Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.     

Synergistic hydrolysis of carboxymethyl cellulose and acid-swollen cellulose by two endoglucanases (CelZ and CelY) from Erwinia chrysanthemi

Erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of E. chrysanthemi. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.     

Pure enzyme cocktails tailored for the saccharification of sugarcane bagasse pretreated by using different methods

The compositions and physical properties of pretreated lignocellulose vary depending on pretreatment methods; therefore, enzyme cocktails specific to pretreatments are desired for efficient saccharification of lignocellulose. Here, enzyme cocktails consisting of three pure lignocellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH) and endoxylanase (XN) with a fixed amount of β-glucosidase were tailored for acid- and alkali-pretreated sugarcane bagasse (ACID and ALKALI, respectively). Based on a mixture design, the optimal mass ratios of EG, CBH, and XN were determined to be 61.25:38.73:0.02 and 53.99:34.60:11.41 for ACID and ALKALI, respectively. The optimized enzyme cocktail yielded a higher or comparable amount of reducing sugars from the hydrolysis of ACID and ALKALI when compared to that obtained using commercial cellulase mixtures. Using the commercial and easily available pure enzymes, this simple method for the in-house preparation of an enzyme cocktail specific to pretreated lignocellulose consisting of only four enzymes with a high level of hydrolysis will be helpful for achieving enzymatic saccharification in the lignocellulose-based biorefinery.     

A mechanistic model for rational design of optimal cellulase mixtures

A model‐based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello‐oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three‐dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model‐based framework for optimizing cellulase mixtures. Biotechnol. Bioeng. 2011;108: 2561–2570. © 2011 Wiley Periodicals, Inc.     

Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.     

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.     

The cellulosome and cellulose degradation by anaerobic bacteria

Despite its simple chemical composition, cellulose exists in a number of crystalline and amorphous topologies. Its insolubility and heterogeneity makes native cellulose a recalcitrant substrate for enzymatic hydrolysis. Microorganisms meet this challenge with the aid of a multi-enzyme system. Aerobic bacteria produce numerous individual, extra-cellular enzymes with binding modules for different cellulose conformations. Specific enzymes act in synergy to elicit effective hydrolysis. In contrast, anaerobic bacteria possess a unique extracellular multi-enzyme complex, called cellulosome. Up to 11 different enzymes are aligned on the non-catalytic scaffolding protein and thus ensure a high local concentration, together with the correct ratio and order of the components. These multi-enzyme complexes attach both to the cell envelope and to the substrate, mediating the proximity of the cells to the cellulose. Binding to the scaffolding stimulates the activity of each individual component towards the crystalline substrate. The most complex and best investigated cellulosome is that of the thermophilic bacterium Clostridium thermocellum, but a scheme for the cellulosomes of the mesophilic clostridia and the ruminococci emerges. Many crucial details of cellulose hydrolysis are still to be uncovered. Yet, a mechanistic model for the action of enzyme complexes on the surface of insoluble substrates becomes apparent and the application of enzymatic hydrolysis of cellulosic biomass can now be addressed.     

Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments

Bioprocess and Biosystems Engineering - High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to...     

Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO₂) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with β‐glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8–116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and β‐glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase–xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the enzyme adsorption and substrate characteristics were assessed during batch and fed-batch hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant original substrate limited their access to the freshly added substrate. After 72-h hydrolysis of the SPCS substrate at low enzyme loadings, ～40-50% of the added cellulases were desorbed into solution, whereas only 20% of the added enzyme was released from the EPCS substrate. Both simultaneous and sequential treatments with xylanases and cellulases resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme loading. Simons' stain measurements indicated that xylanase treatment increased cellulose access, thus facilitating cellulose hydrolysis.     

Benefits from tween during enzymic hydrolysis of corn stover

Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. (The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution.) The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 degrees C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. Copyright 1998 John Wiley & Sons, Inc.     

