Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.     

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.     

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.     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

Endo-exo Synergism in Cellulose Hydrolysis Revisited

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.     

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.     

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.     

Cellulase and Xylanase Production by the Mexican Strain <Emphasis Type="Italic">Talaromyces stollii</Emphasis> LV186 and Its Application in the Saccharification of Pretreated Corn and Sorghum Stover

A Mexican strain of Talaromyces stollii LV186 was isolated from decaying pretreated corn stover. The production of cellulase and xylanase enzyme cocktails was evaluated with corn and sorghum stover used as inducers in a mineral medium. The volumetric and specific activities of T. stollii LV186 were compared with the values produced by Trichoderma reesei ATCC 26921 in a time-course experiment. After the submerged culture and a posterior ultrafiltration stage, the enzyme complexes were evaluated over acid-pretreated corn or sorghum stover in baffled flasks under controlled temperature and agitation conditions, and hydrolysis levels of 30 and 39 % of the theoretical maximum were obtained after only 72-h reactions, for each substrate. A side-by-side comparison showed a better ratio of endoglucanase to cellobiohydrolase to β-glucosidase and of xylanase to β-xylosidase enzymes in T. stollii than in T. reesei ATCC 26921. Furthermore, the hydrolysis of pretreated corn and sorghum stover achieved by T. stollii is significantly higher compared with that of a commercial cocktail from T. reesei ATCC 26921 (Celluclast). Therefore, the T. stollii LV186 strain is a good candidate for the hydrolysis of complex lignocellulose substrates. To the authors’ knowledge, this study is the first to describe the cellulolytic and hemicellulolytic activities produced by a T. stollii strain.     

A mechanistic model for enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition

The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate.     

High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration

The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.     

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme–inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.     

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.     

Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose

Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohy-drolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30-90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased with increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I. (c) 1994 John Wiley & Sons, Inc.     

Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes

Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40 degrees C when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4 degrees C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.     

The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover

Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-β-xylanase III), Xyn 11 (endo-xylanase II), and β-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and β-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity.     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.     

Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis

We discuss the hydrolysis of cellulose using a pure cellulase: endo-1,4-β-D-glucanase (EG) from the fungus, Aspergillus niger, in buffer, the pure ionic liquid (IL), tris-(2-hydroxyethyl)-methylammonium methylsulfate (HEMA), and various mixtures of the two at different temperatures. Steady-state fluorescence and absorbance studies were performed to monitor the stability and activity of EG using cellulose azure as the substrate. EG attains its highest activity at 45°C in buffer and denatures at ～55°C. On the other hand, HEMA imparts substantial stability to the enzyme, permitting the activity to peak at 75°C. The relative roles of temperature, viscosity, pH, polarity, and the constituent ions of the ILs on the hydrolysis reaction are examined. It is demonstrated that pretreatment of cellulose with ILs such as BMIM Cl, MIM Cl, and HEMA results in more rapid conversion to glucose than hydrolysis with cellulose that is not pretreated. The percent conversion to glucose from pretreated cellulose is increased when the temperature is increased from 45 to 60°C. Two different ILs are used to increase the efficiency of cellulose conversion to glucose. Cellulose is pretreated with BMIM Cl. Subsequent hydrolysis of the pretreated cellulose in 10-20% solutions of HEMA in buffer provides higher yields of glucose at 60°C. Finally, to our knowledge, this is the first study dealing with a pure endoglucanase from commercial A. niger. This enzyme not only shows higher tolerance to ILs, such as HEMA, but also has enhanced thermostability in the presence of the IL.     

Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-β-xylanase fused to a GH43 β-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.