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Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail
Accepted Manuscript Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail Bianca Consorti Bussamra, Sindelia Freitas, Aline Carvalho da Costa PII: DOI: Reference:
S0960-8524(15)00449-6 http://dx.doi.org/10.1016/j.biortech.2015.03.117 BITE 14807
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 January 2015 23 March 2015 25 March 2015
Please cite this article as: Bussamra, B.C., Freitas, S., Costa, A.C.d., Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/ j.biortech.2015.03.117
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Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail Bianca Consorti Bussamraa*, Sindelia Freitasa, Aline Carvalho da Costab a
Brazilian Bioethanol Science and Technology Laboratory (CTBE), Brazilian Center for
Research in Energy and Materials, Rua Giuseppe Máximo Scolfaro, 10000, Post Code: 6192, Zip Code: 13083-970, Campinas, São Paulo, Brazil b
School of Chemical Engineering, University of Campinas (Unicamp), Av. Albert
Einstein, 500, Post Code: 6066, Zip Code: 13083-852, Campinas, São Paulo, Brazil Abstract The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5 L converts 49.11% ± 0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Keywords protein purification · glycosyl hydrolases · mixture experimental design · enzymatic hydrolysis · sugar cane bagasse
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* Corresponding author. Tel.: + (55) (15) 991424508; fax: + (55) (15) 32821308. E-mail address: [email protected]
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1. Introduction The energy dependence of some countries and global warming have turned the world’s attention to renewable fuels. The net CO2 generation of renewable fuel combustion is much lower than that of fossil fuel combustion because renewable materials have carbon that has been fixed during photosynthesis (Kheshgi et al., 2000). Lignocellulosic biomass is a sugar source for bioethanol production, and its use avoids the accumulation of this material. Other alternative renewable sources of sugar are agriculture residues, waste papers from chemical pulps, forest wood biomass and municipal waste (Limayem and Ricke, 2012). Brazil produces a large amount of sugar cane bagasse, a residue of sugar cane juice extraction in the production of sugar and ethanol. This lignocellulosic residue is a sustainable, environmentally friendly and promising alternative to fossil fuels (Lynd et al., 2008). The high cost of converting lignocellulose by enzymatic hydrolysis is a recurring problem that limits the production of bioethanol from a renewable and highly energetic source (Hess, 2008). Many factors affect the efficiency of the hydrolysis process, such as the recalcitrance of the lignocellulosic substrates, the pretreatment efficiency, enzyme ratios and loadings, presence of inhibitors and non-productive adsorption. Among the promising approaches to optimize the hydrolysis reaction is the use of well-balanced cocktails involving different classes of enzymes (Zhou et al., 2009). Among the enzymes needed to hydrolyze lignocellulosic biomass are cellulases and hemicellulases. They act synergistically and differ in the hydrolytic site and mechanism of action. Cellulases comprise three classes of enzymes: endoglucanases (EG) (EC 3.2.1.4), cellobiohydrolases (CBHI and CBHII) and β-glucosidases (BGL)
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(EC 3.2.1.21). According to the International Union of Biochemistry and Molecular Biology (IUBM), endoglucanases randomly cleave β-1,4 glucosydic bonds in the cellulose chain. This action exposes new reducing and non-reducing ends to the cellobiohydrolase action, resulting in the production of cellobiose. The presence of cellobiohydrolases and endoglucanase in optimized enzyme cocktails can be explained by the supplementary action between them on cellulose fibers (Väljamäe et al., 1998). β-glucosidase plays a key role in converting the principal cellobiohydrolase inhibitor (cellobiose) into glucose, the unit used to calculate cellulose conversion in the hydrolysis reaction (Holtzapple et al., 1990). Hemicellulases act on the hemicellulose chain and its lateral residues. Endo-1,4-β-D-xylanases (EC 3.2.1.8) cleave β-D-(1-4) xylosidic linkages in xylans, while 1,4-β-D-xylosidases (EC 3.2.1.37) remove successive D-xylose residues from the non-reducing end of xylan. Acetyl xylan esterases (EC 3.1.1.72) and α-L-arabinofuranosidases (EC 3.2.1.55) remove side groups from hemicellulose chains (Gilead and Shoham, 1995). Accessory proteins also improve hydrolysis efficiency. Expansin is a plant cell wall protein that cleaves the glucan-glucan binding by a mechanism that is not yet well established, loosening the fiber and increasing its accessibility to enzymes (Cosgrove et al., 2002). To obtain an optimal enzymatic mixture, a range of factors should be taken into account. For example, different substrates and pretreatments require different enzymatic mixtures to maximize hydrolysis (Meyer et al., 2009). Additionally, the relative proportions of the enzymes may be dependent on the protein load in the reaction. GAO and co-workers (Gao et al., 2010) produced and purified ten enzymes and used mixture design to determine the best proportion among them to maximize substrate conversion. The optimal cocktail composition was 27 – 30% of cellobiohydrolase I, 17 – 20% of
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cellobiohydrolase II, 29 – 35% of endoglucanase I, 14 – 15% of endoxylanase, 2 – 6% of β-xylosidase and 1 – 5% of β-glucosidase. The aim of this work was to evaluate the enzyme supplementation of Trichoderma reesei Rut-C30 extract for the hydrolysis of pretreated sugar cane bagasse. Six enzyme preparations correlated with lignocellulosic activities (T. reesei extract, endoglucanase, β-glucosidase, endoxylanase,
acetyl xylan esterase and α-L-
arabinofuranosidase) and one expansin were considered in a mixture design using the glucose concentration after 48 h of hydrolysis as the response. 2. Materials and Methods 2.1 Biomass The hydrothermally pretreated sugar cane bagasse (190 ºC, 10 min) was kindly provided by Dr. George Jackson da Rocha of the Brazilian Bioethanol Science and Technology Laboratory (CTBE). The composition of the pretreated material was 60.56% cellulose, 3.13% hemicellulose, 29.88% lignin and 6.2% ash. The bagasse was dried at room temperature until constant moisture, and milled (0.08 mm mesh) at the speed of 104 rotations per minute (RPM) (Fritsch, Pulverisette 14). 2.2 Enzymes Commercial enzymes α-L-arabinofuranosidase (ARF) and acetyl xylan esterase (AXE) were obtained from Megazyme (Wicklow, Ireland). Their activities were 12.1 U/mg and 500 U/mL, respectively, and they were diluted in 3.2 mol/L ammonium sulfate. Celluclast 1.5 L, Novozymes (Bagsværd, Denmark) and β-glucosidase C6105 (BGL) from Aspergillus niger, Sigma (St. Louis, USA) were also used in this work.
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The cloned genes for β-1,4-endoglucanase (Gene ID: 938607), endo-β-1,4xylanase (Gene ID: 939861) and expansin (Gene ID: 940108) are from Bacillus subtilis subsp. strain 168 and the sequences were submitted to GenBank. Cloning procedure is described elsewhere for β-1,4-endoglucanase (Santos et al., 2012), endo-β-1,4-xylanase A (Ruller et al., 2006) and expasin-YoaJ (Kerff et al., 2008). Escherichia coli BL21 (DE3) (Novagen), transformed with a pET28-a-c(+) (Novagen) expression vector and individually encoding recombinant gene for β-1,4-endoglucanase, endo-β-1,4-xylanase A and expasin-YoaJ expression, were cultivate in 1,5 L bioreactors (New Brunswick, Bioflo 115) over approximately 10 h. The medium composition was: 13.3 g/L KH2PO4, 4 g/L (NH4)2HPO4, 1.2 g/L MgSO4.7H2O, 1.7 g/L citric acid, 14.1 mg/L EDTA, 1 mL/L trace elements solution, 10.8 mg/L Fe(III) citrate, 4.5 mg/L thiamine HCl and 30 mg/L kanamycin. Trace elements solution were constituted by (per liter): 2.5 g CoCl2.6H2O, 15 g MnCl2.4H2O, 1.5 g CuCl2.2H2O, 3 g H3BO3, 2.1 g Na2MoO4.2H2O and 33.8 g Zn(CH3COO)2.2H2O. A volume of microorganisms was inoculated so that the initial absorbance (600 nm) was 0.2-0.4 in the bioreactor. Operational conditions were: 37 ºC, 1 L/min aeration, 30% dissolved oxygen, 7.0 pH, 200-1200 rpm cascade stirring. To induce expression, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, SigmaAldrich) was added to the media. The final culture was centrifuged (10,000 rpm, 30 min, 2 ºC) for cells recovery. 1 mL resultant pellets (before and after induction) were resuspended in 1 mL lyse buffer (1 mM PMSF, 150 mM NaCl, 20 mM Tris-HCl, 0.3 mg/mL lysozyme) and sonicated (Vibracell,
VCX-750). Supernatant protein
compositions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 12% (w/v) Polyacrylamide gels (Laemmli, 1970) were loaded with 10 µL prepared samples (10 µL sample + 20 µL loading buffer) and stained
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by Coomassie Blue. PageRuler prestained protein ladder (ThermoScientific, Waltham, USA) was used as molecular weight standard. The recombinant proteins from lysates were purified via immobilized metal affinity chromatography (IMAC). Agarose columns with immobilized nickel (Ni) were conditioned with 10 column volumes (CV) of 50 mM sodium phosphate pH 6.5 300 mM NaCl buffer (for endoglucanase) or 50 mM Tris-HCl pH 7.5 300 mM NaCl buffer (for expansin). The samples were injected and the protein recovery was performed through elution with 200 mM imidazole for endoglucanase, and 100 mM imidazole for expansin. The endoxylanase purification was carried out by Fast Protein Liquid Chromatography (FPLC), adapted with a 5 mL HisTrap column (GE, New York, USA). 50 mM phosphate pH 7.4 300 mM NaCl 20 mM imidazole was used as running buffer. To generate the elution buffer, imidazole was added to running buffer to a final concentration of 500 mM. The elution was performed following a linear gradient. 2 mL/min and 0.3 mPa maximum pressure were defined as parameters for purification. The purified enzymes were concentrated and the buffer exchanged to 50 mM 4.8 pH citrate through Vivaspin 20 (GE, New York, USA), 10 molecular weight cut off (MWCO). The Trichoderma reesei Rut C30 (ATCC 56765) extract was the source of cellulases, specially cellobiohydrolases. The microorganism was cultivated in a 6 L bioreactor (New Brunswick, Bioflo Celligen, USA 115). Inoculum was incubated at 200 rpm, 29 ºC, over 72 h. Medium composition for inoculum was (for each 0.5 L): 5 g Celuflok cellulose; 5 g glucose; 0.5 mL Tween 80; 0.5 g peptone; 25 mL salt solution (1 g KH2PO4, 0.7 g (NH4)2SO4, 0.15 g urea, 0.15 g MgSO4.7H2O, 0.15 g CaCl2, 2.5 mg FeSO4.7H2O, 0.7 mg ZnSO4.7H2O, 0.8 mg MnSO4.H2O and 1 mg CoCl2 in the final
