Academic literature on the topic 'Cellulase cocktail'

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s−1 and 0.64 s−1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

The conventional endo–exo synergism model has extensively been supported in literature, which is based on the perception that endoglucanases (EGs) expose or create accessible sites on the cellulose chain to facilitate the action of processive cellobiohydrolases (CBHs). However, there is a lack of information on why some bacterial and fungal CBHs and EGs do not exhibit synergism. Therefore, the present study evaluated and compared the synergistic relationships between cellulases from different microbial sources and provided insights into how different GH families govern synergism. The results showed that CmixA2 (a mixture of TlCel7A and CtCel5A) displayed the highest effect with BaCel5A (degree of synergy for reducing sugars and glucose of 1.47 and 1.41, respectively) in a protein mass ratio of 75–25%. No synergism was detected between CmixB1/B2 (as well as CmixC1/C2) and any of the EGs, and the combinations did not improve the overall cellulose hydrolysis. These findings further support the hypothesis that “not all endo-to exo-cellulase interactions are synergistic”, and that the extent of synergism is dependent on the composition of cellulase systems from various sources and their compatibility in the cellulase cocktail. This method of screening for maximal compatibility between exo- and endo-cellulases constitutes a critical step towards the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation.

Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars.

This study was aimed to evaluate the pattern of cellulase biosynthesis from Aspergillusfumigatus ABK9 under submerged fermentation. Production was increased concomitantly with fungal growth up to 72 h and reached maximum (Xmax -6.72 g/l) with specific growth rate (mu max) of 0.126/h. Highest specific rate of enzyme production (q ) was found at initial medium pH of 5.0 and incubation temperature of 30 degrees C. At the same time, in the presence of 2-deoxy-D-glucose concentration of 0.5 mg/ml, the production of cellulolytic enzymes, viz, carboxymethyl cellulase activity (CMCase), filter paper degrading activity (FPase) and P-glucosidase activity reached maximum of 132.2, 21.3 and 28.9 U/ml, respectively. Cellulase biosynthesis was induced in respect to higher volumetric production rate (Qp), specific rate of enzymes production (qp, U/g biomass/h) and enzyme/biomass yield (YE/X) when grown in carboxymethyl cellulose in comparison to other saccharides as sole carbon source. Induction ratios (IR) of cellulases were between 12.3 and 24.4 in the presence of 1.5% (w/v) CMC in the culture media. The strain was quite resistant to catabolic repression by glucose up to 0.4% (w/v). Cellulases production was greatly influenced in the presence of yeast extract and potassium dihydrogen phosphate (KH2POA) as nitrogen and phosphate sources in the culture media. C/N ratio of 10.0 and C/P ratio of 4.0 proved to be the best for the production of enzyme cocktail. Along with the high production yield, the crude enzymes showed a promising cellulose hydrolyzing efficiency of rice straw, indicating the enzyme could be beneficial for its large scale industrial exploitation.

