Enzymes
Two acetyl xylan esterases, AXE16A_ASPNG and AXE16B_ASPNG; a polygalacturonase (PGA28A_ASPNG), a α-arabinofuranosidase (ABF54B_ASPNG); an endoglucanase GH7 (EGL7A_THITE) and an endoxylanase (XYN11A_THITE) were produced as recombinant proteins in A. niger. Recombinant feruloyl esterase (FAE 1a) was cloned into the pET30a expression vector and overexpressed in Escherichia coli BL21 (DE3). β-glucosidase (E-BGLUC) and β-xylosidase (E-BXSRB) were procured from Megazyme International (Bray, Ireland). Mixed rumen enzymes were prepared from rumen fluid collected from two rumen cannulated cows fed a mixed alfalfa hay (30%), barley silage (50%) and barley grain (20%; DM basis) diet. Rumen contents were collected 2 h after feeding from the reticulum, ventral, caudal and dorsal-ventral sac of the reticulo-rumen of each cow and thoroughly mixed. Contents were strained through 4 layers of cheesecloth and the collected fluid was centrifuged at 38,300 × g for 15 min. The supernatant (500 mL) was lyophilized and reconstituted in 50 mM sodium citrate (pH 5.0, containing 5 μg/mL tetracycline, 5 μg/mL cycloheximide and 0.02% sodium azide) and used as source of rumen mixed enzymes. Three batches of rumen mixed enzymes from three different collections of rumen fluid were prepared. The commercial enzyme preparations of Accellerase 1500 and Accellerase XC were obtained from Genencor (Rochester, NY, US). Accellerase 1500 contained endoglucanase (2200–2800 carboxymethycellulose (CMC U) U/g) and β-glucosidase activities (450–775 p-nitrophenyl-β-D-glucopyranoside (pNPG U) U/g) whilst Accellerase XC contained mainly endoglucanase (1000–1400 CMC U/g) and xylanase activity (2500–3800 acid birchwood xylanase units ABXU/g; Genencor, Rochester, NY, US).
Production of acetyl xylan esterase, polygalacturonase, α-arabinofuranosidase, endoglucanase, endoxylanase in A. niger
Nucleotide sequences corresponding to target genes were obtained from Mycocosm genome resource of the Joint Genome Institute (http://genome.jgi-psf.org/programs/fungi/index.jsf). Additional file 6: Table S1 outlines the origin of the cloned genes and the carbohydrases that they encode. Cloning was accomplished using the Gateway recombination method with Invitrogen enzymes (Life Technologies Inc., Burlington, ON). Forward and reverse primers for cloning possessed at their 3′ end 20–25 nucleotides identical to the N terminal and C-terminal portions of the coding region of the targeted ORF, along with Gateway BP reaction compatible recombination sites [25]. Complementary DNA prepared from poly (A) + RNA was amplified by polymerase chain reaction (PCR). The amplified PCR products were cloned into ANIp7G, a Gateway-compatible vector previously constructed from ANIp7 [26]. Protoplasts of A. niger strain N593 glaA::hisG were transformed with the target genes as previously described [27]. FAE 1a from Anaeromyces mucronatus was cloned, expressed in E. coli BL21 and purified as earlier described [28].
Biochemical characterization of recombinant enzymes
Recombinant proteins produced in A. niger were purified at room temperature by anion exchange chromatography using an ÄKTA chromatography system (Amersham Biosciences, Piscataway, NJ). Prior to chromatography, supernatants from A. niger cultures were concentrated using a Vivaspin ultrafiltration device (GE Healthcare Life Sciences, Baie d’Urfe, PQ) at a 10 kDa cut-off. The concentrated proteins were repeatedly washed in 20 mM Tris–HCl buffer, pH 8.0, by dilution and ultrafiltration, and the retentate was applied to a MonoQ HR 5/10 anion exchange column equilibrated with the same buffer. Bound proteins were eluted using a linear 0 to 1 M KCl gradient in 20 mM Tris–HCl buffer, pH 8.0. Fractions (1 mL) were collected at a flow rate of 1 mL/min and stored on ice until analyzed for enzyme activity. Purified protein was stored at -80°C for further use. Protein purity was checked by SDS-PAGE as previously outlined [29].
