Anna Suurnäkki

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.

Investigations on the laccase-catalyzed polymerization of lignin model compounds using size-exclusion HPLC

The reaction of laccase from Trametes hirsutawith monomeric and dimeric lignin model compounds was studied with special attention to the formation of polymeric reaction products. Different HPLC techniques were evaluated for the analysis of the reaction products. Methods requiring a solvent change from aqueous to organic phase were found not to be adequate for the analysis of product mixtures since evaporation over nitrogen led to the polymerization of reactive monomers and extraction with dichloromethane was not quantitative in all cases. Therefore, aqueous phase size-exclusion chromatography (SEC) at high pH was used to characterize reaction product mixtures. The product molecular weight distribution (MWD) after reaction with laccase was observed to be highly dependent on the type of model compound whereas the enzyme concentration had only a limited impact.

Activity of laccase on unbleached and bleached thermomechanical pulp

The introduction of value-added properties to pulp fibres is an attractive proposition. One interesting option is targeted modification of fibre surfaces by enzymatic activation. In this work, the activity of laccase on pulp and pulp fractions from thermomechanical pulp (TMP) and peroxide bleached TMP was studied on the basis of consumption of the co-substrate oxygen in the reaction and by studying the formation of radicals in the pulp material as analysed by electron paramagnetic resonance spectroscopy (EPR). Laccases obtained from Trametes hirsuta and Myceliophthora thermophila were used in the study. Laccases were found to be active on pulp material of unbleached TMP, whereas only fines from bleached TMP reacted in laccase treatment. Dissolved and colloidal substances (DCS) were assumed to have a mediating role in the laccase-catalysed oxidation of the fibre-bound material.

Fibre porosity development of dissolving pulp during mechanical and enzymatic processing

Dissolving grade pulps are used as raw material for manufacture of regenerated cellulose fibres and their use is constantly growing. Despite intensive research, there is still a need to develop cellulose dissolution-regeneration processes that would be economically viable, fulfil the pre-conditions of sustainability and would be able to meet the strict product quality requirements. The basis for creation of such a process is in deep understanding of the biomass structure and factors affecting the cellulose modification and dissolution. In this paper, the effects of the mechanical and enzymatic pre-treatments on the pore structure and alkaline solubility of dissolving grade pulp are discussed. Formation of micro- and macropores in the pulp fibres during mechanical shredding was found to correlate with the susceptibility of the fibres to enzymatic hydrolysis. The fibre porosity development during the processing was studied by a modified solute exclusion approach, which revealed differences between the effect of mild enzyme or acid hydrolysis on the pore structure of fibres. The dissolution of the modified fibres in NaOH/ZnO was evaluated and found to correlate with overall pore volume and accessible surface area analysed by the modified solute exclusion method.

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions.

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30°C and 50°C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases.

Partial enzymatic hydrolysis of microcrystalline cellulose in ionic liquids by Trichoderma reesei endoglucanases

The enzymatic hydrolysis of cellulose in ionic liquid (IL) containing systems has recently received a lot of interest as a pretreatment in biomass conversion to liquid biofuels and chemicals. In this paper we present a study in which the activity and action of two Trichoderma reesei endoglucanases, Cel7B and Cel5A, were evaluated in aqueous solutions containing 0–90% (v/v) of the ionic liquids 1,3-dimethylimidazolium dimethylphosphate or 1-ethyl-3-methylimidazolium acetate, using microcrystalline cellulose (Avicell) as a model substrate. The degree of hydrolysis was analysed by capillary electrophoresis of the hydrolysates and gel permeation chromatography of the remaining cellulose residues. Both of the employed ionic liquids severely inactivated the T. reesei endoglucanases. Only traces of soluble oligosaccharides were present in hydrolysis mixtures containing 40% (v/v) or more of ionic liquids. The employed ILs were found to have a basic impact on the hydrolysis environment, but it could be concluded that the basicity of the ILs was not the only reason for the cellulase inactivation. The effect of an IL on the cellulose binding module in Cel5A was evaluated by comparing the hydrolysis yields of the intact Cel5A and the Cel5A core lacking the cellulose binding module. In this study the cellulose binding module was found to be the most ionic liquid sensitive part of the enzymes used. Comparative data from the partial hydrolysis of an ionic liquid regenerated cellulose is also reported.

