cellulase and phosphoric-acid

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from

Topics: Cellulase; Cellulose; Chitin; Mixed Function Oxygenases; Polysaccharides

Protocols were developed for efficient release of glucose from the biomass of Senna siamea, one of the highly efficient biomass producing tree legumes. Composition of mature, 1year and 2years coppice biomass were analysed. For the hydrolysis of the glucan, two pretreatments, cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF) and alkali (sodium hydroxide) were used; COSLIF (85% phosphoric acid, 45min incubation at 50°C) pretreated mature biomass exhibited best result in which 88.90% glucose released after 72h of incubation with the use of 5 filter paper units (FPU) of cellulase and 10 international units (IU) of β-glucosidase per gram of glucan. Of the biomass of different particle sizes (40-200mesh) used for saccharification, 40-60mesh shown the maximum glucose release. COSLIF pretreated mature, 1year and 2years coppice biomass showed equivalent glucose release profiles.

Topics: beta-Glucosidase; Biomass; Carbohydrate Metabolism; Cellulase; Cellulose; Chemical Fractionation; Fabaceae; Glucans; Glucose; Hydrolysis; Lignin; Particle Size; Phosphoric Acids; Solvents; Time Factors

Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases, and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli, and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461, and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.

Topics: Cell Wall; Cellulase; Cellulose; DNA Mutational Analysis; Hydrolysis; Mutagenesis, Site-Directed; Mutation; Phosphoric Acids; Plant Cells; Protein Domains; Ruminococcus; Solubility; Substrate Specificity

Pretreatment is of vital importance in the production of ethanol and methane from agricultural residues. In this study, the effects of steam pretreatment with phosphoric acid on enzymatic hydrolysis (EH), simultaneous saccharification and fermentation (SSF), anaerobic digestion (AD) and the total energy output at three different temperatures were investigated. The effect of separating the solids for SSF and the liquid for AD was also studied and compared with using the whole slurry first in SSF and then in AD. Furthermore, the phosphoric acid was compared to previous studies using sulphuric acid or no catalyst. Using phosphoric acid resulted in higher yields than when no catalyst was used. However, compared with sulphuric acid, an improved yield was only seen with phosphoric acid in the case of EH. The higher pretreatment temperatures (200 and 210 °C) resulted in the highest yields after EH and SSF, while the highest methane yield was obtained with the lower pretreatment temperature (190 °C). The highest yield in terms of total energy recovery (78 %) was obtained after pretreatment at 190 °C, but a pretreatment temperature of 200 °C is, however, the best alternative since fewer steps are required (whole slurry in SSF and then in AD) and high product yields were obtained (76 %).

Topics: Anaerobiosis; Biotechnology; Carbohydrate Metabolism; Cellulase; Ethanol; Fermentation; Glucose; Hydrogen-Ion Concentration; Hydrolysis; Methane; Phosphoric Acids; Steam; Temperature; Waste Products; Xylose; Zea mays

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20% inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90%, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH(∙) scavenging, FRAP and in vivo antioxidant capacity against H2O2-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.

Topics: Antioxidants; Biofuels; Biotechnology; Cellulase; Chromatography, Thin Layer; Endo-1,4-beta Xylanases; Fermentation; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Phosphoric Acids; Phylogeny; Saccharomyces cerevisiae; Temperature; Thermoascus; Triticum

Pretreatment of rice straw on pilot scale steam explosion has been attempted to achieve maximum sugar recovery. Three different reaction media viz. water, sulfuric acid and phosphoric acid (0.5%, w/w) were explored for pretreatment by varying operating temperature (160, 180 and 200°C) and reaction time (5 and 10min). Using water and 0.5% SA showed almost similar sugar recovery (∼87%) at 200 and 180°C respectively. However, detailed studies showed that the former caused higher production of oligomeric sugars (13.56g/L) than the later (3.34g/L). Monomeric sugar, followed the reverse trend (7.83 and 11.62g/L respectively). Higher oligomers have a pronounced effect in reducing enzymatic sugar yield as observed in case of water. Mass balance studies for water and SA assisted SE gave total saccharification yield as 81.8% and 77.1% respectively. However, techno-economical viability will have a trade-off between these advantages and disadvantages offered by the pretreatment medium.

