Anikó Várnai

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood.

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes. The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan. When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.

Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs

BackgroundA vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellulases (dockerins). This finding poses the question about the role of the CBMs: why would most cellulases lack CBMs, if they are necessary for the efficient hydrolysis of cellulose?ResultsThe advantage of CBMs, which increase the affinity of cellulases to substrates, was found to be diminished by reducing the amount of water in the hydrolytic system, which increases the probability of enzyme-substrate interaction. At low substrate concentration (1% w/w), CBMs were found to be more important in the catalytic performance of the cellobiohydrolases TrCel7A and TrCel6A of Trichoderma reesei as compared to that of the endoglucanases TrCel5A and TrCel7B. Increasing the substrate concentration while maintaining the enzyme-to-substrate ratio enhanced adsorption of TrCel7A, independent of the presence of the CBM. At 20% (w/w) substrate concentration, the hydrolytic performance of cellulases without CBMs caught up with that of cellulases with CBMs. This phenomenon was more noticeable on the lignin-containing pretreated wheat straw as compared to the cellulosic Avicel, presumably due to unproductive adsorption of enzymes to lignin.ConclusionsHere we propose that the water content in the natural environments of carbohydrate-degrading organisms might have led to the evolution of various substrate-binding structures. In addition, some well recognized problems of economical saccharification such as unproductive binding of cellulases, which reduces the hydrolysis rate and prevents recycling of enzymes, could be partially overcome by omitting CBMs. This finding could help solve bottlenecks of enzymatic hydrolysis of lignocelluloses and speed up commercialization of second generation bioethanol.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

Xylan as limiting factor in enzymatic hydrolysis of nanocellulose

The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture.

Changes in Submicrometer Structure of Enzymatically Hydrolyzed Microcrystalline Cellulose

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10-20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

The role of carbohydrate binding module (CBM) at high substrate consistency: comparison of Trichoderma reesei and Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII).

The role of CBM in two fungal model cellulase systems, consisting of Cel7A and Cel5A, from Trichoderma reesei and Thermoascus aurantiacus, were compared in the hydrolysis of various substrates. For comparison, family-1 CBMs were introduced to the T. aurantiacus and removed from the T. reesei enzymes. Especially at high dry matter consistencies of lignocellulosic substrates, pretreated wheat straw and spruce, the T. aurantiacus enzymes lacking CBM outperformed the enzymes carrying the CBM. In these conditions, the CBM-less enzymes from both organisms obviously recognized and bound to the substrate at higher probability than in dilute systems. Avoiding the unproductive binding to lignin caused by the CBMs obviously enhanced the hydrolytic performance. The lignin binding effect was, however, not entirely caused by the CBM, but also by the different structures and affinities of the core enzymes to lignin. Due to decreased binding, the CBM-less enzymes would allow reuse, potentially decreasing hydrolysis costs.

Cellulases without carbohydrate-binding modules in high consistency ethanol production process

BackgroundEnzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis.ResultsHigh hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (Ct Cel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency.ConclusionsThe results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce

BackgroundThe recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs).ResultsLMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis.ConclusionsLMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

Small-angle scattering study of structural changes in the microfibril network of nanocellulose during enzymatic hydrolysis

The hydrolysis of nanofibrillated cellulose (NFC), consisting of individual cellulose fibrils, was followed using small-angle scattering techniques in order to reveal changes in the substrate structure caused by cellulose degrading enzymes. In particular, the nanoscale structure of the network of cellulose fibrils was characterized with the combination of small-angle neutron scattering and small-angle x-ray scattering. In the nanocellulose with higher xylan content, the interfibrillar distance was shown to remain unchanged during enzymatic degradation, whereas the distance increased in the nanocellulose with lower xylan content. The limiting effect of xylan on the hydrolysis and a faster hydrolysis of the more thoroughly fibrillated segments of the NFC network could be observed. Despite the extensive fibrillation of the raw material, however, the hydrolysis was eventually limited by the aggregated and heterogeneous structure of the substrate.