La production industrielle de bioéthanol à partir de biomasse lignocellulosique nécessite l'amélioration de l'efficacité hydrolytique du cocktail enzymatique de Trichoderma reesei. Ce champignon filamenteux présente l'intérêt de sécréter des enzymes cellulolytiques en grande quantité mais l'analyse de son génome montre que la diversité de gènes codant ces enzymes est restreinte. C'est pourquoi, dans ce travail, une complémentation de ce cocktail par des enzymes auxiliaires a été envisagée. Récemment, la présence de la swollénine, une protéine apparentée aux expansines végétales, a été mise en évidence chez T. Reesei. Sa capacité à écarter les fibres de cellulose et l'induction de son gène parallèle à ceux des cellulases laisse penser que cette protéine peut avoir un rôle auxiliaire pendant la cellulolyse. L'étude in silico de séquences comportant des similarités avec les expansines chez les champignons a montré l'existence de plusieurs familles dont une est absente chez T. Reesei. Un représentant de cette famille, la protéine CELA d'Aspergillus fumigatus ainsi qu'une swollénine de cette espèce, SWOAfu, ont donc été choisies pour une expression hétérologue dans T. Reesei. De plus, des constructions chimériques destinées à rapprocher physiquement SWOAfu de la cellobiohydrolase CBH1 de T. Reesei ont été réalisées. Cette protéine de fusion, qui devrait permettre d'augmenter leur synergie a également été exprimée dans T. Reesei. Enfin, pour étudier une implication éventuelle des protéines Endoglucanase-45/Expansin-Like (EEL) de T. Reesei dans l'hydrolyse de la lignocellulose, des études transcriptionnelles ont été réalisées dans des conditions d’induction de cellulases. Une expression constitutive d'une d'entre elles, semblable à celle de l'endoglucanase Cel5b a pu être montrée. Après analyse de leur structure protéique et de leurs promoteurs, les fonctions potentielles des protéines EEL sont discutées
The industrial production of bioethanol from lignocellulosic biomass requires the increase of the hydrolytic efficiency of the enzymatic pool produced by Trichoderma reesei. This fungus is able to secrete large amounts of cellulolytic enzymes, but its genome shows a low diversity of genes encoding these enzymes. Therefore, in this work, a complementation of this cocktail with auxiliary proteins was envisaged. Recently, the presence of swollenin, a protein related to plant expansins, was brought to light in T. Reesei. Its capacity to loosen cellulose fibers and the induction of its gene parallel to cellulase genes suggests that this protein could have an auxiliary role in cellulose hydrolysis. A database search of sequences presenting similarities with plant expansins in fungi showed that different families exist, one of which is absent in T. Reesei. CELA from Aspergillus fumigatus, a protein belonging to this family, and a swollenin from this species, SWOAfu, were selected for heterologous expression in T. Reesei. Moreover, chimeric protein constructions were realised to approach the catalytic domains of SWOAfu and the cellobiohydrolase CBH1 from T. Reesei. This chimeric protein which should lead to an increase of their synergy was also expressed in T. Reesei. Finally, in order to investigate a potential implication of Endoglucanase-45/Expansin-Like (EEL) proteins of T. Reesei in the cellulolytic process, transcriptional studies were realised under conditions of cellulase induction. A constitutive expression, similarly to the endoglucanase Cel5b, was shown for one of them. Potential functions of the EEL proteins are discussed with regard to their protein and promoter structure

Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost. The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions. Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported. My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway.
Master of Science