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volume). The growth medium was 30 g/L celuflok cellulose, 1.5 g/L peptone, 1.5 mL/L Tween 80, 150 mL/L salt solution, 3 g/L soy bran, 15%v/v inoculum (900 mL) and 4.191 L of water were used to complete the reaction volume (6 L). Salt solution composition at the final culture volume was 6 g/L KH2PO4, 4.2 g/L (NH4)2SO4, 0.9 g/L urea, 0.9/L g MgSO4.7H2O, 0.9 g/L CaCl2, 15 mg/L FeSO4.7H2O, 4.2 mg/L ZnSO4.7H2O, 4.8 mg/L MnSO4.H2O and 6 mg/L de CoCl2. Fermentation was conducted throughout 144 h, at 29 ºC, pH 5.0 and 20% D.O. Agitation and aeration were set up in cascade, corresponding to 200-600 rpm and 0.3-3 L/min, respectively. The supernatant was recovered by centrifugation (10,000 rpm, 30 min, 2 ºC). The T. reesei supernatant was sequentially precipitated with gradient ammonium sulfate saturation aiming at cellobiohydrolase fraction enrichment. Firstly, 20 mL of supernatant was precipitated with 40, 50, 60, 70 and 80% salt saturation. Based on this study, 40% salt saturation was chosen aiming the removal of the precipitated proteins. Ammonium sulfate salt was added to the supernatant to achieve 60% saturation of the sample. The saturated solution was incubated at 4 ºC over-night to precipitate. The proteins precipitated in 60% of salt saturation were recovered by centrifugation (10,000 rpm, 20 min, 4 ºC) and resuspended in 50 mM 4.8 pH citrate buffer (the volume was 10 times lower) originating the T. reesei fraction. 2.3 Analytical methods Protein quantification was determined using the Bio-Rad Protein Assay reagent (Bio-Rad, CA, USA) and bovine serum albumin as a protein standard (Bradford, 1976). The concentration of purified recombinant proteins was measured through absorbance at 280 nm wavelength in spectrophotometer (Termo Scientific, Nanodrop 2000c), using
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their specific extinction coefficient determined by ProtParam tool (Gasteiger et al., 2005). The extinction coefficient (M-1 cm-1) and molecular weights (kDa) were, respectively, 106,925 and 57.75 for endoglucanase, 82,850 and 22.7 for endoxylanase and 41,370 and 25.37 for expansin. Total cellulase activity (filter paper activity) was measured according to Ghose (1987). An international unit of enzymatic activity is defined as the amount of enzyme required to produce 1 µmol of product per minute. Endoglucanase and endoxylanase activities were estimated by the degradation of 0.5% carboxymethyl cellulose (CMC) and 0.5% beechwood xylan, respectively. The reactions were conducted at 50 ºC for 30 min (endoglucanase) or 10 min (endoxylanase). The reaction was comprised of 50 µL of the correspondent substrate, 40 µL of citrate sodium buffer (50 mM) pH 5.0 and 10 µL of the correspondent enzyme. The reducing sugar concentration was estimated by the 3,5-dinitrosalicylic acid method (Miller, 1959).1 mM p-nitrofenil-β-D-glucopiranoside (p-NPG), and 4 mM p-nitrofenil-β-D-cellobioside (p-NPC) (Sigma–Aldrich, St. Louis, EUA) were the substrates for the activity determining reactions for β-glucosidase and cellobiohydrolase, respectively. After 10 min for β-glucosidase and 30 min for cellobiohydrolase activities measurement, at 50 ºC, the reactions (comprised of 80 µL of the substrate and 20 µL of the enzyme) were stopped by addition of 1 mol/L sodium carbonate. Absorbance at 400 nm was used to estimate p-NP concentration release. 2.4 Hydrolysis assays Hydrolysis reactions were carried out in two scales: 1.5 and 50 mL final volume. For hydrolysis in larger scale, 77.7% moisture hydrothermal bagasse and stirred flasks at 200 rpm were used. For 1.5 mL final volume reactions, dried and milled bagasse and
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stirring of 1,000 rpm were used. In both assays, the parameters of reaction were 5% (w/v) water-insoluble substrate (WIS), 50 ºC temperature and 4.8 pH (50 mM citrate buffer). The protein load for hydrolysis was stablished as 10 FPU per gram of dried bagasse for the validation of hydrolysis microassays. Sampling was performed at 6, 24, 48 and 72 h of reaction time. In these reactions, additional 20 UI of β-glucosidase per gram of dried bagasse were loaded. In the experimental design hydrolysis assays, protein load was 10 mg per gram of dried bagasse and the reactions were carried out for 48 h. To pause the enzymatic activity, samples were boiled at 99 ºC, for 5 min, centrifuged (12,000 rpm for 10 min at 4 ºC) and the supernatant analyzed for sugars (glucose, xylose and cellobiose) at high performance liquid chromatography (HPLC). The samples were injected in HPX-87H 300 mm X 7.8 mm column (Bio Rad), with a refractive index (RI) detector. 5 mM sulfuric acid was used as mobile phase, in a flow rate of 0.6 mL/min, at 35 ºC. 2.5 Optimization of enzymatic cocktail The protein proportion that lead to the highest glucose release in microassays hydrolysis was determined through experimental design. The seven factors and their maximum and minimum proportions are listed in Table 1. The total proportion sum for each run was one. To evaluate the response (glucose released at 48 h of hydrolysis- g/L) and how it was affected by the factors, a simplex lattice mixture design, polynomial degree 2 and augmented with interior points and centroid, was performed. The results of the experimental mixture design were analyzed and interpreted using the software Statistica 10 (StatSoft Inc., USA). To validate the mathematical model, analysis of variance (ANOVA) was performed. The validated model was maximized using SOLVER (EXCEL, Microsoft, USA).
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3. Results and Discussion 3.1 Enzyme production Endoglucanase, endoxylanase and expansin were successfully produced by genetically modified E. coli. Induction of the protein expression can be seen in Fig. 1. The growth of microorganisms and enzyme production were performed in batch reactors. The enzymes were purified by immobilized metal affinity chromatography (IMAC). The enzyme-containing supernatants and purified proteins are presented in Fig. 2. To obtain imidazole-free and buffer-conditioned enzyme solutions, the elution fractions were concentrated and washed several times. The protein concentrations of purified recombinant endoglucanase, endoxylanase and expansin were, respectively, 2.83, 2.75 and 1.64 mg/mL. The endoglucanase and endoxylanase enzymatic activities were, respectively, 1.48 and 71.24 UI/mL. The enzymes produced by T. reesei had a cellulose activity of 3.9 PFU/mL after 144 h. The supernatant contains a mixture of essential enzymes for biomass conversion into fermentable sugars. Among them are cellobiohydrolases I and II, the main enzymes produced by this microorganism (Foreman et al., 2003). To concentrate the fungal supernatant and obtain an enriched cellobiohydrolase fraction, a sequential ammonium sulfate precipitation of the T. reesei culture (20 mL) was performed. The 40-60% precipitation fractions presented the greatest specific activity against the cellobiose substrate p-NPC, with values of 0.29 UI/mg for the supernatant and 0.36, 0.71 and 0.36 UI/mg for the 40, 50 and 60% precipitation fractions, respectively. Most of the enzymes concentrated in the 50 and 60% precipitation fractions weighed from 40 to 70 kDa (Fig.
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3). The molecular weights of the cellobiohydrolases are 54.1 kDa (CBHI, GH 7) and 49.7 kDa (CBHII, GH 6) (Ståhlberg et al., 1993). The salt precipitation procedure enables protein concentration, buffer conditioning and total cellulase activity maintenance. The specific cellulase activity of the T. reesei fraction was 2.55 FPU/mg before and 2.7 FPU/mg after the precipitation. Many enzymatic classes are required to convert sugar cane bagasse into fermentable sugars. Because hydrothermally pretreated bagasse contains 60.56% cellulose, T. reesei extract (2.68 FPU/mg cellulase activity) was used to enable cellulose conversion. Endoglucanase, xylanase and expansin were also used as factors in the experimental design, as well as three commercial enzymes (β-glucosidase, α-Larabinofuranosidase and acetyl xylan esterase). The T. reesei extract has other enzymatic activities aside from cellobiohydrolases (Table 2) that could contribute to the hydrolysis of the substrate to some extent, limiting the significant effects of the chosen enzymatic activities on the experimental analysis. Fig. 4 shows the seven enzyme preparations used in the work reported here. 3.2 Validation of hydrolysis microassays To evaluate many enzyme proportions within a hydrolysis reaction, the smaller the volume that can be used, the cheaper the experiment. Therefore, a hydrolysis microassay was validated for 5% (w/v) water-insoluble solids in an experiment described in the Materials and Methods section. In these experiments, 20 UI of βglucosidase per gram of bagasse were added to supplement the T. reesei action in 5% WIS hydrolysis. The validation of the microassay (1.5 mL) was performed by comparison with hydrolysis performed at a larger scale (50 mL), and the results are
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shown in Fig. 5b and Fig. 5a, respectively. Significant differences in the glucose release between the reactions with and without β-glucosidase were observed for each time and scale. The glucose concentrations after 48 h, used to calculate the conversion, are significantly different between reactions with and without additional enzyme (βglucosidase) for the same scale, at the 95% confidence level. This enzymatic effect occurs at both scales, and it validates the hydrolysis microassay. 3.3 Optimization of biomass saccharification To enhance glucose release in hydrothermal bagasse hydrolysis, the T. reesei fraction was used in a supplementation study involving six other proteins/factors. Five of them were enzymes with different actions in lignocellulosic biomass (EG, BGL, EX, AXE and ARF), while the last was expansin (EXP), a protein that cleaves hydrogen bonds between fibers and accessory sugars by a non-hydrolytic and a mechanism that is not well established, loosening the fiber and increasing its accessibility to enzymes (Cosgrove et al., 2002). Fig. 4 shows the SDS-Page electrophoresis of the proteins (factors) studied in the mixture experimental design. Considering that the bagasse composition is primarily cellulose, the lack of cellulase enzymes would prevent hydrolysis and also the perception of gain of other enzymes. Thus, the factors T. reesei, EG and BGL, which are cellulase enzymes, had their minimum fractions in the mixtures fixed at 0.2, 0.1 and 0.1, respectively. The enzymatic mixture composition was optimized through an experimental simplex lattice design. The protein loading was 10 mg per gram of dry bagasse. The released glucose concentration after 48 h of hydrolysis was the response evaluated. The
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mathematical model (Eq. (1)) below presents the coefficients in the coded form of the factors (pseudo-components): = 24.23 + 15.46 + 14.82 + 15.37 + 9.26 + 9.5 + 15.53 + 7.18 ∙ + 6.1 ∙ + 9.42 ∙ + 8.48 ∙ + 9.94 ∙ + 6.81 ∙ (1) where is the predicted response, glucose concentration after 48 h of hydrolysis, = ((. − 0.2)⁄0.6),
= (( − 0.1)⁄0.6),
= ((# − 0.1)⁄0.6),
= ($⁄0.6), = ( $ ⁄0.6), = ( %⁄0.6) e = ($& ⁄0.6). The values of T. reesei, EG, BGL, EX, AXE, ARF e EXP correspond to the real proportion of them in the mixture. The mathematical model was evaluated by analysis of variance (ANOVA) (Table 3) and the F test. For statistically significant models, the calculated F value should be higher than the critical (tabulated) F value. In this case, the calculated F value is 224.32, while '(,*+(,-./0-,12) = 1.92, which implies a significant model. The model equation was used to calculate the values of the factors that maximize the response using SOLVER from Excel. By analyzing the standardized effect estimates (the effect values divided by the standard error of each factor), calculated in terms of the original components, it is possible to rank the factors. T. reesei appears to be the most important factor to glucose release in the hydrolysis reaction (Table 4). Besides the cellulase activity from the T. reesei extract, additional endoglucanase and β-glucosidase were used to supplement the fungi extract, resulting in an optimized cocktail (80% of T. reesei extract, 10% of endoglucanase (EG) and 10% of