Topics: Biotechnology; Carbohydrates; Cellulase; Hydrolysis; Oryza; Phosphoric Acids; Pilot Projects; Steam; Sulfuric Acids; Temperature; Waste Products

The structure of lignin after dilute phosphoric acid plus steam explosion pretreatment process of sugarcane bagasse in a pilot scale and the effect of the lignin extracted by ethanol on subsequent cellulose hydrolysis were investigated. The lignin structural changes caused by pretreatment were identified using advanced nondestructive techniques such as gel permeation chromatography (GPC), quantitative (13)C, and 2-D nuclear magnetic resonance (NMR). The structural analysis revealed that ethanol extractable lignin preserved basic lignin structure, but had relatively lower amount of β-O-4 linkages, syringyl/guaiacyl units ratio (S/G), p-coumarate/ferulate ratio, and other ending structures. The results also indicated that approximately 8% of mass weight was extracted by pure ethanol. The bagasse after ethanol extraction had an approximate 22% higher glucose yield after enzyme hydrolysis compared to pretreated bagasse without extraction.

Topics: Biomass; Biotechnology; Cellulase; Cellulose; Ethanol; Glucose; Hydrolysis; Lignin; Magnetic Resonance Spectroscopy; Molecular Weight; Phosphoric Acids; Saccharum; Steam

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.

Topics: Adsorption; Biotechnology; Cellulase; Cellulose; Glucose; Glycoside Hydrolases; Hydrolysis; Kinetics; Panicum; Phosphoric Acids; Xylans; Xylose

The modified cellulose solvent- (concentrated phosphoric acid) and organic solvent- (95% ethanol) based lignocellulose fractionation (COSLIF) was applied to a naturally-dry moso bamboo sample. The biomass dissolution conditions were 50 degrees C, 1 atm for 60 min. Glucan digestibility was 88.2% at an ultra-low cellulase loading of one filter paper unit per gram of glucan. The overall glucose and xylose yields were 86.0% and 82.6%, respectively. COSLIF efficiently destructed bamboo's fibril structure, resulting in a approximately 33-fold increase in cellulose accessibility to cellulase (CAC) from 0.27 to 9.14 m(2) per gram of biomass. Cost analysis indicated that a 15-fold decrease in use of costly cellulase would be of importance to decrease overall costs of biomass saccharification when cellulase costs are higher than $0.15 per gallon of cellulosic ethanol.

Topics: Bambusa; Biomass; Biotechnology; Carbohydrates; Cellulase; Cellulose; Enzymes; Glucans; Hydrolysis; Lignin; Phosphoric Acids; Solvents; Time Factors

A low level of phosphoric acid (1% w/w on dry bagasse basis, 160 degrees C and above, 10 min) was shown to effectively hydrolyze the hemicellulose in sugar cane bagasse into monomers with minimal side reactions and to serve as an effective pre-treatment for the enzymatic hydrolysis of cellulose. Up to 45% of the remaining water-insoluble solids (WIS) was digested to sugar monomers by a low concentration of Biocellulase W (0.5 filter paper unit/gWIS) supplemented with beta-glucosidase, although much higher levels of cellulase (100-fold) were required for complete hydrolysis. After neutralization and nutrient addition, phosphoric acid syrups of hemicellulose sugars were fermented by ethanologenic Escherichia coli LY160 without further purification. Fermentation of these syrups was preceded by a lag that increased with increased pre-treatment temperature. Further improvements in organisms and optimization of steam treatments may allow the co-fermentation of sugars derived from hemicellulose and cellulose, eliminating need for liquid-solid separation, sugar purification, and separate fermentations.

Topics: Biotechnology; Carbohydrates; Cellulase; Cellulose; Ethanol; Fungi; Hydrolysis; Lignin; Phosphoric Acids; Saccharum; Sulfuric Acids; Temperature; Time Factors; Xylose

