Materials and strains
Chemicals, biochemicals and solvents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were of p.a. (pro-analysis) quality. The oligonucleotides used for DNA amplification, mutagenesis and sequencing were synthesised by Sigma Genosys Ltd. (Pampisford, Cambs, UK). The restriction and modifying enzymes were from New England Biolabs (Beverly, MA, USA). The Ni-NTA His·Bind chromatographic media were from QIAGEN (Hilden, Germany). The E. coli EPI300-T1R strain (Epicentre Biotechnologies; Madison, WI, USA) used for the fosmid library construction and screening and E. coli GigaSingles for the cloning and BL21 (DE3) for the expression using the pET-41 Ek/LIC vector (Novagen, Darmstadt, Germany) were cultured and maintained according to the recommendations of the suppliers. pNP-β-D-glucopyranoside (pNPβG), pNP-β-D-cellobioside (pNPβC), the cello-oligosaccharides (ranging from cellobiose to cellopentaose), the D-glucose assay kit and the almond β-glucosidase G0395, were all provided by Sigma Chemical Co. (St. Louis, MO, USA). Agrobacterium sp. β-glucosidase E-BGOSAG was provided by Megazyme (Bray, Ireland).
Metagenomic library construction and enzyme screening
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.
Total DNA was extracted from LAB and SRF microbial communities as described previously , using the G’NOME® DNA Isolation Kit (Qbiogene, Heidelberg, Germany). Purified and size-fractioned DNA was ligated into the pCCFOS fosmid vector and further cloned in E. coli EPI300-T1R according to the instructions of Epicentre Biotechnologies (WI, USA) and a procedure described earlier . Fosmid clones (16,896) harbouring approximately 600 Mbp of community genomes were arrayed using the QPix2 colony picker (Genetix Co., UK) and grown in 384-microtitre plates containing Luria Bertani (LB) medium with chloramphenicol (12.5 μg ml-1) and 15% (v/v) glycerol and stored at −80°C.
To screen for GH activity, the clones were plated onto large (22.5 × 22.5 cm) Petri plates with LB agar containing chloramphenicol (12.5 μg ml-1) and the induction solution (Epicentre Biotechnologies; WI, USA) as recommended by the supplier to induce a high fosmid copy number. An array of 2,304 clones per plate was set. After overnight incubation, each library was screened for the ability to hydrolyse pNPβG and pNPβC. For screens, the plates (22 × 22 ccm) were covered with an agar buffer substrate solution (40 ml of 50 mM sodium acetate, pH 5.6, 0.4% agarose and 5 mg ml-1 of pNPβG and pNPβC as substrates). Positive clones appeared due to the formation of a yellow colour. The positive clones were selected and their DNA inserts fully sequenced with a Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at Life Sequencing S.L (Valencia, Spain).
Cloning, expression, purification and characterisation of plant polymeric substance hydrolases
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 OD600nm 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.
To produce E. coli BL21 (DE3) cell extracts expressing β-glucosidases, clones were grown overnight at 37°C with shaking at 200 rpm in LB medium containing kanamycin (30 μg ml-1). Afterwards, 0.3 ml of this culture was used to inoculate 30 ml of LB medium in a 250 ml flask, which was then incubated to an OD600nm of ~0.8. Protein expression was induced by 1 mM IPTG followed by incubation for 4 h. The cells were harvested by centrifugation at 5000 × g for 15 min and re-suspended in 10 ml McIlvaine buffer (100 mM pH 5.0) containing lysozyme (0.1 mg ml-1). The cell suspensions were then sonicated until a completed cell lysis was observed by microscope and centrifuged at 15000 × g for 15 min; the supernatants were retained and their protein concentrations and activity levels using pNPβG and cellobiose as substrates, examined.
For the enzyme characterisation, the absorbance was measured using a BioTek Synergy HT spectrophotometer under the following conditions: [Enzyme] ranging from 0 to 300 nM, [substrate] ranging from 0 to 50 mM in 100 mM buffer, T = 40°C. For the hydrolysis of the pNP derivatives, the corresponding volume of a pNP derivative stock solution (150 mM) was incubated for 2–20 min in appropriate buffer and measured at 410 nm in 96-well microtiter plates . The substrates tested included pNPβG and pNPβC. For cello-oligosaccharides the level of released glucose was determined, under the same conditions, using a glucose oxidase kit (Sigma Chemical Co., St. Louis, MO, USA). The initial rates were fitted to the Michaelis–Menten kinetic equation using non-linear regression to determine the apparent K
m and k
cat; kinetic parameter calculations were performed based on the molecular masses listed in Additional file 1: Table S1. Substrate specificity was investigated also using carboxymethylcellulose (CMC), lichenan, barley glucan, laminarin and avicel (all from Sigma Chemical Co., [St. Louis, MO, USA]) and filter paper (Whatman, England). Enzymatic activity was quantified by measuring release of reducing sugars according to Miller  using [enzyme] of 20 nM, [substrate] of 10 mg ml-1 in 50 mM sodium acetate buffer pH 5.6, T = 40°C. In all cases, one unit (U) of enzyme activity was defined as the amount of enzyme producing 1 μmol of reducing sugars in 1 min under the assay conditions. Unless otherwise stated, standard assays were performed using [enzyme] of 20 nM, pNPβG] of 10 mg ml-1 in 50 mM sodium acetate buffer pH 5.6, T = 40°C.