Optimization of Enzymatic Hydrolysis of Steam Pretreated Triticale Straw

Efficient conversion of the carbohydrates into fermentable sugars is crucial for industrial implementation of 2G biofuels such as bioethanol. The main objective of this study was to improve the enzymatic hydrolysis of steam pretreated triticale straw (slurry, pressed-slurry or water insoluble solids (WIS)) by optimal combination of cellulase (Cellic® CTec2) and hemicellulase (Cellic® HTec2) and incubation period for a target glucan conversion of 80 %. Among the three substrates evaluated, pressed-slurry and WIS resulted in similar sugar yields but WIS presented lower enzyme requirements. Different combinations of cellulase and endo-xylanase could provide an 80 % of glucan conversion depending on the weight assigned to constrain. The selected enzyme combination, 0.1 mL Cellic®CTec2/g WIS and 0.2 mL Cellic®HTec2/g WIS, could achieve a glucan conversion of 80 % in 45 h (desirability of 0.9). Doubling the enzyme dosage could further improve the saccharification productivity by reducing the incubation period to 37 h. The optimisation of enzymatic hydrolysis of lignocellulosic substrates, to reduce the cost of sugars production, is a compromise between substrate, enzyme dosage, incubation time and the benchmark yield, although a more favourable response can be generated with an optimised combination of enzymes.     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

Characterization of the interaction between surfactants and enzymes by fluorescence probe

In order to investigate the mechanism of the different stimulatory effects of the biosurfactant rhamnolipid and the chemical surfactant Tween 80 on enzymatic hydrolysis of lignocellulose, the interaction between surfactants and enzymes was analyzed by the fluorescence probe method using pyrene as probe. Based on the evolution law of pyrene fluorescence spectroscopy in the “surfactants-enzymes” systems, the interaction relationship between surfactants and enzymes was analyzed and discussed in this paper. The results show that enzyme molecules bind with rhamnolipid molecules, participate in the formation of rhamnolipid micelles, and increase the inner hydrophobic polarity of micelles, but do not change the properties of rhamnolipid micelles above the CMC (Critical Micelle Concentration). Nevertheless, for Tween 80, enzyme molecules also participate in the forming of micelles, however, they exhibit a stronger interaction with enzymes above the CMC. Both rhamnolipid and Tween 80 bind more strongly with xylanase than cellulase. Considering also previous experimental results, it can be concluded that the interaction between surfactants and enzymes improve enzyme stability and activity, and, therefore, the efficiency of enzymatic hydrolysis of lignocellulose is enhanced. The findings further provide theoretical knowledge about the mechanism of the stimulative effects of surfactants on enzymatic hydrolysis of lignocellulose.     

A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates

Current attempts to produce ethanol from lignocellulosic biomass are focused on the optimization of pretreatment to reduce substrate recalcitrance and the improvement of enzymes for hydrolysis of the cellulose and hemicellulose components to produce fermentable sugars. Research aimed at optimizing both aspects of the bioconversion process involves assessment of the effects of multiple variables on enzyme efficiency, resulting in large factorial experiments with intensive assay requirements. A rapid assay for lignocellulose hydrolysis has been developed to address this need. Pretreated lignocellulose is formed into handsheets, which are then used to prepare small disks that are easily dispensed into microtiter plates. The hydrolysis of cellulose to glucose is estimated using an enzyme-coupled spectrophotometric assay. Using disks prepared from ethanol organosolv pretreated yellow poplar, it is shown that the assay generates data comparable with those produced by hydrolysis of pretreated yellow poplar pulp in Erlenmeyer flasks, followed by HPLC analysis of glucose. The assay shows considerable time and cost benefits over the standard assay protocol and is applicable to a broad range of lignocellulosic substrates.     

Effect of pretreatment on the hydrolysis of cellulose by Penicillium funiculosum cellulase and recovery of enzyme

Penicillium funiculosum produces a complete cellulase which brings about 97% hydrolysis of cotton and has high beta-glucosidase, xylanase, laminarinase, and lichenase activities. This article deals with the effect of different pretreatments on the hydrolysis of sugarcane bagasse by P. funiculosum enzymes and the recovery of enzyme from the insoluble residues. Enzymic saccharification of bagasse pretreated with hot 1N NaOH followed by neutralization with HCI and steam treated under pressure (7 kg/cm(2)) gave 63 and 59% saccharification, respectively, in 48 h. Hemicellulose is not lost in these pretreatments. With a 30% slurry of steam-treated bagasse, a semisolid mass containing 14% sugar was obtained. A 90% recovery of CMCase, beta-glucosidase, and filter paper activity from the hydrolysates was obtained under the following conditions: (1) maintaining the ratio of enzyme to substrate high by stepwise addition of substrate, (2) brief grinding of the residual substrate with glass powder, and (3) addition of 0.4% Tween-80 to the eluting buffer. The high recovery of cellulolytic enzymes indicates that the adsorption of these enzymes on cellulose is not irreversible.     

Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass

Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free‐fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (A_w) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low‐field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism – A key factor for the hydrolysis of galactomannan substrates

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75–25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.     

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.