Folke Tjerneld

Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose

Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.

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.

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

Isolation and characterization of galactoglucomannan from spruce (Picea abies)

Water-soluble hemicelluloses were extracted from spruce chips by microwave heat fractionation. The chips were impregnated with water at different pH values. Screening of heat-fractionation conditions, i.e. impregnation medium, temperature and residence time was performed with the aim to extract O-acetyl-galactoglucomannan. The impregnation and heat fractionation conditions were evaluated on the basis of the yield of dissolved mannan (oligo- and polysaccharides), molecular weight of the carbohydrates and amount of dissolved lignin. Increasing temperature and residence time increases the yield of mannan and decreases the molecular weight of dissolved carbohydrates. For a structural study of the extracted carbohydrates the chips were impregnated with water and treated at 200oC for 2min. Oligo- and polysaccharides were fractionated with preparative size-exclusion chromatography from the filtered extract.The structure of the obtained saccharides in two fractions 8 and 9 was determined by 1H NMR spectroscopy. The polysaccharides in the fractions were O-acetyl-galactoglucomannan with a degree of polymerization ~20 and ~11 for fractions 8 and 9, respectively. The molar ratio for galactose:glucose:mannose was approximately 0.1:1:4. About one-third of the d-mannosyl units are substituted by O-acetyl groups almost equally distributed between C-2 and C-3. (Less)

The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol

Simultaneous saccharification and fermentation (SSF) of steam-pretreated wood constitutes an attractive process configuration for ethanol production from biomass. However, the high enzyme addition in SSF contributes to a high process cost. In this study we explore the effect of the non-ionic surfactant Tween-20 as an additive in SSF. Tween-20 addition at 2.5 g/l had several positive effects on SSF: (i) the ethanol yield was increased by 8%; (ii) the amount of enzyme loading could be reduced by 50%, while maintaining a constant yield; (iii) the enzyme activity increased in the liquid fraction at the end of SSF, probably by preventing unproductive binding of the cellulases to lignin, which could facilitate enzyme recovery; (iv) the time required to attain maximum ethanol concentration was reduced. Surfactants as an additive in SSF can significantly lower the operational cost of the process. However, less expensive surfactants must be investigated.

Driving forces for phase separation and partitioning in aqueous two-phase systems

A set of simple analytical equations, derived from the Flory-Huggins theory, are used to identify the dominant driving forces for phase separation and solute (e.g., protein) partitioning, in the absence and presence of added electrolyte, in every general class of aqueous two-phase systems. The resulting model appears to capture the basic nature of two-phase systems and all trends observed experimentally. Case studies are used to identify fundamental differences in and the magnitudes of enthalpic and entropic contributions to partitioning in polymer-polymer (e.g., PEG-dextran), polymer-salt, and thermoseparating polymer-water (e.g., UCON-water) two-phase systems. The model therefore provides practitioners with a better understanding of partition systems, and industry with a simple, fundamental tool for selecting an appropriate two-phase system for a particular separation.

Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase

The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.

Partition of proteins in aqueous polymer two-phase systems and the effect of molecular weight of the polymer

The partition of substances in aqueous polymer two-phase systems is influenced by the molecular weight of the phase-forming polymers. We investigate how the effect of the molecular weight of the polymers depends on the molecular weight of the partitioned protein. We show that the magnitude of change of the partition is very small for proteins of molecular weights around 10,000, but increases almost linearly up to molecular weights of 250,000.

Fungal cellulolytic enzyme production: A review

Abstract Cellulolytic enzyme production from fungi has been reviewed. Enzyme concentrations, enzyme productivities and enzyme yield have been compared for soluble substrates, purified cellulose and lignocellulosic substrates in relation to the estimated technical-economical goals of 20 filter paper units (FPU) ml−1 and 200 FPU litre−1 h−1. This can be achieved with Trichoderma reesei mutants in fed-batch cultures. The relative contributions from strain development, nature of substrate, substrate concentration and cultivation conditions on the improvements in enzyme production with Trichoderma species were found to be of the same order of magnitude.