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β-glucosidase (BGL)). T. reesei activities for the β-glucosidase specific substrate (pNPG) and endoglucanase substrate (CMC) were 0.18 and 3.76 UI/mg, respectively (Table 2). Xylanase activity was high (96.80 UI/mg) and can be sufficient for providing the
accessibility to
cellulose,
which probably explains why
hemicellulase
supplementation was not necessary (EX, AXE and ARF factors). Another reason is that hydrothermal bagasse has a low percentage of hemicelluloses, only 3.13% of the biomass, which means that the access to cellulose is independent of hemicellulase action. However, there was also no need for xylanase supplementation when the biomass used was steam-exploded bagasse with a higher hemicelluloses percentage (results not shown). It is important to note that there are two distinct families in which xylanases can be classified, GH10 and GH11 (Ruller et al., 2006). In the present study, the endo-1,4-xylanase GH11 was applied. According to Goldbeck et al. (2014), GH11 alone releases more xylose from pretreated sugar cane bagasse hydrolysis, achieving 64.3% hemicellulose conversion. Comparing the α-L-arabinofuranosidases families, GH54 showed a positive effect on xylose production from pretreated sugar cane bagasse, while GH51 presented a negative effect (Goldbeck et al., 2014). When a mixture of hemicellulolytic enzymes was composed of endo-1,4-xylanase GH11, βxylosidase GH43 and α-L-arabinofuranosidase GH51, production of xylobiose and xylotriose was favored. When GH51 was substituted for GH54, there was an increase in xylose production from pretreated bagasse hydrolysis (Goldbeck et al., 2014). The improved enzyme cocktail developed in this study does not contain any additional α-Larabinofuranosidase, despite the verified importance of this enzyme in a cocktail produced by another Trichoderma specie (Delabona et al., 2013). Hu et al. (2011) studied the interaction between xylanase and the commercial Celluclast and stated that
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the lower the cellulase (Celluclast) fraction in the mixture, the higher their synergism, resulting in more hydrolyzed cellulose and xylan from steam-pretreated corn stover. Moreover, when a range of xylanase loads was added to a constant amount of Celluclast (required to hydrolyze 70% of cellulose), no synergism between them was identified (Hu et al., 2011). This suggests that xylanases can contribute synergistically to the hydrolysis when they appear in large proportions in enzymatic mixtures. In the same way, the protein load of T. reesei in the reaction system could have influenced the synergism between it and the other protein preparations studied. Although the T. reesei extract presents activities other than that from cellulase, all the involved factors were significant as main effects, which means that the supplementation of this cocktail with other proteins has a positive statistical effect on the considered response. Furthermore, all the two-interaction factors involving T. reesei extract are statistically significant (Table 4). In the optimal cocktail composition determined (80% T. reesei, 10% EG and 10% BGL), endoglucanase and β-glucosidase were maintained at the lower level defined in the mixture design.
β-glucosidase
supplementation is doubtless important, as seen from the results in Fig. 5, but the real need for endoglucanase supplementation should be evaluated in future works. Gourlay et al. (2013) reported that swollenin, a “disrupting” protein such as expansin, did not present
synergism
with
purified
hydrolytic
enzymes
(exoglucanase
Cel7A,
endoglucanase Cel5A, xylanases GH10 and GH11) when the evaluated response was the glucose release after the incubation of the proteins with the substrate (steampretreated corn stover). However, when the evaluated response was xylose release, swollenin and xylanases presented strong synergism. The loosely ordered xylan structure enables the action of swollenin on the fibers. This makes the hemicellulose
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more accessible to xylanase action, which justifies the fundamental presence of swollenin in the enzymatic mixture, once hemicellulose limits the access of the cellulase to cellulose (Gourlay et al., 2013). In the present work, expansin did not present synergism with the T. reesei fraction, whether the response evaluated was glucose or xylose (data not shown for analysis involving xylose). This inconsistency with the literature can be related to the fact that hydrothermally pretreated sugar cane biomass has only 3.13% hemicellulose, eliminating the need for a swelling protein to enhance the enzymatic accessibility of cellulose. Due to the high value of the T. reesei coefficient in the model equation, the higher its proportion in the mixture, the higher is the predicted response (glucose concentration). For this reason, when the protein load is 10 mg of protein per gram of biomass, the T. reesei factor plays a key role in the cellulose conversion. The protein load influences the ratio of the cocktail components (Gao et al., 2010). Gao et al. (2010) reported that endoglucanase seemed to be more important to glucose release when a low protein load was applied (7.5 mg of protein per gram of glucan). However, Zhou et al. (2009) optimized a cellulase cocktail using a two-level fractional factorial design. In this type of experimental design, the factor levels vary independently from each other, and consequently, each run presents a different protein load. The amount of glucose released (15.5 mg/ml) by hydrolysis under the action of the optimized enzyme mixture was 2.1 times that produced under the action of the multi-enzyme cocktail secreted from the T. viride mutant strain T 100-14 (Zhou et al., 2009). 3.4 Comparison of the optimized cocktail and commercial Celluclast 1.5 L.
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The glucose release predicted by the model (24.23 g/L) when the optimized cocktail is used corresponds to a 72% cellulose conversion. Under the same conditions (10 mg of protein per gram of dry bagasse), the commercial Celluclast cocktail achieved 49.11% ± 0.49 cellulose conversion. This commercial cocktail presents a filter paper activity of 108 FPU/mL. To validate the response predicted by the model, the hydrolysis of hydrothermally pretreated bagasse using the optimized cocktail was carried out. The observed conversion of cellulose was statistically similar to the predicted value, at 68.36% ± 5.57. Without supplementation, the T. reesei fraction presents 58.81% ± 0.71 cellulose conversion. Braga et al. (2014) studied the supplementation of Celluclast 1.5L (10 FPU/g of dry bagasse load in hydrolysis) with 30% (v/v) crude enzymatic complex from Aspergillus oryzae. The conversion of cellulose from the hydrothermally pretreated bagasse increased by 36%, achieving 50.8% at the end of 72 h hydrolysis. The enzymatic load was 6.7 mg protein/g dry bagasse (Braga et al., 2014). The conversion yield of glucose from cellulose was higher under the action of the optimized cocktail developed in this work. In addition, the replacement of a commercial cocktail in an optimized enzyme preparation reduces costs and unnecessary enzymatic activities. Ramos et al. (2015) proposed a reaction condition to obtain high conversion rates and concentration of glucose equivalents (which also takes into account the amount of cellobiose that is released in the reaction medium) after 72 h of hydrolysis of phosphoric-catalyzed steam-exploded sugar cane bagasse. The highest amount of sugar released, which corresponds to a glucan conversion of 69.2%, was achieved when the parameters of substrate total solids (%), agitation (rpm) and enzyme loading of Cellic CTec2 from Novozymes (g/g of cellulose) were maintained at their highest levels, 20 wt%, 200 rpm and 0.1 g/g of cellulose, respectively. The glucose yield achieved by
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Ramos et al. (2015) was similar to that achieved by the optimized cocktail developed in this work, although they used a higher total solid content and lower enzyme loading. However, the surface response presented by Ramos et al. (2015) suggests that the glucose yield would continue rising as the enzyme loading increases. The evaluation of the differences in sugar release as a function of time for the optimized (2.75 FPU/mg) and the commercial Celluclast (2.57 FPU/mg) cocktails during hydrolysis is a way of assessing the performance of the improved cocktail composition. Fig. 6 shows the sugar release profile (glucose, xylose and cellobiose) from hydrolysis for these enzyme preparations. The glucose and xylose concentrations are higher in hydrolyses under the optimized cocktail action after 6 h of reaction and longer. The cellobiose concentration is higher throughout the hydrolysis under the action of Celluclast, as this enzyme preparation does not contain sufficient βglucosidase activity. The optimized cocktail maintained a low cellobiose concentration throughout the reaction, which was 0.41 ± 0.1 g/L at the end of hydrolysis. Around fifty percent of the glucose released was obtained in the first 6 hours of the reaction for both systems (13.32 ± 0.11 g/L for the optimized cocktail and 7.79 ± 0.15 for Celluclast, which represent 39.59% and 23.05% of the cellulose conversion, respectively). However, the rate of conversion was 2 times higher using the optimized cocktail (approximately 2.2 g/L.h) compared with Celluclast (approximately 1.2 g/L.h). This significant difference in the rates in the first 6 hours can be associated with the accumulation of cellobiose in the Celluclast system, which is known as the main cellobiohydrolase inhibitor (Holtzapple et al., 1990). Delabona et al. (2012) cultivated a new strain of Trichoderma harzianum isolated from the Amazon rainforest, aiming for the production of an enzymatic complex to hydrolyze biomass, which presented a
20
glucose yield of 19.4% after 72 h of hydrolysis of steam-pretreated sugar cane bagasse. This value corresponds to a glucose release of 5.5 g/L, while T. reesei hydrolysis under the same conditions released 5.8 g/L of glucose (Delabona et al., 2012). Hydrolysis conversion (%) is related to enzyme efficiency, while the glucose concentration released throughout the hydrolysis determines how viable the process is for the subsequent step of glucose utilization (Ramos et al., 2015). The enzymatic cocktail optimized in this work enables an efficient cellulose conversion, the highest reported among the revised studies involving hydrothermally pretreated bagasse, even though the glucose release is not as high as that reported in some studies listed here. 4. Conclusions This study reported the production and recovery of proteins to be used in hydrolysis assays aiming at cocktail optimization for sugar cane saccharification.
