Tarja Tamminen

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 effect of isolation method on the chemical structure of residual lignin

Two methods are used for the isolation of residual lignin: acidolytic and enzymatic hydrolysis. Recently a two-step procedure that is a combination of enzymatic and acidic hydrolyses was proposed. In this paper, the structures of residual lignins isolated by these three methods are compared. Enzymatic hydrolysis gave lignin with the highest yield (83%); however, it contained high amounts of carbohydrates and protein. The molar mass of enzymatic lignin was the highest, indicating that no cleavage of lignin occurred. Acidolysis gave a significantly lower lignin yield (40%), but this lignin was practically free from impurities. The β-aryl ether and lignin-carbohydrate linkages cleaved during the isolation, which was manifested in the decreased molar mass of the lignin as well as in increased phenolic hydroxyl group content. The new two-step isolation procedure gave properties between the preparations of enzymatic and acidolytic hydrolyses. The lignin yield was high (78%), but it contained some impurities, although less than the enzymatic lignin. The lignin-carbohydrate linkages cleaved to some extent, but the β-aryl ether linkages remained intact.

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.

Cellulose crystallinity and ordering of hemicelluloses in pine and birch pulps as revealed by solid-state NMR spectroscopic methods

Solid-state 13C NMR spectroscopy was used to determine the degree of cellulose crystallinity (CrI) in kraft, flow-through kraft and polysulphide–anthraquinone (PS–AQ) pulps of pine and birch containing various amounts of hemicelluloses. The applicability of acid hydrolysis and the purely spectroscopic proton spin-relaxation based spectral edition (PSRE) method to remove the interfering hemicellulose signals prior to the determination of CrI were also compared. For softwood pulps, the spectroscopic removal of hemicelluloses by PSRE was found to be more efficient than the removal of hemicelluloses by acid hydrolysis. In addition to that, the PSRE method also provides information on the associations between cellulose and hemicelluloses. On the basis of the incomplete removal of xylan from the cellulose subspectra by PSRE, the deposition of xylan on cellulose fibrils and therefore an ordered ultrastructure of xylan in birch pulps was suggested. The ordered structure of xylan in birch pulps was also supported by the observed change of xylan conformation after regeneration. Similarly, glucomannan in pine pulps may have an ordered structure. According to the 13C CPMAS measurements conducted after acid hydrolysis, the degree of cellulose crystallinity was found to be slightly lower in birch pulps than in the pine pulps. Any significant differences in cellulose crystallinity were not found between the pulps obtained by the various pulping methods. Only in pine PS–AQ pulp, the degree of cellulose crystallinity may be slightly lower than in the kraft pulps containing less hemicelluloses.

Accurate and reproducible determination of lignin molar mass by acetobromination.

The accurate and reproducible determination of lignin molar mass by using size exclusion chromatography (SEC) is challenging. The lignin association effects, known to dominate underivatized lignins, have been thoroughly addressed by reaction with acetyl bromide in an excess of glacial acetic acid. The combination of a concerted acetylation with the introduction of bromine within the lignin alkyl side chains is thought to be responsible for the observed excellent solubilization characteristics acetobromination imparts to a variety of lignin samples. The proposed methodology was compared and contrasted to traditional lignin derivatization methods. In addition, side reactions that could possibly be induced under the acetobromination conditions were explored with native softwood (milled wood lignin, MWL) and technical (kraft) lignin. These efforts lend support toward the use of room temperature acetobromination being a facile, effective, and universal lignin derivatization medium proposed to be employed prior to SEC measurements.

Study of Residual Lignin in Pulp by UV Resonance Raman Spectroscopy

Summary A major problem in the development of new bleaching technologies for pulp is the difficulty of analysing the changes induced by bleaching chemicals in the structures of residual lignin. Separation and isolation of the residual lignin before the analysis may modify the constituents of interest, whereas if the pulp is analysed directly, the responses from cellulose and hemicelluloses will overlap the signal from the residual lignin. In this study, UV resonance Raman spectroscopy, a powerful technique for detecting trace components in complex mixtures, was applied to detect the changes in the residual lignin content after bleaching stages. The resonance Raman technique was found to be highly sensitive and selective for lignin structures allowing the detection of trace amounts of lignin after the final bleaching stages. Furthermore, it enabled rapid and easy determination of hexenuronic acid content. UV resonance Raman spectroscopy would appear to be a technique of great potential for pulping and bleaching research.

Carbohydrate structures in residual lignin-carbohydrate complexes of spruce and pine pulp

Abstract Residual lignin carbohydrate complexes (RLCC) were isolated enzymatically from spruce and pine pulp. The RLCCs contained 4.9–9.4% carbohydrates, with an enrichment of galactose and arabinose compared to the original pulp samples. The main carbohydrate units present in all studied RLCCs were 4-substituted xylose, 4-, 3- and 3,6-substituted galactose, 4-substituted glucose and 4 and 4,6-substituted mannose. These units were assigned to carbohydrate residues of xylan, 1,4- and 1,3/6-linked galactan, cellulose and glucomannan. RLCCs of surface material and the inner part of spruce kraft pulp fiber were compared to obtain information on the heterogeneity of layers of the fiber wall. The 1,4-linked galactan was the major galactan in RLCC of fiber surface material of spruce kraft pulp. Towards the inner part of the fiber, the proportion of 1,3/6-linked galactan increased relative to 1,4-linked galactan. This finding is presented for the first time. 1,3/6-Linked galactan structures are suggested to have a role in restricting lignin removal from the secondary fiber wall. RLCCs of three different alkaline pine pulps were studied before and after oxygen delignification to evaluate differences resulting from the cooking method. The pulps were conventional kraft pine pulp (PCK), a polysulfide/anthraquinone pine pulp (PPSAQ) and a soda/anthraquinone pine pulp (PSoAQ); all were cooked to approximately kappa number 30. Small differences were found in the carbohydrate structures of the unbleached pulps. The study indicated that the RLCC of unbleached PSoAQ pulp contained longer oligomeric carbohydrate chains and less branched 1,3/6-linked galactan residues than the RLCCs of unbleached PCK and PPSAQ pulps. The RLCC of the unbleached PSoAQ also contained more 1,4-linked glucose units suggesting a greater number of linkages of lignin to cellulose in the PSoAQ pulp than in the other two pulps. All RLCCs of oxygen-delignified pulps had more non-reducing ends and less 1,3/6-linked galactan than the corresponding RLCCs of the unbleached pulps. The RLCC of the oxygen-delignified PSoAQ pulp had a higher ratio of 1,4-galactan to 1,3/6-linked galactan and shorter xylan residues than the RLCCs of oxygen-delignified PCK and PPSAQ pulps.

Modification of spruce lignans with Trametes hirsuta laccase

Summary The effect of Trametes hirsuta laccase on isolated spruce wood lignans was evaluated. Lignans were isolated from the heartwood of spruce branches and treated with different laccase dosages and treatment times. The effect of the treatment was monitored by gas chromatography, size exclusion chromatography and ionization difference UV spectroscopy. Lignans were efficiently oxidized by T. hirsuta laccase. About half of the phenolic groups present in lignans remained intact during the treatment. The oxidation of phenolic groups in lignans produced oligomeric structures containing approximately 4–5 lignan units (i.e., 8–10 phenyl propane units). Precipitation of the formed oligomeric structures probably prevented further polymerization.

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.