Kaisa Marjamaa

Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin-rich surface

Lignin‐derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin‐rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin‐bound enzymes. Severe inactivation of the lignin‐bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS–PAGE analysis of the lignin‐bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat‐induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature. Biotechnol. Bioeng. 2011;108: 2823–2834.

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.

The role of xylem class III peroxidases in lignification

Lignification is a cell wall fortifying process which occurs in xylem tissue in a scheduled manner during tissue differentiation. In this review, enzymes and the genes responsible for lignin biosynthesis have been studied with an emphasis on lignin polymerizing class III secretable plant peroxidases. Our aim is to understand the cell and molecular biology of the polymerization of lignin especially in tracheids and vessels of woody species but much of the experimental evidence comes from herbaceous plants. Class III peroxidases pose many problems for empirical work as their encoding genes are variable, their substrate specificities are wide and the half-life of many of the isozymes is very long. However, there is some evidence for the role of specific peroxidases in lignin polymerization through antisense mutants in tobacco and poplar and from tissue and cell culture lines of Picea abies and Zinnia elegans. Peroxidase enzyme action has been shown by substrate specificity studies and, for example, RT-PCR results have pointed out that many peroxidases have tissue-specific expression patterns. Tissue-level location of gene expression of some peroxidases has been studied by in situ hybridization and their cellular localization with antibodies and using EGFP-fusion genes. From these, it can be concluded that, although many of the xylem class III peroxidases have the potential for functioning in the synthesis of the lignin polymer, the combined information of catalytic properties, expression, and localization can reveal differences in the significance of different peroxidases in the lignification process.

Cell wall lignin is polymerised by class III secretable plant peroxidases in Norway spruce.

Class III secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class III plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.

Cellulase-lignin interactions-the role of carbohydrate-binding module and pH in non-productive binding.

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme-lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase-lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM-cellulose interaction were also shown to contribute to lignin-binding.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

Preferential Adsorption and Activity of Monocomponent Cellulases on Lignocellulose Thin Films with Varying Lignin Content

Understanding the enzymatic hydrolysis of cellulose and the influence of lignin in the process are critical for viable production of fuels and chemicals from lignocellulosic biomass. The interactions of monocomponent cellulases with cellulose and lignin substrates were investigated by using thin films supported on quartz crystal microgravimetry (QCM) resonators. Trichoderma reesei exoglucanase (CBH-I) and endoglucanase (EG-I) bound strongly to both cellulose and lignin but EG-I exhibited a distinctive higher affinity with lignin, causing a more extensive inhibition of the cellulolytic reactions. CBH-I was found to penetrate into the bulk of the cellulose substrate increasing the extent of hydrolysis and film deconstruction. In the absence of a cellulose binding domain (CBD) and a linker, the CBH-I core adsorbed slowly and was not able to penetrate into the film. Conversely to CBH-I, EG-I exhibited activity only on the surface of the lignocellulose substrate even when containing a CBD and a linker. Interestingly, EG-I displayed a clearly different interaction profile as a function of contact time registered by QCM.

Impact of hydrothermal pre-treatment to chemical composition, enzymatic digestibility and spatial distribution of cell wall polymers

The effect of hydrothermal pretreatment on chemical composition, microscopic structure and enzymatic digestibility of wheat straw was studied. Wheat straw was pretreated with increasing severity to obtain series of samples with altered chemistry and structure. The hydrothermal pretreatment caused solubilisation of arabinoxylan and phenolic acids and their dimers in a temperature dependent manner with minor effects on the cellulose and Klason lignin content. In the cell wall level, the pretreatment intensified staining of cellulose and relocalised xylan in the cell walls. The distribution, properties and content of the cell wall phenolic compounds was altered as observed with phloroglucinol and autofluorescence imaging. In the enzymatic hydrolysis, the highest yields were obtained from the samples with a low xylan and diferulate content. On the cell wall structural level, the sample with the highest digestibility was observed to have intensified cellulose staining, possibly reflecting the increased accessibility of cellulose.

Novel Penicillium cellulases for total hydrolysis of lignocellulosics.

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35°C while at 45°C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.

Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a β-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B>TrCel6A>TrCel5A>AnCel3A>TrCel7A>TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.