bagasse
The hydrolysis microassays validated in this work were used to
evaluate the glucose release during the mixture design experiments. The optimized cocktail, composed of 80% T. reesei CBH enriched fraction, 10% endoglucanase and 10% β-glucosidase, increased the conversion of cellulose by approximately 40% compared to Celluclast 1.5L. Thus, the approach of identifying the necessary activities to improve a fungal extract through mixture experiments can contribute to optimized cocktail development for different substrates and various applications. The experimental matrix of the simplex lattice design and the response for each run are presented in Table S1 (Supplementary Material). Acknowledgements
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The authors would like to thank the São Paulo Research Foundation (FAPESP, process numbers 2008/57873-8, 2010/08089-2 and 2012/23223-2) for financial support, the know-how in protein purification of the CBMEG/UNICAMP, and the Fungal Hydrolases Biosynthesis Laboratory, CTBE, Campinas, Brazil, for the assistance in Trichoderma reesei Rut C30 cultivation. Moreover, the authors are grateful to Dr. George Jackson da Rocha (CTBE/Brazil) for the hydrothermally pretreated bagasse, Dr. José Geraldo da Cruz Pradella (CTBE/Brazil) for the T. reesei Rut C30 strain and Dr. Roberto Ruller (CTBE/Brazil) for the cloned genes, kindly provided to us. References
1. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–54. 2. Braga, C.M.P., Delabona, P. da S., Lima, D.J. da S., Paixão, D.A.A., Pradella, J.G. da C., Farinas, C.S., 2014. Addition of feruloyl esterase and xylanase produced onsite improves sugarcane bagasse hydrolysis. Bioresour. Technol. 170, 316–324. doi:10.1016/j.biortech.2014.07.115 3. Cosgrove, D.J., Li, L.C., Cho, H., Hoffmann-Benning, S., Richard, C., Blecker, D., 2002. The Growing World of Expansins. Plant Cell Physiol. 43, 1436–1444. 4. Delabona, P. da S., Farinas, C.S., Ribeiro, M., Freitas, S., Pradella, J.G. da C., 2012. Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production. Bioresour. Technol. 107, 517–521. doi:10.1016/j.biortech.2011.12.048
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5. Delabona, P.D.S., Cota, J., Hoffmam, Z.B., Paixão, D.A.A., Farinas, C.S., Cairo, J.P.L.F., Lima, D.J., Squina, F.M., Ruller, R., Pradella, J.G.D.C., 2013. Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase. Bioresour. Technol. 131, 500–7. doi:10.1016/j.biortech.2012.12.105 6. Foreman, P.K., Brown, D., Dean, R., Diener, S., Dunn-coleman, N.S., Goedegebuur, F., Houfek, T.D., England, G.J., Kelley, S., Meerman, H.J., Mitchinson, C., Heather, A., Teunissen, P.J.M., Yao, J., Ward, M., Dankmeyer, L., Kelley, A.S., Mitchell, T., Olivares, H.A., 2003. Transcriptional Regulation of Biomass-degrading Enzymes in the Filamentous Fungus Trichoderma reesei. J. Biol. Chem. 278, 31988–31997. doi:10.1074/jbc.M304750200 7. Gao, D., Chundawat, S.P.S., Krishnan, C., Balan, V., Dale, B.E., 2010. Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour. Technol. 101, 2770–81. doi:10.1016/j.biortech.2009.10.056 8. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., Bairoch, A., 2005. Protein Identification and Analysis Tools on the ExPASy Server, in: Walker, J.M. (Ed.), The Proteomics Protocols Handbook. pp. 571–607. 9. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257– 268. 10. Gilead, S., Shoham, Y., 1995. Purification and Characterization of α-LArabinofuranosidase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 61, 170–174.
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11. Goldbeck, R., Damásio, A.R.L., Gonçalves, T. a, Machado, C.B., Paixão, D. a a, Wolf, L.D., Mandelli, F., Rocha, G.J.M., Ruller, R., Squina, F.M., 2014. Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications. Appl. Microbiol. Biotechnol. 98, 8513–25. doi:10.1007/s00253-014-5946-6 12. Gourlay, K., Hu, J., Arantes, V., Andberg, M., Saloheimo, M., Penttilä, M., Saddler, J., 2013. Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass. Bioresour. Technol. 142, 498–503. doi:10.1016/j.biortech.2013.05.053 13. Hess, M., 2008. Thermoacidophilic proteins for biofuel production. Trends Microbiol. 16, 414–419. doi:10.1016/j.tim.2008.06.001 14. Holtzapple, M., Cognata, M., Shu, Y., Hendrickson, C., 1990. Inhibition of Trichoderma reesei Cellulase by Sugars and Solvents. Biotechnol. Bioeng. 36, 275–287. 15. Hu, J., Arantes, V., Saddler, J.N., 2011. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? Biotechnol. Biofuels 4, 36. doi:10.1186/17546834-4-36 16. Kerff, F., Amoroso, A., Herman, R., Sauvage, E., Petrella, S., Filée, P., Charlier, P., Joris, B., Tabuchi, A., Nikolaidis, N., Cosgrove, D.J., 2008. Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization. Proc. Natl. Acad. Sci. U. S. A. 105, 16876–81. doi:10.1073/pnas.0809382105
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17. Kheshgi, H.S., Prince, R.C., Marland, G., 2000. The potential of biomass fuels in the context of climate change: Focus on Transportation Fuels. Annu. Rev. Energy Environ. 25, 199–244. 18. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 19. Limayem, A., Ricke, S.C., 2012. Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Prog. Energy Combust. Sci. 38, 449–467. doi:10.1016/j.pecs.2012.03.002 20. Lynd, L.R., Laser, M.S., Bransby, D., Dale, B.E., Davison, B., Hamilton, R., Himmel, M., Keller, M., McMillan, J.D., Sheehan, J., Wyman, C.E., 2008. How biotech can transform biofuels. Nat. Biotechnol. 26, 169–72. doi:10.1038/nbt0208169 21. Meyer, A.S., Rosgaard, L., Sørensen, H.R., 2009. The minimal enzyme cocktail concept for biomass processing. J. Cereal Sci. 50, 337–344. doi:10.1016/j.jcs.2009.01.010 22. Miller, G.L., 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 31, 426–428. 23. Ramos, L.P., Silva, L., Ballem, A.C., Pitarelo, A.P., Chiarello, L.M., Silveira, M.H.L., Silveira, L., 2015. Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids and low enzyme loadings. Bioresour. Technol. 175, 195–202. doi:10.1016/j.biortech.2014.10.087 24. Ruller, R., Rosa, J.C., Faça, V.M., Greene, L.J., Ward, R.J., 2006. Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli
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DH5alpha under the control of the Bacillus BsXA promoter. Biotechnol. Appl. Biochem. 43, 9–15. doi:10.1042/BA20050016 25. Santos, C.R., Paiva, J.H., Sforça, M.L., Neves, J.L., Navarro, R.Z., Cota, J., Akao, P.K., Hoffmam, Z.B., Meza, A.N., Smetana, J.H., Nogueira, M.L., Polikarpov, I., Xavier-Neto, J., Squina, F.M., Ward, R.J., Ruller, R., Zeri, A.C., Murakami, M.T., 2012. Dissecting structure-function-stability relationships of a thermostable GH5CBM3 cellulase from Bacillus subtilis 168. Biochem. J. 441, 95–104. doi:10.1042/BJ20110869 26. Ståhlberg, J., Johansson, G., Pettersson, G., 1993. Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose. Biochim. Biophys. Acta 1157, 107–13. 27. Väljamäe, P., Sild, V., Pettersson, G., Johansson, G., 1998. The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surfaceerosion model. Eur. J. Biochem. 253, 469–75. 28. Zhou, J., Wang, Y.-H., Chu, J., Luo, L.-Z., Zhuang, Y.-P., Zhang, S.-L., 2009. Optimization of cellulase mixture for efficient hydrolysis of steam-exploded corn stover by statistically designed experiments. Bioresour. Technol. 100, 819–25. doi:10.1016/j.biortech.2008.06.068
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Figure Captions Fig. 1. SDS-PAGE electrophoresis of recombinant protein expression in E. coli. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lanes a, c and e: before induction of the expression system encoding for endoglucanase, endoxylanase and expansin, respectively; lanes b, d and f: after induction of the expression system encoding for endoglucanase, endoxylanase and expansin, respectively. Fig. 2. SDS-PAGE electrophoresis of expression and chromatograph purification of recombinant proteins. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: endoglucanase-containing supernatant; lane b: purified endoglucanase; lane c: endoxylanase-containing supernatant; lane d: purified endoxylanase; lane e: expansin-containing supernatant; lane f: purified expansin. Fig. 3. SDS-PAGE electrophoresis of supernatant and ammonium sulfate precipitation fractions of T. reesei culture. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: T. reesei culture supernatant; lane b: 40% ammonium sulfate precipitation fraction; lane c: 50% ammonium sulfate precipitation fraction; lane d: 60% ammonium sulfate precipitation fraction; lane e: 70% ammonium sulfate precipitation fraction; lane f: 80% ammonium sulfate precipitation fraction. Fig. 4. SDS-PAGE electrophoresis of the proteins (factors) used in the mixture experimental design. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: T. reesei; lane b: endoglucanase (EG); lane c:
27
β-glucosidase (BGL); lane d: endoxylanase (EX); lane e: acetyl xylan esterase (AXE); lane f: α-L-arabinofuranosidase (ARF); lane g: expansin (EXP). Fig. 5. Effect of β-glucosidase addition on the glucose release in different times of hydrothermally bagasse hydrolysis. TR indicates hydrolysis under the T. reesei action only. TR+BLG indicates hydrolysis with β-glucosidase supplementation. The experiments were carried out in 50 mL (a) and 1.5 mL (b) reaction volumes. Different letters indicate statistic difference between averages (Tukey, 0.05%). Fig. 6. Concentration (g/L) of sugar (glucose, xylose and cellobiose) throughout 48 h of hydrolyses of hydrothermally pretreated bagasse due to action of the optimized cocktail and commercial cocktail (Celluclast 1.5L) in reaction volume of 1.5 mL.
M.W.
a
b
c
d
Fig. 1
M.W.
e
f
M.W.
a
b
c
Fig. 2
d
e
f
M.W.
a
b
c
Fig. 3
d
e
f
M.W.
