The four putative GHF3 β-glucosidases (SRF2g14, SRF2g18, LAB20g4 and LAB25g2) were cloned, expressed in Escherichia coli BL21 (DE3) and purified. Furthermore, their activities were tested with a battery of substrates and the half-saturation (Michaelis) coefficient (K_m), the catalytic rate constant (k_cat), the catalytic efficiency (k_cat/K_m) values and/or specific activities, were determined.

The optimum activity for GHF3 β-glucosidases was observed within a narrow range of temperatures, between 45 and 55°C, and within a narrow pH range (4.0-7.0) being most active at pH close to 5.0 (Figure ​(Figure1).1). Stability assays (measured at 50°C and pH 5.2 in conditions reassembling the supplementation assays – see details below) revealed LAB25g2 to be substantially stable, with only 18% loss of activity after 5 days of incubation; under similar conditions, the half life for SRF2g14, SRF2g18 and LAB20g4 was 18.06 ± 1.9 h, 37.5 ± 3.9 h and 136.1 ± 9.4 h, respectively (Figure ​(Figure1C).1C). The stability features, namely for LAB25g2 enzyme, are better than those of many other microbial β-glucosidases (provided in Additional file 2: Table S​S22).

Saccharification of the cellulose in biomass results in sugar-rich liquid streams useful for the production of bio-ethanol [5]. To estimate the capability of herein discovered β-glucosidases from cow rumen in such process, crude extracts of E. coli BL21 (DE3) cells expressing β-glucosidases were produced as described in Methods and used in saccharification of pre-treated corn stover biomass. First, their protein concentrations and activity levels using pNPβG and cellobiose as substrates, were examined. As shown in Table ​Table22 the cell extract containing β-glucosidase LAB25g2 showed the higher activity level toward both substrates which agrees with activity tests using pure proteins; using cellobiose (58 g l^-1) as substrate, β-glucosidase LAB25g2 produced approx. 2.5 g glucose mg^-1 cell extract h^-1 at 40°C and pH 5.0. The differences in activity performance for the three other β-glucosidases when using cell extracts as compared to assays using pure proteins may be due to differences in expression level and protein solubility during production in E. coli BL21 (DE3), which was evidenced by SDS-PAGE analysis (not shown).

Rumen contents were collected from four rumen-fistulated, non-lactating Holstein cows (average weight of 731 kg) housed at Trawsgoed experimental farm (Aberystwyth, Ceredigion, Wales). Samples were retrieved under the authorities of the UK Animal (Scientific Procedures) Act (1986). The animals were fed a diet composed of a mixture of grass silage and straw (75:25) ad libitum and ~1 kg of sugar beet nuts at 07:00 am; the cows had constant access to fresh water. Sampling was done 2 h after concentrate feeding. Approximately 1 liter of mixed liquid and solid ruminal content was sampled from each cow pooled together to give a composite sample from all cows. The samples were then processed to produce two fractions: strained ruminal fluid (SRF) and liquid-attached bacteria (LAB). For SRF retrieval, total ruminal content was strained through four layers of muslin in order to remove large particles, and SRF was then frozen at −80°C until use. For LAB retrieval, approximately 1 liter of mixed total rumen content was hand squeezed to get rumen liquor, and the solid fraction was put in a large foil tray. The liquid fraction was spun at 2,000 × g, 10 min, 4°C (MSE Europa 24 M, Berthold Hermle KG, Weisbaden, Germany); the supernatant was then strained through a 1 mm² pore-sized nylon mesh to remove feed particles, and spun again at 13,000 × g, 25 min, 4°C. The pellet was washed in a saline solution (made from 180 g NaCl dissolved in 20 L distilled water), and subsequently centrifuged at 13,000 x g, 25 min, 4°C. The pellet was re-washed with distilled water, and spun down at 13,000 x g, 15 min, 4°C. The pellet, containing LAB, was then transferred into a sterile jar and kept at −80°C until DNA extraction.