Polymerization of guaiacol and a phenolic β-O-4-substructure by Trametes hirsuta laccase in the presence of ABTS

The reaction of the monomeric lignin model compound guaiacol and the β‐O‐4‐type dimer erol (1–(4‐hydroxy‐3‐methoxyphenyl)–2(2‐methoxyphenoxy)‐propane‐1,3‐diol with laccase from Trametes hirsuta was studied in the presence of the mediator ABTS (2,2′‐azino‐di[3ethylbenzothiazoline‐6‐sulfonic acid]). The product mixtures were analyzed by means of aqueous‐phase size exclusion chromatography (SEC) with 50 mM NaOH as eluent. Interestingly, in the laccase‐catalyzed reaction with both substrates, the mediator not only functioned as an electron carrier but underwent coupling reactions with the substrate to give polymeric coupling products. The molecular weight of these copolymeric products was significantly higher than the molecular weight of products obtained without ABTS. After ultrafiltration, 33% and 21% of the initially applied ABTS could be found in the polymeric product fraction for the substrates guaiacol and erol, respectively, on the basis of nitrogen analysis. When ABTS was added to substrates after full laccase‐catalyzed polymerization, the reaction proceeded toward higher molecular weights.

Enzyme-aided alkaline extraction of oligosaccharides and polymeric xylan from hardwood kraft pulp

In this paper we describe the effect of enzyme treatments on the production of polymeric xylan, oligosaccharides and hemicellulose lean pulp by alkaline extraction of bleached hardwood kraft pulp. Enzyme treatments were carried out before one or in between two subsequent alkaline extractions by purified Trichoderma reesei xylanase and endoglucanase II (Cel 5a) as well as by a commercial monocomponent endoglucanase (FibreCareR). Without enzyme pre-treatment 61% and 7% of the pulp xylan was extracted in high purity in the first and second alkaline stage, respectively. Higher molecular mass xylan was obtained in the second than in the first alkaline extraction. Xylanase treatment before alkaline extraction hydrolyzed up to 12% of xylan to xylooligosaccharides. According to our results, preparation of polymeric xylan, and/or oligosaccharides as well as hemicellulose lean pulp with cellulose content of 93-94%, is possible by enzyme-aided alkaline extraction process.

Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions

Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto the cellulosic substrate were studied in the presence of cellulose‐dissolving ILs. Two different ILs, 1,3‐dimethylimidazolium dimethylphosphate ([DMIM]DMP) and 1‐ethyl‐3‐methylimidazolium acetate ([EMIM]AcO), and two monocomponent cellulases, Trichoderma reesei cellobiohydrolase Cel7A and endoglucanase Cel5A, were used in the study. The role and IL sensitivity of the carbohydrate‐binding module (CBM) were studied by performing hydrolysis and binding experiments with both the intact cellulases, and their respective core domains (CDs). Based on hydrolysis yields and substrate binding experiments for the intact enzymes and their CDs in the presence of ILs, the function of the CBM appeared to be very IL sensitive. Binding data suggested that the CBM was more important for the substrate binding of endoglucanase Cel5A than for the binding of cellobiohydrolase Cel7A. The CD of Cel7A was able to bind well to cellulose even without a CBM, whereas Cel5A CD had very low binding affinity. Hydrolysis also occurred with Cel5A CD even if this protein had very low binding affinity in all the studied matrices. Binding and hydrolysis were less affected by the studied ILs for Cel7A than for Cel5A. To our knowledge, this is the first systematic study of IL effects on cellulase substrate binding. Biotechnol. Bioeng. 2014;111: 726–733.

Cellulose hydrolysis with thermo- and alkali-tolerant cellulases in cellulose-dissolving superbase ionic liquids

Pretreatment with ionic liquids (ILs) is known to greatly increase the subsequent biomass hydrolysis with enzymes. However, the presence of even low amounts of ILs has negative effects on cellulase action. Most studies on cellulase inactivation by ILs have focused on imidazolium-based ILs, which until recently were one of the few IL classes known to dissolve cellulose. In this article we describe results of cellulase action in matrices containing ILs belonging to two IL classes recently reported as cellulose solvents. These ILs are based on the organic superbases 1,1,3,3-tetramethylguanidine (TMG) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN). In this study commercial thermo- and alkaline stabile cellulase products were employed, as these were anticipated to also have a higher stability in ILs. For comparison, hydrolysis experiments were also carried out with a well-characterised endoglucanase (Cel5A) from Trichoderma reesei and in matrices containing 1-ethyl-3-methylimidazolium acetate, [EMIM]AcO. Two different substrates were used, microcrystalline cellulose (MCC) and eucalyptus pre-hydrolysis kraft dissolving grade pulp. The hydrolysis yields were on the same level for both of these substrates, but decreases in molecular weight of the cellulose was observed only for the dissolving grade pulp. By using commercial cellulases with good thermo- and alkali-stability some benefits were obtained in terms of IL compatibility. Enzyme thermostability correlated with higher hydrolysis yields in IL-containing matrices, whereas activity at high pH values did not offer benefits in terms of IL tolerance. The new classes of cellulose-dissolving superbase ILs did not differ in terms of cellulase compatibility from the well-studied imidazolium-based ILs. Of the novel superbase ILs tested, [TMGH]AcO was found to inhibit the enzymatic hydrolysis the least.