a
b
c
d
Fig. 4
e
f
g
a
A
TR TR + BGL
25
B B/C
20
C/D
Glucose (g/L)
D 15
E
E
10
5
F
0
6
24
48
72
Time (h)
b
A TR TR + BGL
25
A A
20
Glucose (g/L)
B B/C
15
C
C
10
5
A
0
6
24
48
Time (h)
Fig. 5
72
50
20
14
20
1.8
26
6
24
4 1.6 10
22
15
2.8 10
2.4 5
2
2.0
0
20
18
40
Time (h)
16
60
1.2 1.0
14
Xylose (g/L)
1.4
20
Cellobiose (g/L)
10
2.0
20
0
1.6 1.2
0.8
12
0.8
10
0.6 0.4
8
0.4
6 0.0 0
20
40
Time (h) Fig. 6
60
Cellobiose (g/L)
12
8
28
Glucose (g/L)
30
25
16
Xylose (g/L)
Glucose (g/L)
30
18
Glucose_Optimized cocktail Glucose_Celluclast 1.5 Xylose_Optimized cocktail Xylose_Celluclast 1.5 Cellobiose_Optimized cocktail Cellobiose_Celluclast 1.5
40
30
Table 1 Proportion range (maximum and minimum) of each factor: T. reesei extract (T. reesei), endoglucanase (EG), β-glucosidase (BGL), endoxylanase (EX), acetyl xylan esterase (AXE), α-Larabinofuranosidase (ARF) and expansin (EXP). Total proportion sum for each run=1 Factors Proportion
T. reesei
EG
BGL
EX
AXE
ARF
EXP
Minimum
0.2
0.1
0.1
0
0
0
0
Maximum
0.8
0.7
0.7
0.6
0.6
0.6
0.6
Table 2 Enzymatic activities of 60% ammonium sulfate precipitation fraction from the T. reesei supernatant Substrates
Volumetric activity (UI/mL) Specific activity (UI/mg)
p-NPC 4 mM
5.99
0.60
p-NPG 1 mM
1.78
0.18
p-NPX 1 mM
0.18
0.02
Beech wood xylan 0.5%
968
96.80
CMC 0.5%
37.57
3.76
1,4-β-D-manan 0.5%
9.96
1.00
Lichenan 0.5%
27.81
2.78
Wheat arabinoxylan 0.5%
30.76
3.08
Table 3 ANOVA analysis for the mixture design. Confidence interval of 95% Sum of
Degrees of
Mean
squares
freedom
square
995.57
12
83
21.817
59
0.37
9.449
23
0.41
Pure error
12.368
36
0.34
Total
1017.93
71
Source Model Total error Lack of fit
% explained variation: 97.8%
Table 4 Mathematical model effects, residual errors, t-student and p-value to the original components Effect
Error
t(44)
p-value*
(A)T. reesei
24.31
1.12
21.71
0.000
(B)EG
9.44
1.65
5.73
0.000
(C)BGL
7.79
1.65
4.73
0.000
(D)EX
6.76
2.08
3.25
0.002
(E)AXE
-0.32
2.08
-0.15
0.878
(F)ARF
0.31
2.08
0.15
0.884
(G)EXP
9.21
2.08
4.43
0.000
AB
19.18
5.91
3.24
0.002
AC
18.48
5.91
3.13
0.003
AD
27.42
5.91
4.64
0.000
AE
22.85
5.91
3.86
0.000
AF
25.58
5.91
4.33
0.000
AG
19.19
5.91
3.25
0.002
* p-values below 0.05 were significant and above were not significant
28
Highlights
•
Well-balanced enzymatic cocktails as promising approach to optimize biomass hydrolysis
•
Sugar cane bagasse 5% (W/V) hydrolysis microassay was validated
•
Identification of necessary activities to improve fungal extract performance
•
Mixture experimental design was used to develop an optimized enzyme cocktail
•
Cellulose conversion was increased by approximately 40% in respect of Celluclast 1.5L
S0960-8524(15)00449-6 http://dx.doi.org/10.1016/j.biortech.2015.03.117 BITE 14807
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 January 2015 23 March 2015 25 March 2015
Please cite this article as: Bussamra, B.C., Freitas, S., Costa, A.C.d., Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/ j.biortech.2015.03.117
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Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail Bianca Consorti Bussamraa*, Sindelia Freitasa, Aline Carvalho da Costab a
Brazilian Bioethanol Science and Technology Laboratory (CTBE), Brazilian Center for
Research in Energy and Materials, Rua Giuseppe Máximo Scolfaro, 10000, Post Code: 6192, Zip Code: 13083-970, Campinas, São Paulo, Brazil b
School of Chemical Engineering, University of Campinas (Unicamp), Av. Albert
Einstein, 500, Post Code: 6066, Zip Code: 13083-852, Campinas, São Paulo, Brazil Abstract The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5 L converts 49.11% ± 0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Keywords protein purification · glycosyl hydrolases · mixture experimental design · enzymatic hydrolysis · sugar cane bagasse
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* Corresponding author. Tel.: + (55) (15) 991424508; fax: + (55) (15) 32821308. E-mail address: [email protected]
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1. Introduction The energy dependence of some countries and global warming have turned the world’s attention to renewable fuels. The net CO2 generation of renewable fuel combustion is much lower than that of fossil fuel combustion because renewable materials have carbon that has been fixed during photosynthesis (Kheshgi et al., 2000). Lignocellulosic biomass is a sugar source for bioethanol production, and its use avoids the accumulation of this material. Other alternative renewable sources of sugar are agriculture residues, waste papers from chemical pulps, forest wood biomass and municipal waste (Limayem and Ricke, 2012). Brazil produces a large amount of sugar cane bagasse, a residue of sugar cane juice extraction in the production of sugar and ethanol. This lignocellulosic residue is a sustainable, environmentally friendly and promising alternative to fossil fuels (Lynd et al., 2008). The high cost of converting lignocellulose by enzymatic hydrolysis is a recurring problem that limits the production of bioethanol from a renewable and highly energetic source (Hess, 2008). Many factors affect the efficiency of the hydrolysis process, such as the recalcitrance of the lignocellulosic substrates, the pretreatment efficiency, enzyme ratios and loadings, presence of inhibitors and non-productive adsorption. Among the promising approaches to optimize the hydrolysis reaction is the use of well-balanced cocktails involving different classes of enzymes (Zhou et al., 2009). Among the enzymes needed to hydrolyze lignocellulosic biomass are cellulases and hemicellulases. They act synergistically and differ in the hydrolytic site and mechanism of action. Cellulases comprise three classes of enzymes: endoglucanases (EG) (EC 3.2.1.4), cellobiohydrolases (CBHI and CBHII) and β-glucosidases (BGL)
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(EC 3.2.1.21). According to the International Union of Biochemistry and Molecular Biology (IUBM), endoglucanases randomly cleave β-1,4 glucosydic bonds in the cellulose chain. This action exposes new reducing and non-reducing ends to the cellobiohydrolase action, resulting in the production of cellobiose. The presence of cellobiohydrolases and endoglucanase in optimized enzyme cocktails can be explained by the supplementary action between them on cellulose fibers (Väljamäe et al., 1998). β-glucosidase plays a key role in converting the principal cellobiohydrolase inhibitor (cellobiose) into glucose, the unit used to calculate cellulose conversion in the hydrolysis reaction (Holtzapple et al., 1990). Hemicellulases act on the hemicellulose chain and its lateral residues. Endo-1,4-β-D-xylanases (EC 3.2.1.8) cleave β-D-(1-4) xylosidic linkages in xylans, while 1,4-β-D-xylosidases (EC 3.2.1.37) remove successive D-xylose residues from the non-reducing end of xylan. Acetyl xylan esterases (EC 3.1.1.72) and α-L-arabinofuranosidases (EC 3.2.1.55) remove side groups from hemicellulose chains (Gilead and Shoham, 1995). Accessory proteins also improve hydrolysis efficiency. Expansin is a plant cell wall protein that cleaves the glucan-glucan binding by a mechanism that is not yet well established, loosening the fiber and increasing its accessibility to enzymes (Cosgrove et al., 2002). To obtain an optimal enzymatic mixture, a range of factors should be taken into account. For example, different substrates and pretreatments require different enzymatic mixtures to maximize hydrolysis (Meyer et al., 2009). Additionally, the relative proportions of the enzymes may be dependent on the protein load in the reaction. GAO and co-workers (Gao et al., 2010) produced and purified ten enzymes and used mixture design to determine the best proportion among them to maximize substrate conversion. The optimal cocktail composition was 27 – 30% of cellobiohydrolase I, 17 – 20% of
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cellobiohydrolase II, 29 – 35% of endoglucanase I, 14 – 15% of endoxylanase, 2 – 6% of β-xylosidase and 1 – 5% of β-glucosidase. The aim of this work was to evaluate the enzyme supplementation of Trichoderma reesei Rut-C30 extract for the hydrolysis of pretreated sugar cane bagasse. Six enzyme preparations correlated with lignocellulosic activities (T. reesei extract, endoglucanase, β-glucosidase, endoxylanase,
acetyl xylan esterase and α-L-
arabinofuranosidase) and one expansin were considered in a mixture design using the glucose concentration after 48 h of hydrolysis as the response. 2. Materials and Methods 2.1 Biomass The hydrothermally pretreated sugar cane bagasse (190 ºC, 10 min) was kindly provided by Dr. George Jackson da Rocha of the Brazilian Bioethanol Science and Technology Laboratory (CTBE). The composition of the pretreated material was 60.56% cellulose, 3.13% hemicellulose, 29.88% lignin and 6.2% ash. The bagasse was dried at room temperature until constant moisture, and milled (0.08 mm mesh) at the speed of 104 rotations per minute (RPM) (Fritsch, Pulverisette 14). 2.2 Enzymes Commercial enzymes α-L-arabinofuranosidase (ARF) and acetyl xylan esterase (AXE) were obtained from Megazyme (Wicklow, Ireland). Their activities were 12.1 U/mg and 500 U/mL, respectively, and they were diluted in 3.2 mol/L ammonium sulfate. Celluclast 1.5 L, Novozymes (Bagsværd, Denmark) and β-glucosidase C6105 (BGL) from Aspergillus niger, Sigma (St. Louis, USA) were also used in this work.