The gene cloning was performed by PCR using goTaq® DNA polymerase and custom oligonucleotide primers. To amplify the hydrolase genes, the corresponding fosmid was used as the template; the vectors and the pairs of primers are described as follows: SRF2g14 Fwd 5′-gacgacgacaagATGAAGAAGACTCTGTTTTTCGCCTTTGGC-3′ and SRF2g14 Rev 5′-gaggagaagcccggTTATCGTTTCAGGAGGTTCATCTGAACCTGTGG-3′ (total gene length 2361 bp); SRF2g18 Fwd 5′-gacgacgacaagATGAGAAAATCGATTCATCAGATTAGTTTGG-3′ and SRF2g18 Rev 5′-gaggagaagcccggTTATCGCTTCAGTAGGTTGAGTTTCAATTTG-3′ (total gene length 2343 bp); LAB20g4 Fwd 5′-gacgacgacaagATGAAGAAAATCATGCTCCTCTCCGCCACC-3′ and LAB20g4 Rev 5′-gaggagaagcccggTTATTTCATTAAGAGCACGCGGTTGGCGGGC-3′ (total gene length 2295 bp); LAB25g2 Fwd 5′gacgacgacaagATGAAAAAATTACTAACAATTTGCTTCGTAGC-3′ and LAB25g2 Rev 5′-gaggagaagcccggTTACTGTTTCAGAAGATTGAGTTTCTGTTTTG-3′ (total gene length 2340 bp). The PCR conditions were as follows: 95°C for 120 s, followed by 30 cycles of 95°C for 30s, 55°C for 45 s and 72°C for 120 s, with a final annealing at 72°C for 500 s. The PCR products were analysed from agarose gel-purified using the Mini Elute Gel Purification Kit (Qiagen, Hilden, Germany) and cloned into pET-41-Ek/LIC (Novagen, Darmstadt, Germany) according to the manufacturer’s instructions. The resulting plasmids were introduced into the non-expressiong E. coli GigaSingles host and then, after plasmid extraction, into E. coli BL21 (DE3) for protein expression; the clones were selected on LB agar supplemented with kanamycin (30 μg ml^-1). For the enzyme expression and purification, clones were grown overnight at 37°C with shaking at 200 rpm in 100 ml of LB medium containing kanamycin. Afterwards, 25 ml of this culture was used to inoculate 1 l of LB medium, which was then incubated for 4 hours to an OD_600nm of ~0.6 at 37°C. Protein expression was induced by 1 mM isopropyl-β-D-galactopyranoside (IPTG) followed by incubation for 16 h at 16°C. The cells were harvested by centrifugation at 5000 × g for 15 min to yield 2–3 g l^-1 of pellet (wet weight). The cell pellet was frozen at −80°C overnight, thawed and resuspended in 10 ml 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) [pH 7.0] g^-1 wet cells; Lysonase Bioprocessing Reagent (Novagen, Darmstadt, Germany) was then added (4 μl g^-1 wet cells) and incubated for 30 min on ice with rotating mixing. The cell suspension was then sonicated for a total of 1.2 min and centrifuged at 15000 × g for 15 min at 4°C; the supernatant was retained. The His₆-tagged enzymes were purified at 25°C after binding to a Ni-NTA His·Bind resin (Novagen, Darmstadt, Germany). The columns were pre-washed with 20 mM HEPES (pH 7.0), 0.15 M NaCl and 50 mM imidazole, and the enzymes were eluted with 20 mM HEPES (pH 7.0), 0.15 M NaCl and 250 mM imidazole. The monitoring of the enzyme elution was performed by SDS-PAGE and/or activity measurements, using standard assays. Pure enzyme thus obtained was treated with enterokinase, as recommended by the supplier (Novagen, Darmstadt, Germany) to remove the His tags. The purity was assessed as >95% using SDS-PAGE, which was performed with 12% (v/v) polyacrylamide gels, using a Bio-Rad Mini Protean system.