The enzymatic hydrolysis of cellulose encounters various limitations that are both substrate- and enzyme-related. Although the crystallinity of pure cellulosic Avicel plays a major role in determining the rate of hydrolysis by cellulases from Trichoderma reesei, we show that it stays constant during enzymatic conversion. The mode of action of cellulases was investigated by studying their kinetics on cellulose samples. A convenient method for reaching intermediate degrees of crystallinity with Avicel was therefore developed and the initial rate of the cellulase-catalyzed hydrolysis of cellulose was demonstrated to be linearly proportional to the crystallinity index of Avicel. Despite correlation with the adsorption capacity of cellulases onto cellulose, at a given enzyme loading, the initial enzymatic rate continued to increase with a decreasing crystallinity index, even though the bound enzyme concentration stayed constant. This finding supports the determinant role of crystallinity rather than adsorption on the enzymatic rate. Thus, the cellulase activity and initial rate data obtained from various samples may provide valuable information about the details of the mechanistic action of cellulase and the hydrolysable/reactive fractions of cellulose chains. X-ray diffraction provides insight into the mode of action of Cel7A from T. reesei. In the conversion of cellulose, the (021) face of the cellulose crystal was shown to be preferentially attacked by Cel7A from T. reesei.

Topics: Adsorption; Cellulase; Cellulose; Crystallography, X-Ray; Hydrolysis; Magnetic Resonance Spectroscopy; Phosphoric Acids; Trichoderma

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.

Topics: Aspergillus; Bioelectric Energy Sources; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Ethanol; Kinetics; Phosphoric Acids; Recombinant Proteins; Saccharomyces cerevisiae; Trichoderma

Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.

Topics: Bioreactors; Carbohydrate Metabolism; Cellulase; Cellulose; Hydrogen-Ion Concentration; Models, Biological; Phosphoric Acids; Polysaccharides; Rheology; Saccharum; Solubility; Viscosity

Corbicula japonica is a typical brackish water bivalve species belonging to the order Veneroida, and it is the most important inland fishery resource in Japan. Corbicula japonica has been suggested to assimilate organic matter from terrestrial plants, unlike Ruditapes philippinarum and Mactra veneriformis, which selectively assimilate organic matter of marine origin. This led us to hypothesize that C. japonica, despite being a suspension feeder, could assimilate cellulosic materials derived from terrestrial plants. In the present study, we measured cellulase and hemicellulase activities in the crystalline styles of C. japonica and other commercially important Veneroida bivalve species in Japan: Ruditapes philippinarum, Meretrix lamarckii and Meretrix lusoria. Corbicula japonica demonstrated notably higher cellulase, xylanase and beta-mannanase activities than the other marine bivalves, suggesting that this species possesses a far greater biochemical capacity to break down the structural polysaccharides of plant cell walls than the other species. In contrast, the beta-1,3-glucanase and pectinase activities of C. japonica were similar to or even lower than those of the others. This is possibly due to the presence of these polysaccharides in the cell walls of diatoms, a principal food of most marine bivalves. Although direct evidence is lacking, the high cellulase, xylanase and beta-mannanase activities of C. japonica may result from adaptation to an upstream estuarine environment where phytoplankton and diatoms are scarce, but plant-derived substances are abundant.

Topics: Animals; beta-Mannosidase; Bivalvia; Cellulase; Cellulose; Corbicula; Endo-1,4-beta Xylanases; Glucan 1,3-beta-Glucosidase; Glycoside Hydrolases; Phosphoric Acids; Polygalacturonase; Water

Efficient liberation of fermentable soluble sugars from lignocellulosic biomass waste not only decreases solid waste handling but also produces value-added biofuels and biobased products. Industrial hemp, a special economic crop, is cultivated for its high-quality fibers and high-value seed oil, but its hollow stalk cords (hurds) are a cellulosic waste. The cellulose-solvent-based lignocellulose fractionation (CSLF) technology has been developed to separate lignocellulose components under modest reaction conditions (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng. 2007, 97 (2), 214- 223). Three pretreatment conditions (acid concentration, reaction temperature, and reaction time) were investigated to treat industrial hemp hurds for a maximal sugar release: a combinatorial result of a maximal retention of solid cellulose and a maximal enzymatic cellulose hydrolysis. At the best treatment condition (84.0% H3PO4 at 50 degrees C for 60 min), the glucan digestibility was 96% at hour 24 at a cellulase loading of 15 filter paper units of cellulase per gram of glucan. The scanning electron microscopic images were presented for the CSLF-pretreated biomass for the first time, suggesting that CSLF can completely destruct the plant cell-wall structure, in a good agreement with the highest enzymatic cellulose digestibility and fastest hydrolysis rate. It was found that phosphoric acid only above a critical concentration (83%) with a sufficient reaction time can efficiently disrupt recalcitrant lignocellulose structures.

