The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.     

Influence of lignin and its degradation products on enzymatic hydrolysis of xylan

The influence of lignin, lignin model compounds, and black liquor from the kraft pulping process on the hydrolysis of xylan by xylanase was investigated. Addition of vanillic acid, acetovanillone, and protocatechuic acid increased the rate of hydrolysis of xylan by as much as 18–50% at low concentrations, but reached maxima at about 0.05% concentration. Addition of vanillin caused a 15% improvement in xylan hydrolysis, while addition of guaiacol more than doubled the hydrolysis rate. Increasing concentrations of either lignin or black liquor also increased the hydrolysis rate of xylan. Circular dichroism spectroscopy indicated a change in the structure of xylanase in the presence of black liquor.     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Structural modification of lignocellulosics by pretreatments to enhance enzymatic hydrolysis

In this work an evaluation was made of a wide variety of single and multiple pretreatment methods for enhancing the rate of enzymatic hydrolysis of wheat straw. A multiple pretreatment consisted of a physical pretreatment followed by a chemical pretreatment. The structural features of wheat straw, including the specific surface area, crystallinity index, and lignin content, were measured to understand the mechanism of the enhancement in the hydrolysis rate upon pretrement. It has been found that, in general, multiple pretreatments were not promising, since the hydrolysis rates rarely exceeded those achieved by single pretreatments. Ballmilling pretreatment was found to be effective in increasing the specific surface area and decreasing the crystallinity index. Treatment with ethylene glycol was highly effective in increasing the specific surface area, in addition to a high degree of delignification. Peracetic acid pretreatment was highly effective in delignifying substrate. Among multiple pretreatments, those involving peracetic acid treatment generally had lower crystallinity indices and lignin content values. The relationship between the hydrolysis rate and the set of structural features indicated that an increase in surface area and a decrease in the crystallinity and lignin content enhance the hydrolysis; the specific surface area is the most influential of the structural features, followed by the lignin content.     

Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover

Ethanol can be produced from lignocellulosic biomass using steam pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields, from both hemicellulose and cellulose are critical parameters for an economically-feasible ethanol production process. This study shows that a near-theoretical glucose yield (96-104%) from acid-catalysed steam pretreated corn stover can be obtained if xylanases are used to supplement cellulases during hydrolysis. Xylanases hydrolyse residual hemicellulose, thereby improving the access of enzymes to cellulose. Under these conditions, xylose yields reached 70-74%. When pre-treatment severity was reduced by using autocatalysis instead of acid-catalysed steam pretreatment, xylose yields were increased to 80-86%. Partial delignification of pretreated material was also evaluated as a way to increase the overall sugar yield. The overall glucose yield increased slightly due to delignification but the overall xylose yield decreased due to hemicellulose loss in the delignification step. The data also demonstrate that steam pretreatment is a robust process: corn stover from Europe and North America showed only minor differences in behaviour.     

Comparison of lignin derivatives as substrates for laccase-catalyzed scavenging of oxygen in coatings and films

Background

Lignin derivatives are phenylpropanoid biopolymers derived from pulping and biorefinery processes. The possibility to utilize lignin derivatives from different types of processes in advanced enzyme-catalyzed oxygen-scavenging systems intended for active packaging was explored. Laccase-catalyzed oxidation of alkali lignin (LA), hydrolytic lignin (LH), organosolv lignin (LO), and lignosulfonates (LS) was compared using oxygen-scavenging coatings and films in liquid and gas phase systems.

Results

When coatings containing lignin derivatives and laccase were immersed in a buffered aqueous solution, the oxygen-scavenging capability increased in the order LO?<?LH?<?LA?<?LS. Experiments with coatings containing laccase and LO, LH or LA incubated in oxygen-containing gas in air-tight chambers and at a relative humidity (RH) of 100% showed that paperboard coated with LO and laccase reduced the oxygen content from 1.0% to 0.4% during a four-day period, which was far better than the results obtained with LA or LH. LO-containing coatings incubated at 92% RH also displayed activity, with a decrease in oxygen from 1.0% to 0.7% during a four-day period. The oxygen scavenging was not related to the content of free phenolic hydroxyl groups, which increased in the order LO?<?LS?<?LH?<?LA. LO and LS were selected for further studies and films containing starch, clay, glycerol, laccase and LO or LS were characterized using gel permeation chromatograpy, dynamic mechanical analysis, and wet stability.