6
The cloned genes for β-1,4-endoglucanase (Gene ID: 938607), endo-β-1,4xylanase (Gene ID: 939861) and expansin (Gene ID: 940108) are from Bacillus subtilis subsp. strain 168 and the sequences were submitted to GenBank. Cloning procedure is described elsewhere for β-1,4-endoglucanase (Santos et al., 2012), endo-β-1,4-xylanase A (Ruller et al., 2006) and expasin-YoaJ (Kerff et al., 2008). Escherichia coli BL21 (DE3) (Novagen), transformed with a pET28-a-c(+) (Novagen) expression vector and individually encoding recombinant gene for β-1,4-endoglucanase, endo-β-1,4-xylanase A and expasin-YoaJ expression, were cultivate in 1,5 L bioreactors (New Brunswick, Bioflo 115) over approximately 10 h. The medium composition was: 13.3 g/L KH2PO4, 4 g/L (NH4)2HPO4, 1.2 g/L MgSO4.7H2O, 1.7 g/L citric acid, 14.1 mg/L EDTA, 1 mL/L trace elements solution, 10.8 mg/L Fe(III) citrate, 4.5 mg/L thiamine HCl and 30 mg/L kanamycin. Trace elements solution were constituted by (per liter): 2.5 g CoCl2.6H2O, 15 g MnCl2.4H2O, 1.5 g CuCl2.2H2O, 3 g H3BO3, 2.1 g Na2MoO4.2H2O and 33.8 g Zn(CH3COO)2.2H2O. A volume of microorganisms was inoculated so that the initial absorbance (600 nm) was 0.2-0.4 in the bioreactor. Operational conditions were: 37 ºC, 1 L/min aeration, 30% dissolved oxygen, 7.0 pH, 200-1200 rpm cascade stirring. To induce expression, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, SigmaAldrich) was added to the media. The final culture was centrifuged (10,000 rpm, 30 min, 2 ºC) for cells recovery. 1 mL resultant pellets (before and after induction) were resuspended in 1 mL lyse buffer (1 mM PMSF, 150 mM NaCl, 20 mM Tris-HCl, 0.3 mg/mL lysozyme) and sonicated (Vibracell,
VCX-750). Supernatant protein
compositions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 12% (w/v) Polyacrylamide gels (Laemmli, 1970) were loaded with 10 µL prepared samples (10 µL sample + 20 µL loading buffer) and stained
7
by Coomassie Blue. PageRuler prestained protein ladder (ThermoScientific, Waltham, USA) was used as molecular weight standard. The recombinant proteins from lysates were purified via immobilized metal affinity chromatography (IMAC). Agarose columns with immobilized nickel (Ni) were conditioned with 10 column volumes (CV) of 50 mM sodium phosphate pH 6.5 300 mM NaCl buffer (for endoglucanase) or 50 mM Tris-HCl pH 7.5 300 mM NaCl buffer (for expansin). The samples were injected and the protein recovery was performed through elution with 200 mM imidazole for endoglucanase, and 100 mM imidazole for expansin. The endoxylanase purification was carried out by Fast Protein Liquid Chromatography (FPLC), adapted with a 5 mL HisTrap column (GE, New York, USA). 50 mM phosphate pH 7.4 300 mM NaCl 20 mM imidazole was used as running buffer. To generate the elution buffer, imidazole was added to running buffer to a final concentration of 500 mM. The elution was performed following a linear gradient. 2 mL/min and 0.3 mPa maximum pressure were defined as parameters for purification. The purified enzymes were concentrated and the buffer exchanged to 50 mM 4.8 pH citrate through Vivaspin 20 (GE, New York, USA), 10 molecular weight cut off (MWCO). The Trichoderma reesei Rut C30 (ATCC 56765) extract was the source of cellulases, specially cellobiohydrolases. The microorganism was cultivated in a 6 L bioreactor (New Brunswick, Bioflo Celligen, USA 115). Inoculum was incubated at 200 rpm, 29 ºC, over 72 h. Medium composition for inoculum was (for each 0.5 L): 5 g Celuflok cellulose; 5 g glucose; 0.5 mL Tween 80; 0.5 g peptone; 25 mL salt solution (1 g KH2PO4, 0.7 g (NH4)2SO4, 0.15 g urea, 0.15 g MgSO4.7H2O, 0.15 g CaCl2, 2.5 mg FeSO4.7H2O, 0.7 mg ZnSO4.7H2O, 0.8 mg MnSO4.H2O and 1 mg CoCl2 in the final
8
volume). The growth medium was 30 g/L celuflok cellulose, 1.5 g/L peptone, 1.5 mL/L Tween 80, 150 mL/L salt solution, 3 g/L soy bran, 15%v/v inoculum (900 mL) and 4.191 L of water were used to complete the reaction volume (6 L). Salt solution composition at the final culture volume was 6 g/L KH2PO4, 4.2 g/L (NH4)2SO4, 0.9 g/L urea, 0.9/L g MgSO4.7H2O, 0.9 g/L CaCl2, 15 mg/L FeSO4.7H2O, 4.2 mg/L ZnSO4.7H2O, 4.8 mg/L MnSO4.H2O and 6 mg/L de CoCl2. Fermentation was conducted throughout 144 h, at 29 ºC, pH 5.0 and 20% D.O. Agitation and aeration were set up in cascade, corresponding to 200-600 rpm and 0.3-3 L/min, respectively. The supernatant was recovered by centrifugation (10,000 rpm, 30 min, 2 ºC). The T. reesei supernatant was sequentially precipitated with gradient ammonium sulfate saturation aiming at cellobiohydrolase fraction enrichment. Firstly, 20 mL of supernatant was precipitated with 40, 50, 60, 70 and 80% salt saturation. Based on this study, 40% salt saturation was chosen aiming the removal of the precipitated proteins. Ammonium sulfate salt was added to the supernatant to achieve 60% saturation of the sample. The saturated solution was incubated at 4 ºC over-night to precipitate. The proteins precipitated in 60% of salt saturation were recovered by centrifugation (10,000 rpm, 20 min, 4 ºC) and resuspended in 50 mM 4.8 pH citrate buffer (the volume was 10 times lower) originating the T. reesei fraction. 2.3 Analytical methods Protein quantification was determined using the Bio-Rad Protein Assay reagent (Bio-Rad, CA, USA) and bovine serum albumin as a protein standard (Bradford, 1976). The concentration of purified recombinant proteins was measured through absorbance at 280 nm wavelength in spectrophotometer (Termo Scientific, Nanodrop 2000c), using
9
their specific extinction coefficient determined by ProtParam tool (Gasteiger et al., 2005). The extinction coefficient (M-1 cm-1) and molecular weights (kDa) were, respectively, 106,925 and 57.75 for endoglucanase, 82,850 and 22.7 for endoxylanase and 41,370 and 25.37 for expansin. Total cellulase activity (filter paper activity) was measured according to Ghose (1987). An international unit of enzymatic activity is defined as the amount of enzyme required to produce 1 µmol of product per minute. Endoglucanase and endoxylanase activities were estimated by the degradation of 0.5% carboxymethyl cellulose (CMC) and 0.5% beechwood xylan, respectively. The reactions were conducted at 50 ºC for 30 min (endoglucanase) or 10 min (endoxylanase). The reaction was comprised of 50 µL of the correspondent substrate, 40 µL of citrate sodium buffer (50 mM) pH 5.0 and 10 µL of the correspondent enzyme. The reducing sugar concentration was estimated by the 3,5-dinitrosalicylic acid method (Miller, 1959).1 mM p-nitrofenil-β-D-glucopiranoside (p-NPG), and 4 mM p-nitrofenil-β-D-cellobioside (p-NPC) (Sigma–Aldrich, St. Louis, EUA) were the substrates for the activity determining reactions for β-glucosidase and cellobiohydrolase, respectively. After 10 min for β-glucosidase and 30 min for cellobiohydrolase activities measurement, at 50 ºC, the reactions (comprised of 80 µL of the substrate and 20 µL of the enzyme) were stopped by addition of 1 mol/L sodium carbonate. Absorbance at 400 nm was used to estimate p-NP concentration release. 2.4 Hydrolysis assays Hydrolysis reactions were carried out in two scales: 1.5 and 50 mL final volume. For hydrolysis in larger scale, 77.7% moisture hydrothermal bagasse and stirred flasks at 200 rpm were used. For 1.5 mL final volume reactions, dried and milled bagasse and
10
stirring of 1,000 rpm were used. In both assays, the parameters of reaction were 5% (w/v) water-insoluble substrate (WIS), 50 ºC temperature and 4.8 pH (50 mM citrate buffer). The protein load for hydrolysis was stablished as 10 FPU per gram of dried bagasse for the validation of hydrolysis microassays. Sampling was performed at 6, 24, 48 and 72 h of reaction time. In these reactions, additional 20 UI of β-glucosidase per gram of dried bagasse were loaded. In the experimental design hydrolysis assays, protein load was 10 mg per gram of dried bagasse and the reactions were carried out for 48 h. To pause the enzymatic activity, samples were boiled at 99 ºC, for 5 min, centrifuged (12,000 rpm for 10 min at 4 ºC) and the supernatant analyzed for sugars (glucose, xylose and cellobiose) at high performance liquid chromatography (HPLC). The samples were injected in HPX-87H 300 mm X 7.8 mm column (Bio Rad), with a refractive index (RI) detector. 5 mM sulfuric acid was used as mobile phase, in a flow rate of 0.6 mL/min, at 35 ºC. 2.5 Optimization of enzymatic cocktail The protein proportion that lead to the highest glucose release in microassays hydrolysis was determined through experimental design. The seven factors and their maximum and minimum proportions are listed in Table 1. The total proportion sum for each run was one. To evaluate the response (glucose released at 48 h of hydrolysis- g/L) and how it was affected by the factors, a simplex lattice mixture design, polynomial degree 2 and augmented with interior points and centroid, was performed. The results of the experimental mixture design were analyzed and interpreted using the software Statistica 10 (StatSoft Inc., USA). To validate the mathematical model, analysis of variance (ANOVA) was performed. The validated model was maximized using SOLVER (EXCEL, Microsoft, USA).
11
3. Results and Discussion 3.1 Enzyme production Endoglucanase, endoxylanase and expansin were successfully produced by genetically modified E. coli. Induction of the protein expression can be seen in Fig. 1. The growth of microorganisms and enzyme production were performed in batch reactors. The enzymes were purified by immobilized metal affinity chromatography (IMAC). The enzyme-containing supernatants and purified proteins are presented in Fig. 2. To obtain imidazole-free and buffer-conditioned enzyme solutions, the elution fractions were concentrated and washed several times. The protein concentrations of purified recombinant endoglucanase, endoxylanase and expansin were, respectively, 2.83, 2.75 and 1.64 mg/mL. The endoglucanase and endoxylanase enzymatic activities were, respectively, 1.48 and 71.24 UI/mL. The enzymes produced by T. reesei had a cellulose activity of 3.9 PFU/mL after 144 h. The supernatant contains a mixture of essential enzymes for biomass conversion into fermentable sugars. Among them are cellobiohydrolases I and II, the main enzymes produced by this microorganism (Foreman et al., 2003). To concentrate the fungal supernatant and obtain an enriched cellobiohydrolase fraction, a sequential ammonium sulfate precipitation of the T. reesei culture (20 mL) was performed. The 40-60% precipitation fractions presented the greatest specific activity against the cellobiose substrate p-NPC, with values of 0.29 UI/mg for the supernatant and 0.36, 0.71 and 0.36 UI/mg for the 40, 50 and 60% precipitation fractions, respectively. Most of the enzymes concentrated in the 50 and 60% precipitation fractions weighed from 40 to 70 kDa (Fig.
