Inhibitory Effect Of Lignin Research Articles

Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases

BackgroundEnzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared.ResultsLignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22–39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified.ConclusionsHigh thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions.Graphical

Structure characterization and inhibitory effect of lignin from the outer and inner layers of bamboo after alkali pretreatment

In this work, structural changes and inhibitory effects of lignin from the outer and inner layers of bamboo after alkali pretreatment were investigated. The results revealed that the carbohydrates contents in lignin samples from bamboo outer and inner layers decreased from 22.2% and 21.4 to 10.1% and 10.4% by alkali pretreatment, respectively. Alkali pretreatment could increase the molecular polydispersity index and syringyl/guaiacyl ratio of lignin, decrease lignin molecular weights and cleave the esterified p-coumarate in bamboo lignin. Inhibitory effect of bamboo lignin on cellulose hydrolysis decreased after alkali pretreatment, which could be explained by the lower adsorption capacity of lignin toward cellulases after alkali pretreatment. These findings discovered the structure of alkali pretreated bamboo lignin and enhanced the understanding of the mechanism of alkali pretreatment in increasing enzymatic hydrolysis of bamboo.

Effect of hydrothermal pretreatment severity on lignin inhibition in enzymatic hydrolysis

Hydrothermal pretreatment is commonly used for enhancing enzymatic hydrolysis of lignocellulosics. Spruce and wheat straw were pretreated with increasing severity and lignin characteristics were analysed. The effect of enzymatically isolated lignin on the hydrolysis of Avicel and the adsorption of a cellobiohydrolase onto lignin was measured. Non-pretreated lignins had only a minor effect on Avicel hydrolysis. The structural changes in lignin accompanying hydrothermal pretreatment were associated with increased binding and inactivation of the cellulase on the lignin surface. The inhibitory effect was more pronounced in spruce than in wheat straw lignin. However, similar pretreatment severities caused similar levels of inhibition in Avicel hydrolysis for both biomass sources. The combined severity factor of the pretreatment correlated well with the inhibitory effect of lignin.

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

BackgroundThe non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.ResultsEight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.ConclusionsOur results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.

Inhibitory effects of lignin on enzymatic hydrolysis: The role of lignin chemistry and molecular weight

Abstract Lignocellulose is a promising feedstock for biofuel production, while lignin poses a grand challenge on the entire process, especially enzymatic hydrolysis. In this study, different types of lignin inhibited enzymatic hydrolysis by different mechanisms. Organosolv lignin from Loblolly pine adsorbed enzyme nonproductively and reduced the available enzyme for cellulose, therefore decreasing hydrolysis rate and ultimate sugar yield. Kraft pine lignin precipitated on the surface of cellulose, preventing it from contacting with enzyme. The molecular weight influenced the inhibition effect of lignin. Lignin of lower molecular weight could bind enzyme nonproductively and when the molecular weight increased, the steric repulsion caused by lignin deposition on cellulose became more significant. The NMR analysis revealed that lignin structural features, e.g., functional groups, S/G ratio, determined the behaviors of lignin in enzymatic hydrolysis. High content of aliphatic hydroxyl groups, or low content of carboxylic groups led to high surface hydrophobicity, increasing the adsorption between lignin and enzyme. In addition, the substrate reactivity is also an important factor that affects enzymatic hydrolysis. Cellulose with higher crystallinity exhibited slower hydrolysis rate and lower conversion. When the crystallinity index increased from 0.43 to 0.72 and 0.81, the ultimate conversion decreased from 80 to 68% and 57%, respectively.

Assessment of the Lignin-Derived Inhibition of Enzymatic Hydrolysis by Adding Untreated and Ammonia-Fiber-Expansion-Treated Lignin Isolated from Switchgrass

Lignin, which is considered one of the most recalcitrant components, limits the enzymatic hydrolysis of cellulose. When ball-milled lignin from switchgrass ranging from 21.4 to 64.3 mg/mL was added to a cellulose substrate (Avicel), the glucose release decreased from 17.4 to 22.4% compared to the hydrolysis rate of zero lignin addition. However, the cellulose decreasing conversion was reduced to 8.5% with 21.4 mg/mL ammonia fiber expansion (AFEX)-treated lignin addition, as a result of the structural modification by AFEX pretreatment. The inhibitory effect of lignin was stronger on cellulose hydrolysis than on hemicellulose hydrolysis. The results showed that the free lignin released from biomass reduced the impact of the added lignin and that the AFEX pretreatment intensity was the key factor in increasing the enzymatic efficiency. The lignin structural modifications by AFEX pretreatment, such as slight increases in the molecular weight, degree of polymerization, and syringyl/guaiacyl ratio, reduced the ...

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and β-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

Expression of a fungal glucuronoyl esterase in Populus: Effects on wood properties and saccharification efficiency

The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-d-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency.The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood.Diffuse reflectance FT-IR spectroscopy and pyrolysis–GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content.These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition, the inhibitory effects of lignin are efficiently removed during acid pretreatment, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.

Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption

The effect of lignin as an inhibitory biopolymer for the enzymatic hydrolysis of lignocellulosic biomass was studied; specially addressing the role of lignin in non-productive enzyme adsorption. Botanical origin and biomass pre-treatment give rise to differences in lignin structure and the effect of these differences on enzyme binding and inhibition were elucidated. Lignin was isolated from steam explosion (SE) pre-treated and non-treated spruce and wheat straw and used for the preparation of ultrathin films for enzyme binding studies. Binding of Trichoderma reesei Cel7A (CBHI) and the corresponding Cel7A-core, lacking the linker and the cellulose-binding domain, to the lignin films was monitored using a quartz crystal microbalance (QCM). SE pre-treatment altered the lignin structure, leading to increased enzyme adsorption. Thus, the positive effect of SE pre-treatment, opening the cell wall matrix to make polysaccharides more accessible, may be compromised by the structural changes of lignin that increase non-productive enzyme adsorption.