Parmi les ressources d'origines agricole et forestière utilisables aujourd'hui en tant que biomasse à destination énergétique, le miscanthus apparait comme l'une des espèces de graminées les plus prometteuses pour la production de bioéthanol de seconde génération grâce à son haut potentiel en biomasse. Ce procédé dit "2G" convertit la cellulose contenue dans ces biomasses lignocellulosiques en bioéthanol et ce via un procédé intégrant prétraitement physico-chimique, hydrolyse enzymatique et fermentation. Le principal objectif de ce projet de thèse visait à étudier l'impact de l'hétérogénéité tissulaire et structurale du miscanthus sur sa saccharification et s'est décliné en différents volets liés à l'étude de l'efficacité des prétraitements et à l'analyse des performances de différents cocktails enzymatiques de Trichoderma reesei. L'hydrolyse enzymatique est essentiellement limitée par la structure et la porosité des complexes pariétaux qui réduisent l'accessibilité de la cellulose aux cellulases. En plus des constituants hémicelluloses et lignines qui recouvrent la cellulose, les parois cellulaires du miscanthus sont riches en acides hydroxycinnamiques (pCA et FA) qui jouent un rôle important dans la cohésion du réseau pariétal complexe. L'application de prétraitements acide et alcalin sur le miscanthus a ainsi révélé une différence de réactivité en fonction des types cellulaires. Les parois secondaires du sclérenchyme sont plus facilement dégradées par les cellulases fongiques après prétraitement acide. L'étude de la distribution des composés phénoliques au niveau cellulaire par micro spectrophotométrie UV a rapporté une nette diminution de l'absorbance UV dans tous les tissus après chaque prétraitement. Ceci n'expliquant pas totalement les différences de réactivité observées, d'autres facteurs physicochimiques seraient donc impliqués. Une approche visant à évaluer la progression des cellulases au sein des parois par immunocytochimie a également été initiée mais elle s'est heurtée à des problématiques techniques liées à la nature des tissus et aux anticorps employés. Les performances en terme de conversion de la cellulose ont été évaluées avec des cocktails enzymatiques de T. reesei comprenant des activités (hemi-)cellulolytiques variables. Une meilleure efficacité du prétraitement par explosion à la vapeur a ainsi pu être montrée par réduction de la quantité d'enzymes mises en œuvre. Comme c'est le cas pour d'autres graminées, ces travaux ont permis de confirmer le rôle crucial de l'enzyme β-glucosidase, permettant de limiter l'inhibition par le cellobiose et améliorant la cinétique initiale de saccharification. L'amélioration du rendement d'hydrolyse par l'utilisation d'un sécrétome comprenant une bonne activité hémicellulolytique a pu être ensuite démontrée. L'utilisation de cocktails enzymatiques reconstitués à partir d'enzymes pures a enfin permis de définir un mélange "optimal" composé des quatre principales cellulases de T. reesei (CBH1, CBH2, EG1 et EG2) associées à une hémicellulase (XYN1)
Among agricultural and forest resources, the grass specie miscanthus has emerged as one of the most promising feedstock candidates for 2G-biofuel production due to its high biomass yield. The biofuels 2G-production process is based on cellulose conversion into bioethanol via physicochemical pretreatment, enzymatic hydrolysis and fermentation. The main objective of this Ph.D. project was to evaluate the effect of tissue and structure heterogeneity of miscanthus on its saccharification by evaluating pretreatment efficiency and analyzing the performance of different Trichoderma reesei cellulolytic cocktails.Enzymatic hydrolysis is mainly hindered by cell wall structure and porosity which limit cellulose accessibility to cellulase. In addition to hemicelluloses and lignin polymers, miscanthus cell walls, contain high amounts of hydroxycinnamic acids (pCA and FA) that play a significant role in cross-linking polymers into cohesive network. Applying acid and alkali pretreatments on miscanthus revealed a distinctive reactivity depending on cell types. Secondary cell walls of sclerenchyma appeared more digested by fungal cellulases after acid pretreatment. Addressing phenolics distribution (lignin and hydroxycinnamic acids) at cell level by UV micro spectrophotometry highlighted a significant decrease in UV absorbance after both pretreatments irrespective to cell type indicating that other physicochemical and structural features are involved in distinct cell wall reactivity. We have also attempted to evaluate cellulase progression into miscanthus cell walls by immunocytochemistry but we have had many technical problems due to the nature of miscanthus tissues and used antibodies. Cellulose conversion ability was then evaluated using enzymatic cocktails of T. reesei which vary in their (hemi-)cellulolytic activities. Higher efficiency of the steam explosion pretreatment was demonstrated by reducing enzymes loading. As reported previously on other grasses, β-glucosidase plays a crucial role by limiting the inhibiting effect of cellobiose and improving the initial saccharification step. We furthermore showed that the use of hemicellulases-improved cocktails allowed significant increase in saccharification yields. We finally identified an optimal reconstituted enzyme mixture composed of four major cellulases of T. reesei (CBH1, CBH2, EG1 and EG2) and the hemicellulase XYN-1

Strategies for cartilage tissue engineering and repair have recently focused on cell sources from the surrounding joint tissue as an alternative to chondrocytes. Synovium-derived stem cells (SDSCs) are found in the intimal layer of the synovium, the thin overlying capsule surrounding the joint space [1] and have been found to exhibit a greater chondrogenic potential than stem cells from other origins such as bone marrow stem cells or adipose derived stem cells [2–4]. Under directed cues, these cells have been shown to be capable of migrating from the synovium membrane into articular cartilage defects, though the mechanism behind such movement is unclear. As a first step, we have previously shown that SDSCs expanded in 2D monolayer culture in a growth factor cocktail of TGF-β1, FGF, and PDGF-ββ exhibit directed cathodal migration with perpendicular alignment when under the influence of an applied DC electric field [5]. As cellular behavior and response to an external stimulus can change with exposure to growth factors and passage number, we look here to characterize the effects of passaging on the migration response of SDSCs to an applied electric field. We hypothesize that if these cells develop more chondrocyte-like characteristics with growth factor passaging, their response will mimic that which has previously been reported for chondrocytes, notably directed cathodal (negative pole) migration and perpendicular realignment of the long axis to the direction of applied field [6].