12
3). The molecular weights of the cellobiohydrolases are 54.1 kDa (CBHI, GH 7) and 49.7 kDa (CBHII, GH 6) (Ståhlberg et al., 1993). The salt precipitation procedure enables protein concentration, buffer conditioning and total cellulase activity maintenance. The specific cellulase activity of the T. reesei fraction was 2.55 FPU/mg before and 2.7 FPU/mg after the precipitation. Many enzymatic classes are required to convert sugar cane bagasse into fermentable sugars. Because hydrothermally pretreated bagasse contains 60.56% cellulose, T. reesei extract (2.68 FPU/mg cellulase activity) was used to enable cellulose conversion. Endoglucanase, xylanase and expansin were also used as factors in the experimental design, as well as three commercial enzymes (β-glucosidase, α-Larabinofuranosidase and acetyl xylan esterase). The T. reesei extract has other enzymatic activities aside from cellobiohydrolases (Table 2) that could contribute to the hydrolysis of the substrate to some extent, limiting the significant effects of the chosen enzymatic activities on the experimental analysis. Fig. 4 shows the seven enzyme preparations used in the work reported here. 3.2 Validation of hydrolysis microassays To evaluate many enzyme proportions within a hydrolysis reaction, the smaller the volume that can be used, the cheaper the experiment. Therefore, a hydrolysis microassay was validated for 5% (w/v) water-insoluble solids in an experiment described in the Materials and Methods section. In these experiments, 20 UI of βglucosidase per gram of bagasse were added to supplement the T. reesei action in 5% WIS hydrolysis. The validation of the microassay (1.5 mL) was performed by comparison with hydrolysis performed at a larger scale (50 mL), and the results are
13
shown in Fig. 5b and Fig. 5a, respectively. Significant differences in the glucose release between the reactions with and without β-glucosidase were observed for each time and scale. The glucose concentrations after 48 h, used to calculate the conversion, are significantly different between reactions with and without additional enzyme (βglucosidase) for the same scale, at the 95% confidence level. This enzymatic effect occurs at both scales, and it validates the hydrolysis microassay. 3.3 Optimization of biomass saccharification To enhance glucose release in hydrothermal bagasse hydrolysis, the T. reesei fraction was used in a supplementation study involving six other proteins/factors. Five of them were enzymes with different actions in lignocellulosic biomass (EG, BGL, EX, AXE and ARF), while the last was expansin (EXP), a protein that cleaves hydrogen bonds between fibers and accessory sugars by a non-hydrolytic and a mechanism that is not well established, loosening the fiber and increasing its accessibility to enzymes (Cosgrove et al., 2002). Fig. 4 shows the SDS-Page electrophoresis of the proteins (factors) studied in the mixture experimental design. Considering that the bagasse composition is primarily cellulose, the lack of cellulase enzymes would prevent hydrolysis and also the perception of gain of other enzymes. Thus, the factors T. reesei, EG and BGL, which are cellulase enzymes, had their minimum fractions in the mixtures fixed at 0.2, 0.1 and 0.1, respectively. The enzymatic mixture composition was optimized through an experimental simplex lattice design. The protein loading was 10 mg per gram of dry bagasse. The released glucose concentration after 48 h of hydrolysis was the response evaluated. The
14
mathematical model (Eq. (1)) below presents the coefficients in the coded form of the factors (pseudo-components): = 24.23 + 15.46 + 14.82 + 15.37 + 9.26 + 9.5 + 15.53 + 7.18 ∙ + 6.1 ∙ + 9.42 ∙ + 8.48 ∙ + 9.94 ∙ + 6.81 ∙ (1) where is the predicted response, glucose concentration after 48 h of hydrolysis, = ((. − 0.2)⁄0.6),
= (( − 0.1)⁄0.6),
= ((# − 0.1)⁄0.6),
= ($⁄0.6), = ( $ ⁄0.6), = ( %⁄0.6) e = ($& ⁄0.6). The values of T. reesei, EG, BGL, EX, AXE, ARF e EXP correspond to the real proportion of them in the mixture. The mathematical model was evaluated by analysis of variance (ANOVA) (Table 3) and the F test. For statistically significant models, the calculated F value should be higher than the critical (tabulated) F value. In this case, the calculated F value is 224.32, while '(,*+(,-./0-,12) = 1.92, which implies a significant model. The model equation was used to calculate the values of the factors that maximize the response using SOLVER from Excel. By analyzing the standardized effect estimates (the effect values divided by the standard error of each factor), calculated in terms of the original components, it is possible to rank the factors. T. reesei appears to be the most important factor to glucose release in the hydrolysis reaction (Table 4). Besides the cellulase activity from the T. reesei extract, additional endoglucanase and β-glucosidase were used to supplement the fungi extract, resulting in an optimized cocktail (80% of T. reesei extract, 10% of endoglucanase (EG) and 10% of
15
β-glucosidase (BGL)). T. reesei activities for the β-glucosidase specific substrate (pNPG) and endoglucanase substrate (CMC) were 0.18 and 3.76 UI/mg, respectively (Table 2). Xylanase activity was high (96.80 UI/mg) and can be sufficient for providing the
accessibility to
cellulose,
which probably explains why
hemicellulase
supplementation was not necessary (EX, AXE and ARF factors). Another reason is that hydrothermal bagasse has a low percentage of hemicelluloses, only 3.13% of the biomass, which means that the access to cellulose is independent of hemicellulase action. However, there was also no need for xylanase supplementation when the biomass used was steam-exploded bagasse with a higher hemicelluloses percentage (results not shown). It is important to note that there are two distinct families in which xylanases can be classified, GH10 and GH11 (Ruller et al., 2006). In the present study, the endo-1,4-xylanase GH11 was applied. According to Goldbeck et al. (2014), GH11 alone releases more xylose from pretreated sugar cane bagasse hydrolysis, achieving 64.3% hemicellulose conversion. Comparing the α-L-arabinofuranosidases families, GH54 showed a positive effect on xylose production from pretreated sugar cane bagasse, while GH51 presented a negative effect (Goldbeck et al., 2014). When a mixture of hemicellulolytic enzymes was composed of endo-1,4-xylanase GH11, βxylosidase GH43 and α-L-arabinofuranosidase GH51, production of xylobiose and xylotriose was favored. When GH51 was substituted for GH54, there was an increase in xylose production from pretreated bagasse hydrolysis (Goldbeck et al., 2014). The improved enzyme cocktail developed in this study does not contain any additional α-Larabinofuranosidase, despite the verified importance of this enzyme in a cocktail produced by another Trichoderma specie (Delabona et al., 2013). Hu et al. (2011) studied the interaction between xylanase and the commercial Celluclast and stated that
16
the lower the cellulase (Celluclast) fraction in the mixture, the higher their synergism, resulting in more hydrolyzed cellulose and xylan from steam-pretreated corn stover. Moreover, when a range of xylanase loads was added to a constant amount of Celluclast (required to hydrolyze 70% of cellulose), no synergism between them was identified (Hu et al., 2011). This suggests that xylanases can contribute synergistically to the hydrolysis when they appear in large proportions in enzymatic mixtures. In the same way, the protein load of T. reesei in the reaction system could have influenced the synergism between it and the other protein preparations studied. Although the T. reesei extract presents activities other than that from cellulase, all the involved factors were significant as main effects, which means that the supplementation of this cocktail with other proteins has a positive statistical effect on the considered response. Furthermore, all the two-interaction factors involving T. reesei extract are statistically significant (Table 4). In the optimal cocktail composition determined (80% T. reesei, 10% EG and 10% BGL), endoglucanase and β-glucosidase were maintained at the lower level defined in the mixture design.
β-glucosidase
supplementation is doubtless important, as seen from the results in Fig. 5, but the real need for endoglucanase supplementation should be evaluated in future works. Gourlay et al. (2013) reported that swollenin, a “disrupting” protein such as expansin, did not present
synergism
with
purified
hydrolytic
enzymes
(exoglucanase
Cel7A,
endoglucanase Cel5A, xylanases GH10 and GH11) when the evaluated response was the glucose release after the incubation of the proteins with the substrate (steampretreated corn stover). However, when the evaluated response was xylose release, swollenin and xylanases presented strong synergism. The loosely ordered xylan structure enables the action of swollenin on the fibers. This makes the hemicellulose
17
more accessible to xylanase action, which justifies the fundamental presence of swollenin in the enzymatic mixture, once hemicellulose limits the access of the cellulase to cellulose (Gourlay et al., 2013). In the present work, expansin did not present synergism with the T. reesei fraction, whether the response evaluated was glucose or xylose (data not shown for analysis involving xylose). This inconsistency with the literature can be related to the fact that hydrothermally pretreated sugar cane biomass has only 3.13% hemicellulose, eliminating the need for a swelling protein to enhance the enzymatic accessibility of cellulose. Due to the high value of the T. reesei coefficient in the model equation, the higher its proportion in the mixture, the higher is the predicted response (glucose concentration). For this reason, when the protein load is 10 mg of protein per gram of biomass, the T. reesei factor plays a key role in the cellulose conversion. The protein load influences the ratio of the cocktail components (Gao et al., 2010). Gao et al. (2010) reported that endoglucanase seemed to be more important to glucose release when a low protein load was applied (7.5 mg of protein per gram of glucan). However, Zhou et al. (2009) optimized a cellulase cocktail using a two-level fractional factorial design. In this type of experimental design, the factor levels vary independently from each other, and consequently, each run presents a different protein load. The amount of glucose released (15.5 mg/ml) by hydrolysis under the action of the optimized enzyme mixture was 2.1 times that produced under the action of the multi-enzyme cocktail secreted from the T. viride mutant strain T 100-14 (Zhou et al., 2009). 3.4 Comparison of the optimized cocktail and commercial Celluclast 1.5 L.
18
The glucose release predicted by the model (24.23 g/L) when the optimized cocktail is used corresponds to a 72% cellulose conversion. Under the same conditions (10 mg of protein per gram of dry bagasse), the commercial Celluclast cocktail achieved 49.11% ± 0.49 cellulose conversion. This commercial cocktail presents a filter paper activity of 108 FPU/mL. To validate the response predicted by the model, the hydrolysis of hydrothermally pretreated bagasse using the optimized cocktail was carried out. The observed conversion of cellulose was statistically similar to the predicted value, at 68.36% ± 5.57. Without supplementation, the T. reesei fraction presents 58.81% ± 0.71 cellulose conversion. Braga et al. (2014) studied the supplementation of Celluclast 1.5L (10 FPU/g of dry bagasse load in hydrolysis) with 30% (v/v) crude enzymatic complex from Aspergillus oryzae. The conversion of cellulose from the hydrothermally pretreated bagasse increased by 36%, achieving 50.8% at the end of 72 h hydrolysis. The enzymatic load was 6.7 mg protein/g dry bagasse (Braga et al., 2014). The conversion yield of glucose from cellulose was higher under the action of the optimized cocktail developed in this work. In addition, the replacement of a commercial cocktail in an optimized enzyme preparation reduces costs and unnecessary enzymatic activities. Ramos et al. (2015) proposed a reaction condition to obtain high conversion rates and concentration of glucose equivalents (which also takes into account the amount of cellobiose that is released in the reaction medium) after 72 h of hydrolysis of phosphoric-catalyzed steam-exploded sugar cane bagasse. The highest amount of sugar released, which corresponds to a glucan conversion of 69.2%, was achieved when the parameters of substrate total solids (%), agitation (rpm) and enzyme loading of Cellic CTec2 from Novozymes (g/g of cellulose) were maintained at their highest levels, 20 wt%, 200 rpm and 0.1 g/g of cellulose, respectively. The glucose yield achieved by
19
Ramos et al. (2015) was similar to that achieved by the optimized cocktail developed in this work, although they used a higher total solid content and lower enzyme loading. However, the surface response presented by Ramos et al. (2015) suggests that the glucose yield would continue rising as the enzyme loading increases. The evaluation of the differences in sugar release as a function of time for the optimized (2.75 FPU/mg) and the commercial Celluclast (2.57 FPU/mg) cocktails during hydrolysis is a way of assessing the performance of the improved cocktail composition. Fig. 6 shows the sugar release profile (glucose, xylose and cellobiose) from hydrolysis for these enzyme preparations. The glucose and xylose concentrations are higher in hydrolyses under the optimized cocktail action after 6 h of reaction and longer. The cellobiose concentration is higher throughout the hydrolysis under the action of Celluclast, as this enzyme preparation does not contain sufficient βglucosidase activity. The optimized cocktail maintained a low cellobiose concentration throughout the reaction, which was 0.41 ± 0.1 g/L at the end of hydrolysis. Around fifty percent of the glucose released was obtained in the first 6 hours of the reaction for both systems (13.32 ± 0.11 g/L for the optimized cocktail and 7.79 ± 0.15 for Celluclast, which represent 39.59% and 23.05% of the cellulose conversion, respectively). However, the rate of conversion was 2 times higher using the optimized cocktail (approximately 2.2 g/L.h) compared with Celluclast (approximately 1.2 g/L.h). This significant difference in the rates in the first 6 hours can be associated with the accumulation of cellobiose in the Celluclast system, which is known as the main cellobiohydrolase inhibitor (Holtzapple et al., 1990). Delabona et al. (2012) cultivated a new strain of Trichoderma harzianum isolated from the Amazon rainforest, aiming for the production of an enzymatic complex to hydrolyze biomass, which presented a
20
glucose yield of 19.4% after 72 h of hydrolysis of steam-pretreated sugar cane bagasse. This value corresponds to a glucose release of 5.5 g/L, while T. reesei hydrolysis under the same conditions released 5.8 g/L of glucose (Delabona et al., 2012). Hydrolysis conversion (%) is related to enzyme efficiency, while the glucose concentration released throughout the hydrolysis determines how viable the process is for the subsequent step of glucose utilization (Ramos et al., 2015). The enzymatic cocktail optimized in this work enables an efficient cellulose conversion, the highest reported among the revised studies involving hydrothermally pretreated bagasse, even though the glucose release is not as high as that reported in some studies listed here. 4. Conclusions This study reported the production and recovery of proteins to be used in hydrolysis assays aiming at cocktail optimization for sugar cane saccharification.