Fracionamento dos carboidratos pelas equações do Cornell Net Carbohydrate and Protein System de três cultivares de girassol na presença ou não de irrigação

Objetivou-se quantificar as frações de carboidratos pelas equações do Cornell Net Carbohydrate and Protein System (CNCPS) de três cultivares de girassol (Helianthus annuus L.) cultivados na presença ou não de irrigação. A utilização de uma preparação fibrosa, denominada parede celular (PC), nas equações da CNCPS, em substituição à fibra em detergente neutro (FDN) não promoveu diferenças nas frações de carboidratos B1 e C, mas influenciou as frações A e B2. Como os valores da fração B1, obtidos pelo modelo CNCPS foram menores que os teores de amido e pectina determinados em laboratório, supõe-se que a pectina e outros oligossacarídeos da parede celular, solubilizados pela solução de detergente neutro (fibra solúvel), nunca fizeram parte da fração B1, e sim da fração A. Apesar de os carboidratos da fibra solúvel apresentarem elevadas taxas de degradação, não parece adequada a caracterização da fibra solúvel na fração A. Parece mais adequado que a fibra solúvel (que inclui a pectina) seja alocada a uma fração exclusivamente sua, que pode ser a fração B2, e que seja criada uma nova fração, a B3, para os carboidratos digeríveis da parede celular. Assim, a fração B1 seria composta apenas de amido. A equação da fração C, que estima os carboidratos indigeríveis da parede celular, pode ser simplificada, relacionando a fração indigerível ao teor de lignina na matéria seca, e não à FDN isenta de cinzas e proteína, como atualmente utilizado. Esta proposta tem implicações práticas, uma vez que a fração indigerível da parede celular tem sido expressa em relação à FDN, e não na MS, com base no fato de que os efeitos inibitórios da lignina ocorrem sobre os componentes fibrosos da parede celular vegetal, e não sobre o conteúdo celular.

Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose

There is a need to develop the enzymatic hydrolysis of cellulose for production of ethanol from biomass. In recent years the inhibitory effects of lignin in lignocellulosic substrates has been the focus of several studies. This points to the importance of understanding the interactions between cellulose degrading enzymes and lignin. Surface active substances have been shown to adsorb to lignin surfaces resulting in reduction of unproductive enzyme binding. It is essential to understand the surface properties of both enzymes and lignin to develop pretreatment methods, surface active additives and engineering of cellulose degrading enzyme systems. This study investigates the PEG–lignin interaction as well as interactions between lignin and the enzyme modules of the Hypocrea jecorina ( Trichoderma reesei) enzymes Cel7A and Cel7B. Interactions were monitored with 14C labelled PEG 4000 and by measuring the enzymatic activity in solution. It was found that the dominating driving force of PEG adsorption on lignin is hydrophobic interaction. The effect of PEG addition on enzyme conversion of lignocellulose increased with higher temperature due to increased adsorption of PEG on lignin, thus resulting in a higher surface density of PEG on the surface. The hydrophobic adsorption of enzymes to lignin induces denaturation of enzymes on lignin surfaces. The addition of PEG to the enzyme hydrolysis at a temperature of 50 °C is suggested to hinder deactivation of enzymes by exclusion of enzymes from lignin surfaces. The adsorption of full-length Cel7B to lignin was stronger than for Cel7A. A more hydrophobic surface on the flat face of the cellulose binding module (CBM) together with an additional exposed aromatic residue on the rough face of Cel7B CBM compared to Cel7A CBM gives a higher affinity to lignin for the Cel7B enzyme.

Updates on Softwood-to-Ethanol Process Development

Softwoods are generally considered to be one of the most difficult lignocellulosic feedstocks to hydrolyze to sugars for fermentation, primarily owing to the nature and amount of lignin. If the inhibitory effect of lignin can be significantly reduced, softwoods may become a more useful feedstock for the bioconversion processes. Moreover, strategies developed to reduce problems with softwood lignin may also provide a means to enhance the processing of other lignocellulosic substrates. The Forest Products Biotechnology Group at the University of British Columbia has been developing softwood-to-ethanol processes with SO2-catalyzed steam explosion and ethanol organosolv pretreatments. Lignin from the steam explosion process has relatively low reactivity and, consequently, low product value, compared with the high-value coproduct that can be obtained through organosolv. The technical and economic challenges of both processes are presented, together with suggestions for future process development.

Effect of delignification upon in vitro digestion of forage cellulose.

Orchardgrass forages harvested at two maturities (early and late) were ground through two screens (1 and 8 mm) and digested in vitro as intact forage and forage delignified by permanganate oxidation. Initial and residual cell wall, initial and residual cellulose and potentially digestible cellulose were greater in late intact forage than in the early. In the delignified forage, late cut forage had less residual cellulose than did the early, but initial and potentially digestible cellulose were similar. Particle size had less consistent and smaller effects upon cell wall and cellulose than did maturity. Cellulose of intact orchardgrass was 64% digested at 72 h vs 94% for cellulose of delignified orchardgrass. Digestion rate of cellulose was .0197 and .0220 logn units/hr for early and late cut intact forage and .0554 and .0719 logn units/hr for early and late cut delignified forage. Removal of the inhibitory effects of lignin increased the amount of digestible cellulose, increased the rate at which cellulose degraded and decreased the indigestible cellulose residue. Reduction in lignin could greatly improve forage intake and utilization at moderate levels of animal production.