The pH and temperature optima were determined in the range of pH 4.0–8.5 and 20–70°C in assays containing [enzyme] of 12 nM and [pNPβG]o of 10 mg ml-1. Optimal pH was measured at 40°C; the following buffers (20 mM) were used: acetate (pH 4.0-6.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0-7.0), HEPES (pH 7.0-8.0), Tris–HCl (pH 8.0-8.5). pH was always adjusted at 25°C. Optimal temperature was determined in 50 mM sodium acetate buffer pH 5.6. All of the values were determined in triplicate and were corrected for the spontaneous hydrolysis of the substrate. The results shown are the averages of three independent assays ± the standard deviation.
pH and thermal stability assays were performed in 100 ml flasks with 12 g of pre-treated biomass (see below) and 5 g β-glucosidase solution at 25 U ml-1 (sodium acetate 20 mM), 50°C, pH 5.2). For activity test at different time points, protein solution was separated from soil particles by ultrafiltration through low-adsorption hydrophilic 500000 NMWL cutoff membranes (regenerated cellulose, Amicon) and directly used for activity tests using pNPβG as the substrate, using the standard protocol. All measurements were analysed in triplicate.
In silico analysis of proteins
The MetaGeneMark tool with refined heuristic models for metagenomes (http://exon.gatech.edu/GeneMark/metagenome/index.cgi; ) was used to predict genes in the cloned DNA fragments. The deduced proteins were analysed using blastp and psi-blast  against the non-redundant databases. The translation products were further analysed for protein domains using the Pfam-A database . Models of rumen β-glucosidases were obtained from the SWISS-MODEL server (http://swissmodel.expasy.org/) using the coordinates of Thermotoga napolitana β-glucosidase structure  (PDB identity: 2×41) as the template. Figures were created with PyMOL (http://www.pymol.org/).
Supplementation of commercial cellulases with β-glucosidase LAB25g2
The activity of LAB25g2 on the hydrolysis of pre-treated corn stover biomass was tested by the supplementation of enzymatic hydrolysis reactions using a β-glucosidase-deficient commercial cocktail (Celluclast, Novozymes A/S, Denmark). Pre-treatment of the biomass was performed by diluted acid soaking followed by steam explosion as described previously . Hydrolysis reactions were performed at 50°C in 100 ml flasks with 12 g of pre-treated biomass, 3 g of enzymatic mixture and 5 g LAB25g2 β-glucosidase solution at 25 U ml-1 (for details see Results), to reach 20 g final reaction mass. Pre-treated corn stover was adjusted to an initial pH of 5.2 by the addition of 2 N NaOH, reaching a dry matter content of 20% after addition of enzymes. Concentration of β-glucosidase activity during enzymatic hydrolysis was calculated in the basis of pNPβG assay (the standard substrate used in supplementation assays), and corresponds to 5 U g-1 dry biomass in the absence of LAB25g2 enzyme, and a supplementation of 31.25 U g-1 dry biomass in the presence of the enzyme. This was the minimum dose of LAB25g2 enzyme to achieve saccharification of pre-treated corn stover at a dry matter content of 20%, under conditions reassembling industrial operations. Three control reactions were considered: (i) assay in the absence of LAB25g2 enzyme; (ii) assay in the presence of commercial β-glucosidases G0395 (from almond; Sigma Chemical Co.) with specific activity, measured using pNPβG at 50°C and pH 5.0, of 0.042 ± 0.008 mU mg-1; (iii) assay in the presence of commercial β-glucosidases E-BGOSAG (from Agrobacterium sp.; Megazyme) with specific activity, measured using pNPβG at 50°C and pH 5.0, of 119 ± 8 mU mg-1. For control tests protein solutions at 25 U ml-1 were prepared and added to reaction mixtures as for the LAB25g2, to achieve a final activity value of 31.25 units g-1 dry biomass (in the basis of pNPβG assay). Enzymatic hydrolysis reactions were followed by determining glucose and cellobiose concentrations by HPLC (Agilent Technologies, 1200 Series) using a Refractive Index Detector (RID) and a Aminex HPX-87 H column. The system was operated at 65°C and a flow rate of 6 ml min-1 of 5 mM H2SO4 for product elution as described elsewhere . Glucose concentration was also measured by the glucose oxidase-peroxidase D-Glucose Assay Kit (Megazyme; Bray, Ireland).
Note: the synthetic substrate pNPβG is specific for β-glucosidases, while not being hydrolyzed by cellobiohydrolases or endoglucanases. The numerous assays comparing activity towards pNPβG and cellobiose have demonstrated that both activities are, overall, linear functions one to another (β-glucosidases with high activity towards pNPβG also have high activity for cellobiose; for examples see Additional file 2: Table S2). Thus, supplementation per units of activity towards synthetic pNPβG for all enzymes used in saccharification ensured the same activity towards cellobiose in all cases, and for this reason, this substrate was used in our supplementation assays.