Conclusions

The investigation shows that different lignin derivatives exhibit widely different properties as a part of active coatings and films. Results indicate that LS and LO were most suitable for the application studied and differences between them were attributed to a higher degree of laccase-catalyzed cross-linking of LS than of LO. Inclusion in active-packaging systems offers a new way to utilize some types of lignin derivatives from biorefining processes.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

An approach to cellulase recovery from enzymatic hydrolysis of pretreated sugarcane bagasse with high lignin content

The possibility of recovering the cellulases used for enzymatic hydrolysis of sugarcane bagasse was evaluated. A strategy was adopted to maximize the enzyme recovery: desorption of the enzymes adsorbed in the solid residue after hydrolysis, and re-adsorption of the enzymes from the liquid medium onto a fresh substrate. The use of surfactant during the enzymatic hydrolysis was important to improve the glucose release from the material structure and also to facilitate the enzyme desorption from the solid residue after hydrolysis. The temperature and pH used during desorption influenced the enzymes recovery, with the best results (90% adsorbed cellulase) being achieved at 45?°C and pH 5.5. The enzymes present in the liquid medium after enzymatic hydrolysis were partially recovered (77%) by adsorption onto the fresh substrate and used in new enzymatic hydrolysis batches. It was concluded that it is possible to recycle cellulases from an enzymatic medium for use in subsequent hydrolysis processes.     

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.     

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.     

An alkali-stable enzyme with laccase activity from entophytic fungus and the enzymatic modification of alkali lignin

Mycelia Sterilia YY-5, an entophytic fungus, was isolated from Rhus chinensis Mill and its extracellular enzyme had a higher laccase activity (MS-Lac). After been purified by anion exchange and gel filtration chromatography, MS-Lac, which had a molecular mass of 45 kDa, was found to be an alkali-stable enzyme with an optimum pH of 10.0 and capable of retaining 80% activity after incubation for 72 h with syringaldazine as substrate. It was also found that syringaldazine had a higher affinity than 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as substrate for MS-Lac, which was determined in sodium phosphate buffer (pH 6.0, 0.1 M) at 30 °C. Meanwhile, the lignin modification, catalyzed by MS-Lac, indicated that it could oxidize the phenolic hydroxyl, side chain substituent or carbonyl group of spruce alkali lignin in cetyltrimethylammonium bromide (CTAB) reversed micelles (20 mM, pH 6.0, W/O = 40) and steam-exploded wheat straw alkali lignin in NaOH solution (20 mM, pH 10.0).     

Membrane reactor for enzymatic hydrolysis of cellobiose

A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor, Cellobiose hydrolysis by cellobiase was carried out and theoretically analyzed in terms of steady-state conversion and flow rate through the membrane. When the flow rate exceeds a critical value, a significant fraction of the enzyme inside the reactor is localized in the concentration polarization layer where shear from stirring is high. Consequently, enzyme deactivation inside the concentration polarization layer is accelerated and the conversion decreases due to an exchange of active enzyme in bulk with deactivated enzyme in the polarization layer via convection and back diffusion. Successful operation can be obtained at flow rates lower than the critical point to avoid the polarization and thus the deactivation. It is shown that 6.5 L of 2 mg/mL of cellobiose solution is hydrolyzed to glucose with a conversion of 91% in 20 h with 1.617 mg of cellobiase enzyme, in a reactor attached with a PM 10 membrane of an effective surface area of 39.2 cm².     

Ionic liquid‐mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis

Lignocellulose represents a key sustainable source of biomass for transformation into biofuels and bio‐based products. Unfortunately, lignocellulosic biomass is highly recalcitrant to biotransformation, both microbial and enzymatic, which limits its use and prevents economically viable conversion into value‐added products. As a result, effective pretreatment strategies are necessary, which invariably involves high energy processing or results in the degradation of key components of lignocellulose. In this work, the ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([Emim][CH₃COO]), was used as a pretreatment solvent to extract lignin from wood flour. The cellulose in the pretreated wood flour becomes far less crystalline without undergoing solubilization. When 40% of the lignin was removed, the cellulose crystallinity index dropped below 45, resulting in >90% of the cellulose in wood flour to be hydrolyzed by Trichoderma viride cellulase. [Emim] [CH₃COO] was easily reused, thereby resulting in a highly concentrated solution of chemically unmodified lignin, which may serve as a valuable source of a polyaromatic material as a value‐added product. Biotechnol. Bioeng. 2009;102: 1368–1376. © 2008 Wiley Periodicals, Inc.