Topics: Cannabis; Carbohydrate Metabolism; Carbohydrates; Cellulase; Cellulose; Chemical Fractionation; Hydrogen-Ion Concentration; Hydrolysis; Lignin; Phosphoric Acids; Solvents; Temperature; Time Factors

Topics: beta-Glucosidase; Cellulase; Cellulose; Hydrolysis; Macromolecular Substances; Particle Size; Phosphoric Acids; Solubility; Surface Properties; Time Factors

Endocellulase E2 from the thermophilic bacterium Thermomonospora fusca is a member of glycosyl-hydrolase family 6 and is active from pH 4 to 10. Enzymes in this family hydrolyze beta-1,4-glycosidic bonds with inversion of the stereochemistry at the anomeric carbon. The X-ray crystal structures of two family 6 enzymes have been determined, and four conserved aspartic acid residues are found in or near the active sites of both. These residues have been mutated in another family 6 enzyme, Cellulomonas fimi CenA, and evidence was found for both a catalytic acid and a catalytic base. The corresponding residues in E2 (D79, D117, D156, and D265) were mutated, and the mutant genes were expressed in Streptomyces lividans. The mutant enzymes were purified and assayed for activity on three cellulosic substrates and 2, 4-dinitrophenyl-beta-D-cellobioside. Activity on phosphoric acid-swollen cellulose was measured as a function of pH for selected mutant enzymes. Binding affinities for each mutant enzyme were measured for two fluorescent ligands and cellotriose, and circular dichroism spectra were recorded. The results show that the roles of D117 and D156 are the same as those for the corresponding residues in CenA; D117 is the catalytic acid, and D156 raises the pK(a) of D117. No specific function was assigned to the CenA residue corresponding to D79, but in E2, this residue also assists in raising the pK(a) of D117 and is important for catalytic activity. The D265N mutant retained 7% of the wild-type activity, indicating that this residue is not playing the role of the catalytic base. Experiments were conducted to rule out contamination of the D265 enzymes by either wild-type E2 or an endogenous S. lividans CMCase.

Topics: Actinomycetales; Binding Sites; Carboxymethylcellulose Sodium; Cellobiose; Cellulase; Circular Dichroism; Deuterium Oxide; Filtration; Glucosides; Hydrogen-Ion Concentration; Hymecromone; Mutagenesis, Site-Directed; Paper; Phosphoric Acids; Solvents; Trisaccharides

In cultures of Bacteroides succinogenes, in which cellulose was the source of carbohydrate, from 70 to 80% of the carboxymethylcellulase (CMCase) activity was present in the culture fluid. The crude extracellular enzyme readily hydrolyzed acid-swollen cellulose with the production of glucose and cellobiose. Of this extracellular CMCase, 50-62% was associated with sedimentable membrane fragments, 9-13% with nonsedimentable material with a molecular weight greater than 4 X 10(6), and 28-38% with molecules having a molecular weight of approximately 45 000. Polyacrylamide gel electrophoresis (PAGE), in the presence of sodium dodecyl sulfate, revealed that both the nonsedimentable and the sedimentable fraction had complex protein compositions. The nonsedimentable and sedimentable CMCase fractions, after treatment with Triton X-100, were subjected to PAGE in the presence of 0.2% (w/v) Triton X-100. The results indicated the presence of fast- and slow-migrating CMCases in the former, and of a slow-migrating CMCase in the latter. An apparently uncharged CMCase, which probably corresponded to the slow-migrating component by PAGE, was partially purified from the concentrated culture supernate by solubilization in Triton X-100 and chromatography on DEAE--Sepharose, CM--Sepharose, and Phenyl--Sepharose. The partially purified CMCase had a pH optimum of 5.6-6.6 and a temperature optimum of 50 degrees C.

Topics: Bacteroides; Cellulase; Cellulose; Detergents; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Membranes; Phosphoric Acids; Solubility; Temperature

The release of dye from phosphoric acid-swollen cellulose azure by endocellulases and exocellulases is the basis of a technique for locating these enzymes on gels.

Topics: Cellulase; Cellulose 1,4-beta-Cellobiosidase; Electrophoresis, Polyacrylamide Gel; Glycoside Hydrolases; Isoenzymes; Penicillium; Phosphoric Acids