All enzyme reactions were carried out in triplicate. Glycoside hydrolase activity was determined using a reducing sugar assay performed in a 96-well micro plate format with an assay volume of 50 μL. Briefly, for a 50 μL assay, 10 μL of substrate (1%) was added to 30 μL of 50 mM Britton-Robinson buffer (50 mM boric acid, 50 mM acetic acid and 50 mM phosphoric acid) pH 5.0 and the reaction initiated by the addition of the appropriate enzyme diluted in 10 μL of Britton-Robinson buffer [30]. The reaction mixture was immediately incubated at 40˚C for 30 min. Reaction mixtures were placed on ice and 10 μL was withdrawn and mixed with 190 μL of ice-cold BCA reagent (Bicinchoninic acid assay, Sigma-Aldrich, Oakville, ON) and incubated at 80˚C for 40 min for colour development. The resultant mixture (160 μL) was loaded onto a flat bottom-micro plate and the optical density read at 562 nm. The monosaccharide subunits constituting the polysaccharide substrates were used to prepare standard curves. Polysaccharide substrates (all from Sigma-Aldrich unless otherwise specified) were: birchwood xylan (xylanases); carboxymethylcellulose (endoglucanase); and polygalacturonic acid, sodium salt (polygalacturonase). Arabinofuranosidase activity was determined using 4-nitrophenyl-L-arabinofuranoside as a substrate in 50 mM Britton-Robinson buffer, pH 5.0, as described previously [30]. Acetyl-esterase activity was assessed by measuring released acetic acid using a kit purchased from Megazyme.
Statistical design
Design-Expert® software (Version 8.0; Stat-Ease, Inc., Minneapolis, MN; http://www.statease.com) was used to create the simplex-lattice designs and to analyze responses. All experiments were performed as mixtures at 15 mg protein loadings per g cellulose of the pretreated alfalfa hay or barley straw. The number of mixtures in the simplex-lattice depended on both the number of components in the mixture and the degree of the polynomial. Using an augmented special quadratic design, ten component designs resulted in 66 separate assays. In order to optimize the hydrolysis efficiency of enzyme cocktails the relative abundance of each component was varied using the experimental design outlined in Additional file 7: Table S2. The lower and upper limits of each component were determined with an aim to analyze synergetic interaction among fungal enzymes (AXE16A_ASPNG, AXE16B_ASPNG, PGA28A_ASPNG, ABF54B_ASPNG, EGL7A_THITE, XYN11A_THITE, FAE 1a, E-BGLUC and E-BXSRB) with mixed rumen enzymes or with commercial enzyme preparation to achieve enhanced biomass conversion to fermentable sugar. Therefore, relative abundance of core enzymes (i.e., mixed rumen enzymes/Accellerase 1500/Accellerase XC) was set to vary from 50% to 100%, while upper limit and lower limits for fungal enzymes were set between 50% - 0% in assay mixtures.
Formulation of enzyme mixtures for effective conversion of alkaline peroxide pre-treated (AP) alfalfa hay and barley straw
Alkaline peroxide pre-treatment of alfalfa hay and barley straw
Alfalfa and barley straw were each obtained from a single source of parental material and ground to pass 1.0 mm screen sieve and the resultant particles were pretreated with alkaline peroxide using the procedure described by [8]. Briefly, 50-mL, 1% H2O2 was adjusted to pH = 11.5 with 5 M NaOH and mixed with 1.0 g of alfalfa hay or barley straw in a 250-mL Erlenmeyer flask. Final concentrations were 1% H2O2 (300 mM), 0.8% NaOH (200 mM) and 2% (w/v) substrate. The flasks were incubated in a shaking incubator at 24°C for 24 h at 90 rpm. The slurries were neutralized to pH 7 by drop-wise addition of 12-N HCl. Residual H2O2 was inactivated by addition of 59 μL of catalase (28 mg protein/mL, Sigma–Aldrich). Following inactivation of catalase by heating at 90°C for 15 min, the entire content of the flasks were lyophilized before use in enzymatic assays. Two independent batches of pretreated alfalfa and barley straw were generated.
