Improving enzymatic conversion of lignocellulose to platform sugars

Increasing demand and uncertain availability of fossil fuels urge us to find alternative resources available in large quantities especially for the petrol-based transportation sector. Lignocellulosic biomass, available worldwide in plant cell walls, is a promising alternative feedstock. It can be depolymerised to sugar monomers, which provide potential raw material for sugar platform-based production of fuels and chemicals. However, the enzymatic saccharification of lignocellulose to platform sugars is hindered primarily by the complexity of lignocellulosic substrates as well as by the performance of the hydrolytic enzymes involved. This study focuses on various rate limiting factors such as the decrease in the reactivity and accessibility of the substrates which slow down the hydrolysis, on auxiliary enzymes needed for the efficient solubilisation of cellulose, as well as on the adsorption of enzymes. Consequently, solutions to these limitations were sought to improve the efficiency of biomass conversion processes. Following the morphological and structural changes in the substrate during hydrolysis revealed that the average crystal size and crystallinity of cellulose remained constant while particle size generally decreased (Paper I). In particular, cellulose microfibrils were proposed to be hydrolysed one-by-one in fibre aggregates by peeling off cellulose chains layer-by-layer from the outer crystals of microfibril aggregates. Microscopic observation showed that almost intact particles remained in the residue even after 60% conversion. Lignocellulose is a complex network of lignin and polysaccharides. Lignin was found to impede the hydrolysis of cellulose, and its extensive removal doubled the conversion yields of softwood (Paper II). On the other hand, accumulation of lignin during hydrolysis did not affect hydrolysability by commercial cellulase preparations. Residual hemicelluloses, especially glucomannan, were resistant to enzymatic hydrolysis but could be removed together with lignin during delignification. This suggests that especially glucomannans are bound to lignin as lignin-carbohydrate complexes. In addition, cellulose, xylan and glucomannan were shown to be structurally interlinked in softwood (Paper IV). The hydrolysis yield of these polysaccharides remained below 50% without the simultaneous hydrolysis of all polysaccharides. Synergism between the solubilisation of cellulose and hemicelluloses was found, and the release of glucose, xylose and mannose was in linear correlation. The adsorption and desorption of enzymes were followed during hydrolysis (Paper III). After a quick initial adsorption, slow desorption and re-adsorption of enzymes was observed in alkaline delignified spruce. On the other hand, unproductive adsorption to lignin as well as enzyme inactivation was predicted to play a primary role in the irreversible adsorption of cellulases in steam pretreated spruce or Avicel during hydrolysis when no desorption of cellulases could be detected. This study showed for the first time that increasing substrate concentration could compensate for the absence of carbohydrate binding modules (CBMs) in hydrolytic enzymes (Paper V). The performance of cellulases lacking CBMs was comparable to that of cellulases comprising CBM at 20% substrate concentration. At the same time, over 60% of the enzymes without CBMs could be recovered at the end of the hydrolysis. Thus, the major part of hydrolytic enzymes without CBMs could potentially be recovered in industrial high consistency processes.Increasing demand and uncertain availability of fossil fuels urge us to find alternative resources available in large quantities especially for the petrol-based transportation sector. At present, first generation bioethanol and biodiesel are produced worldwide from cornstarch, sugarcane and rapeseed oil. However, fuels produced from these raw materials are not considered sustainable. Thus, recent efforts have been directed towards the use of sustainable raw materials, such as residues from forestry and agriculture as well as municipal wastes. Lignocellulosic biomass, available worldwide in plant cell walls, is a promising alternative feedstock for the production of second generation biofuels. However, the enzymatic saccharification of lignocellulose to platform sugars is hindered primarily by the complexity of lignocellulosic substrates as well as by the performance of the hydrolytic enzymes involved. Therefore, this work focuses on various rate limiting factors such as the decrease in the reactivity and accessibility of the substrates which slow down the hydrolysis, on auxiliary enzymes needed for the efficient solubilisation of cellulose, as well as on the adsorption of enzymes. Consequently, solutions to these limitations were sought to improve the efficiency of biomass conversion processes

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

Abstract Background The 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). Results LMTs 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. Conclusions LMTs, 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

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation

MSTO1 is a cytoplasmic pro-mitochondrial fusion protein, whose mutation induces myopathy and ataxia in humans.

The protein MSTO1 has been localized to mitochondria and linked to mitochondrial morphology, but its specific role has remained unclear. We identified a c.22G > A (p.Val8Met) mutation of MSTO1 in patients with minor physical abnormalities, myopathy, ataxia, and neurodevelopmental impairments. Lactate stress test and myopathological results suggest mitochondrial dysfunction. In patient fibroblasts, MSTO1 mRNA and protein abundance are decreased, mitochondria display fragmentation, aggregation, and decreased network continuity and fusion activity. These characteristics can be reversed by genetic rescue. Short-term silencing of MSTO1 in HeLa cells reproduced the impairment of mitochondrial morphology and dynamics observed in the fibroblasts without damaging bioenergetics. At variance with a previous report, we find MSTO1 to be localized in the cytoplasmic area with limited colocalization with mitochondria. MSTO1 interacts with the fusion machinery as a soluble factor at the cytoplasm-mitochondrial outer membrane interface. After plasma membrane permeabilization, MSTO1 is released from the cells. Thus, an MSTO1 loss-of-function mutation is associated with a human disorder showing mitochondrial involvement. MSTO1 likely has a physiologically relevant role in mitochondrial morphogenesis by supporting mitochondrial fusion

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities

Abstract Background The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. Results An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). Conclusions This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

Background: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics

Demonstration-scale enzymatic saccharification of sulfite-pulped spruce with addition of hydrogen peroxide for LPMO activation

The saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite-pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co-substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration-scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale