 0.05 M sodium citrate (pH 4) buffer at 4°C for 4 h. Varying the concentration of CMC solution as a substrate from 7.5 to 20 mg/mL was applied to calculate theoretical kinetic values of recombinant Cel5A (rCel5A). The Km and Vmax were calculated using Michaelis-Menten equation at pH 4 and 50°C. All enzyme assays were done in 3 replicates and average values were presented.

The data of enzyme activity were analyzed using the MIXED procedure of SAS (SAS institute Inc., NC, USA) with an appropriate statistical model for each analysis. Differences among different groups were compared with the Tukey’s range test if a significant effect was observed. A statistical significance was declared at p<0.05.

The diluted rumen fluid was spread on the Dehority agar plate contaning 0.5% of Azo-CMC (w/v) and incubated at 39°C for 2 to 3 days. A total of 18 colonies which had clear zones around their area (Figure 1) were randomly selected from the plate and inoculated into the sterilized fresh Dehority liquid medium in Hungate tubes and subcultured. The enzyme activity assay results of final cultivation tubes showed that 8 isolates (1, 2, 3, 6, 12, 16, 17, and 18) had remarkable activity (Figure 2). From those isolates, genomic DNA was extracted and used for the identification study. All isolates had highest similarity (98% to 99%) with E. cellulosolvens Ce2 (Accession number: AY178842.1).

The Cel5A gene′s ORF was amplified from the genomic DNA of isolated E. cellulosolvens using the primers (EC-f, EC-r) which had NdeI, XhoI restriction enzyme sites. The amplified PCR product was digested using adequate restriction enzyme, and ligated with NdeI, XhoI treated pET41b vector (Novagen’s pET Systems, Merck Millipore Corp., Germany) and transformed into E. coli T10F′. The cloned gene was sequenced at NICEM (Seoul National University, Seoul, Korea). The amino-acid sequence was characterized and aligned with the previous published Cel5A gene (Accession number: AB179780.1) using Genbank database (data not shown).

The constructed plasmid was purified and transformed into E. coli BL21 for the production of recombinant enzyme. Isolation of the recombinant clones was done from the LB media containing kanamycin. After colony PCR with the isolated clones, electrophoresis result of the PCR product is presented at Figure 3. The clones having the size 3.5 kb band were selected and used as the host for the expression of the recombinant enzyme. The IPTG induced rCel5A was purified with Ni-NTA His binding resin and used for further characterization.

To determine kinetics of this enzyme, the endoglucanase activity was measured with fixed amount of purified enzyme and 0.5 to 20 mg/mL CMC as a substrate. Theoretical maximal reaction velocities (Vmax) and Michaelis-Menten constant (Km) were calculated against CMC using Lineweaver-Bulk plot (Table 1). Turn over number (Kcat) and catalytic efficiency (Kcat/Km) values of the purified enzyme were 96.69 and 6.88, respectively. The enzyme activity under various conditions was measured to determine the optimal pH, temperature and its stability. The results showed that the purified enzyme had maximum activity at pH 4 and stayed active over pH 3 to 5, showing at least 70% of its optimal activity (Figure 4). It was observed that purified enzyme was highly stable at pH 3 to 9 after 24 h pre-incubation in each pH buffer, and it could maintain its original activity up to almost 90% to 100% (Figure 5). The purified enzyme was active over a broad range of temperature in between 20°C to 80°C. The maximal activity was obtained at temperature of 50°C (Figure 6). However, the enzyme activity dropped sharply as the temperature increased over 40°C after pre-incubation for 30 or 60 min at each temperature (Figure 7). The effect of metal ions and non-ionic reagents after 4 h pre-incubation, is presented in Figure 8. The relative activity was calculated in contrast to the observed maximum activity at no chemical addition. The addition of K⁺ (potassium), Ni²⁺ (nickel), and Fe²⁺ (Iron) cations enhanced enzyme activity by more than 15%. While, the addition of Mn²⁺ (manganese), Cu²⁺ (copper), Zn²⁺ (zinc), divalent cations exhibited negative effect on enzyme activity by about 20% to 50%. Other divalent cations (Ca²⁺, Co²⁺, and Mg²⁺) did not showed any noticeable effect to the enzyme activity. The presence of SDS inhibited the enzyme activity by about 20% while no negative effect was observed with other nonionic reagents.