Enzymatic digestion of alkaline peroxide treated alfalfa hay or barley straw
Treated alfalfa hay or barley straw was first suspended at a final concentration of 0.5% in 50 mM sodium citrate (pH = 5.0) containing 5 μg/mL of tetracycline, 5 μg/mL of cycloheximide and 0.02% sodium azide. A total of 200 μL (duplicate) of substrate slurry was dispensed into a mini-eppendorf while the slurry was kept in suspension using a paddle reservoir designed for dispensing pharmaceutical beads on the Biomek FXP (Model VP 756C-1P100, V&P Scientific, Inc., San Diego, CA). Accuracy and precision of the biomass dispensing was tested by drying and weighing a series of dispensed aliquots, each of 0.2 mL.
Respective enzyme volumes for each reaction mixture were calculated according to statistical design (Additional file 7: Table S2) and dispensed into 200 μL of substrate slurry prepared as described above. Final volume was adjusted to 250 μL with 50 mM sodium citrate buffer (pH 5.0, containing 5 μg/mL tetracycline, 5 μg/mL cycloheximide and 0.02% sodium azide), and the reaction mixture was incubated at 50°C for 48 h in an oven on a platform rotating at 10 rpm. The tubes were then centrifuged at 1,500 × g for 3 min to separate the solid residue from the digested mixture. The supernatants (100 μL) were transferred into microplate wells (Thermo Fisher Scientific, Rochester, NY) and heated at 90°C for 10 min to inactivate enzymes prior determination of liberated glucose and xylose.
Glucose and xylose assays
Free glucose and xylose were determined colorimetrically using enzyme-coupled assays kits supplied by Megazyme (catalog K-GLUC and K-Xylose respectively). Assays were performed in 96-well plates using 12 μL of sample (obtained above) and 194 and 297 μL of assay reagent for glucose and xylose, respectively. Plates containing hydrolysis products and reagent were incubated at 50°C for 20 min and read at 510 nm and 340 nm for glucose and xylose respectively, using a Synergy-HT multi detection micro plate reader (Biotek Instruments, Inc. Winooski, VT). All reactions were replicated once, sampled twice, and assayed twice (n = 8).
For calculating total cellulose content of AP treated alfalfa and barley straw, triplicate alcohol insoluble residues from each feed stock was de-starched using Type-II A Bacillus α-amylase (Sigma-Aldrich; ~1000 units/100 mg cell wall alcohol-insoluble residue) in 50 mM sodium phosphate buffer (pH 7.0) at 25°C in a shaking incubator for 48 h. De-starched samples were centrifuged (3660× g for 10 min at 25°C) and the pellet was subsequently washed thrice with deionized water followed by centrifugation (3660 × g for 10 min at 25°C) and decanting of the water. The resulting pellets were suspended in 500 μl of acetone and evaporated with a stream of air at 36°C until dry. De-starched residue (5 mg) was hydrolyzed with 72% H2SO4. The released sugars were quantitated by combination of Gas Chromatography/mass spectroscopy (GC/MS) of alditol acetate derivatives [31].
Data analysis
Absorbance values from the glucose and xylose assays were converted to relative percent yields (relative to controls i.e., commercial enzyme or rumen enzyme mix only) and these values served as responses in the experimental design. For all experiments, ANOVA calculations of F value, P value, R2, Adjusted R2, Predicted R2 and Adequate Precision were computed by the Design-Expert software as shown in Additional file 8: Table S3 and Additional file 9: Table S4). The F value indicated the effects (if any) of the individual components on the model. An F value close to 1 implied that the components of the mixture did not interact and hence had little effect on the model. Enzyme synergism was deemed to be present at a P value <0.0001. Adjusted R2 and Predicted R2 were estimated and if the difference between these values was > 0.2 the model was considered to be over-parameterized and a different order polynomial was tested and/or a backward or stepwise elimination regression was conducted with an ‘alpha out’ value set to 0.1 as suggested by Banerjee et al., 2010 [8]. This statistical approach eliminated all of the terms in the model that were insignificant (P > 0.0001) and the model that gave a difference between the Adjusted and Predicted R2 values <0.2 was used to navigate the design space. Adequate precision estimated the signal-to-noise ratio, with a value >4 indicating adequate model discrimination. Once all the criteria for a robust model were fulfilled, the model was used to determine the enzyme mixtures that resulted in optimal glucose and xylose release. The predicted vs. actual estimates for each design are shown in Additional file 10.