bagasse
The hydrolysis microassays validated in this work were used to
evaluate the glucose release during the mixture design experiments. The optimized cocktail, composed of 80% T. reesei CBH enriched fraction, 10% endoglucanase and 10% β-glucosidase, increased the conversion of cellulose by approximately 40% compared to Celluclast 1.5L. Thus, the approach of identifying the necessary activities to improve a fungal extract through mixture experiments can contribute to optimized cocktail development for different substrates and various applications. The experimental matrix of the simplex lattice design and the response for each run are presented in Table S1 (Supplementary Material). Acknowledgements
21
The authors would like to thank the São Paulo Research Foundation (FAPESP, process numbers 2008/57873-8, 2010/08089-2 and 2012/23223-2) for financial support, the know-how in protein purification of the CBMEG/UNICAMP, and the Fungal Hydrolases Biosynthesis Laboratory, CTBE, Campinas, Brazil, for the assistance in Trichoderma reesei Rut C30 cultivation. Moreover, the authors are grateful to Dr. George Jackson da Rocha (CTBE/Brazil) for the hydrothermally pretreated bagasse, Dr. José Geraldo da Cruz Pradella (CTBE/Brazil) for the T. reesei Rut C30 strain and Dr. Roberto Ruller (CTBE/Brazil) for the cloned genes, kindly provided to us. References
1. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–54. 2. Braga, C.M.P., Delabona, P. da S., Lima, D.J. da S., Paixão, D.A.A., Pradella, J.G. da C., Farinas, C.S., 2014. Addition of feruloyl esterase and xylanase produced onsite improves sugarcane bagasse hydrolysis. Bioresour. Technol. 170, 316–324. doi:10.1016/j.biortech.2014.07.115 3. Cosgrove, D.J., Li, L.C., Cho, H., Hoffmann-Benning, S., Richard, C., Blecker, D., 2002. The Growing World of Expansins. Plant Cell Physiol. 43, 1436–1444. 4. Delabona, P. da S., Farinas, C.S., Ribeiro, M., Freitas, S., Pradella, J.G. da C., 2012. Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production. Bioresour. Technol. 107, 517–521. doi:10.1016/j.biortech.2011.12.048
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5. Delabona, P.D.S., Cota, J., Hoffmam, Z.B., Paixão, D.A.A., Farinas, C.S., Cairo, J.P.L.F., Lima, D.J., Squina, F.M., Ruller, R., Pradella, J.G.D.C., 2013. Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase. Bioresour. Technol. 131, 500–7. doi:10.1016/j.biortech.2012.12.105 6. Foreman, P.K., Brown, D., Dean, R., Diener, S., Dunn-coleman, N.S., Goedegebuur, F., Houfek, T.D., England, G.J., Kelley, S., Meerman, H.J., Mitchinson, C., Heather, A., Teunissen, P.J.M., Yao, J., Ward, M., Dankmeyer, L., Kelley, A.S., Mitchell, T., Olivares, H.A., 2003. Transcriptional Regulation of Biomass-degrading Enzymes in the Filamentous Fungus Trichoderma reesei. J. Biol. Chem. 278, 31988–31997. doi:10.1074/jbc.M304750200 7. Gao, D., Chundawat, S.P.S., Krishnan, C., Balan, V., Dale, B.E., 2010. Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour. Technol. 101, 2770–81. doi:10.1016/j.biortech.2009.10.056 8. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., Bairoch, A., 2005. Protein Identification and Analysis Tools on the ExPASy Server, in: Walker, J.M. (Ed.), The Proteomics Protocols Handbook. pp. 571–607. 9. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257– 268. 10. Gilead, S., Shoham, Y., 1995. Purification and Characterization of α-LArabinofuranosidase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 61, 170–174.
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Figure Captions Fig. 1. SDS-PAGE electrophoresis of recombinant protein expression in E. coli. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lanes a, c and e: before induction of the expression system encoding for endoglucanase, endoxylanase and expansin, respectively; lanes b, d and f: after induction of the expression system encoding for endoglucanase, endoxylanase and expansin, respectively. Fig. 2. SDS-PAGE electrophoresis of expression and chromatograph purification of recombinant proteins. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: endoglucanase-containing supernatant; lane b: purified endoglucanase; lane c: endoxylanase-containing supernatant; lane d: purified endoxylanase; lane e: expansin-containing supernatant; lane f: purified expansin. Fig. 3. SDS-PAGE electrophoresis of supernatant and ammonium sulfate precipitation fractions of T. reesei culture. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: T. reesei culture supernatant; lane b: 40% ammonium sulfate precipitation fraction; lane c: 50% ammonium sulfate precipitation fraction; lane d: 60% ammonium sulfate precipitation fraction; lane e: 70% ammonium sulfate precipitation fraction; lane f: 80% ammonium sulfate precipitation fraction. Fig. 4. SDS-PAGE electrophoresis of the proteins (factors) used in the mixture experimental design. Molecular weights (M.W.) in decreasing order (kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; lane a: T. reesei; lane b: endoglucanase (EG); lane c:
27
β-glucosidase (BGL); lane d: endoxylanase (EX); lane e: acetyl xylan esterase (AXE); lane f: α-L-arabinofuranosidase (ARF); lane g: expansin (EXP). Fig. 5. Effect of β-glucosidase addition on the glucose release in different times of hydrothermally bagasse hydrolysis. TR indicates hydrolysis under the T. reesei action only. TR+BLG indicates hydrolysis with β-glucosidase supplementation. The experiments were carried out in 50 mL (a) and 1.5 mL (b) reaction volumes. Different letters indicate statistic difference between averages (Tukey, 0.05%). Fig. 6. Concentration (g/L) of sugar (glucose, xylose and cellobiose) throughout 48 h of hydrolyses of hydrothermally pretreated bagasse due to action of the optimized cocktail and commercial cocktail (Celluclast 1.5L) in reaction volume of 1.5 mL.
M.W.
a
b
c
d
Fig. 1
M.W.
e
f
M.W.
a
b
c
Fig. 2
d
e
f
M.W.
a
b
c
Fig. 3
d
e
f
M.W.
a
b
c
d
Fig. 4
e
f
g
a
A
TR TR + BGL
25
B B/C
20
C/D
Glucose (g/L)
D 15
E
E
10
5
F
0
6
24
48
72
Time (h)
b
A TR TR + BGL
25
A A
20
Glucose (g/L)
B B/C
15
C
C
10
5
A
0
6
24
48
Time (h)
Fig. 5
72
50
20
14
20
1.8
26
6
24
4 1.6 10
22
15
2.8 10
2.4 5
2
2.0
0
20
18
40
Time (h)
16
60
1.2 1.0
14
Xylose (g/L)
1.4
20
Cellobiose (g/L)
10
2.0
20
0
1.6 1.2
0.8
12
0.8
10
0.6 0.4
8
0.4
6 0.0 0
20
40
Time (h) Fig. 6
60
Cellobiose (g/L)
12
8
28
Glucose (g/L)
30
25
16
Xylose (g/L)
Glucose (g/L)
30
18
Glucose_Optimized cocktail Glucose_Celluclast 1.5 Xylose_Optimized cocktail Xylose_Celluclast 1.5 Cellobiose_Optimized cocktail Cellobiose_Celluclast 1.5
40
30
Table 1 Proportion range (maximum and minimum) of each factor: T. reesei extract (T. reesei), endoglucanase (EG), β-glucosidase (BGL), endoxylanase (EX), acetyl xylan esterase (AXE), α-Larabinofuranosidase (ARF) and expansin (EXP). Total proportion sum for each run=1 Factors Proportion
T. reesei
EG
BGL
EX
AXE
ARF
EXP
Minimum
0.2
0.1
0.1
0
0
0
0
Maximum
0.8
0.7
0.7
0.6
0.6
0.6
0.6
Table 2 Enzymatic activities of 60% ammonium sulfate precipitation fraction from the T. reesei supernatant Substrates
Volumetric activity (UI/mL) Specific activity (UI/mg)
p-NPC 4 mM
5.99
0.60
p-NPG 1 mM
1.78
0.18
p-NPX 1 mM
0.18
0.02
Beech wood xylan 0.5%
968
96.80
CMC 0.5%
37.57
3.76
1,4-β-D-manan 0.5%
9.96
1.00
Lichenan 0.5%
27.81
2.78
Wheat arabinoxylan 0.5%
30.76
3.08
Table 3 ANOVA analysis for the mixture design. Confidence interval of 95% Sum of
Degrees of
Mean
squares
freedom
square
995.57
12
83
21.817
59
0.37
9.449
23
0.41
Pure error
12.368
36
0.34
Total
1017.93
71
Source Model Total error Lack of fit
% explained variation: 97.8%
Table 4 Mathematical model effects, residual errors, t-student and p-value to the original components Effect
Error
t(44)
p-value*
(A)T. reesei
24.31
1.12
21.71
0.000
(B)EG
9.44
1.65
5.73
0.000
(C)BGL
7.79
1.65
4.73
0.000
(D)EX
6.76
2.08
3.25
0.002
(E)AXE
-0.32
2.08
-0.15
0.878
(F)ARF
0.31
2.08
0.15
0.884
(G)EXP
9.21
2.08
4.43
0.000
AB
19.18
5.91
3.24
0.002
AC
18.48
5.91
3.13
0.003
AD
27.42
5.91
4.64
0.000
AE
22.85
5.91
3.86
0.000
AF
25.58
5.91
4.33
0.000
AG
19.19
5.91
3.25
0.002
* p-values below 0.05 were significant and above were not significant
28
Highlights
•
Well-balanced enzymatic cocktails as promising approach to optimize biomass hydrolysis
•
Sugar cane bagasse 5% (W/V) hydrolysis microassay was validated
•
Identification of necessary activities to improve fungal extract performance
•
Mixture experimental design was used to develop an optimized enzyme cocktail
•
Cellulose conversion was increased by approximately 40% in respect of Celluclast 1.5L