Mechanistic investigation of the effect of endoglucanases related to pulp refining

Endoglucanases are increasingly being touted as the ultimate solution for reducing energy consumption during the refining process in the pulp and paper industry. However, due to the high variety of endoglucanases in different enzyme formulations, these perform heterogeneously when applied to different pulps. In this study, the effect of four endoglucanases on softwood and hardwood pulp was studied using confocal laser scanning microscopy (CLSM) after addition of fluorescently labelled carbohydrate binding modules (CBMs). Nuclear magnetic resonance (NMR) analysis and high-performance liquid chromatography quantification of released oligo- and monosaccharides was performed for in-depth mechanistical investigation. Changes in the crystallinity levels caused by enzymatic degradation of amorphous regions were monitored by incubation with two different CBMs from Caldicellulosiruptor bescii and from Thermobifida fusca with high preference to either amorphous or crystalline regions of cellulose, respectively. When dosed at identical activity on the endoglucanase specific CellG5 substrate, CLSM analysis indicated the highest decrease of amorphous regions for those endoglucanases which were also most active in laboratory refining trials and which released highest amounts of cellooligomers from pulp. Using 13C-NMR analysis, an increase in para-crystalline cellulose caused by enzyme application was observed. Release of reducing sugars was determined at identical CellG5 dosage, indicating a high variance between the enzymes, especially when complex enzyme formulations were used. Scanning electron microscopy images were obtained for visualization of the endoglucanase activity. The results of mechanistical studies indicate that reduction of amorphous moieties of pulp by endoglucanases is especially beneficial for the refining proces

Environmentally friendly covalent coupling of proteins onto oxidized cellulosic materials

Cellulose is a biodegradable and renewable material that is one of the most abundant biopolymers with many different applications from low value newsprint products to high value biomedical sensor devices. In the last years, the demand of functionalized cellulose for the development of new packaging materials was constantly rising. In this study, a new two-step method for surface functionalization of cellulose sheets and fibers involving oxidation by 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) followed by coupling of different proteins was investigated. The cellulose oxidation was monitored via FT-IR at 1610 cm 121, photometrically via toluidine blue staining and via titration for the determination of the \u2013COOH group concentration. TEMPO oxidation increased the amount of \u2013COOH groups from around 0.2 to more than 1.4 mmol g 121 when NaClO2/NaClO regeneration was used. The TEMPO/laccase system instead led to 0.7 mmol g 121 of \u2013COOH groups (determined via HCl titration). The oxidation was monitored over time and showed that 50% of the reaction were completed within the first 60 min of reaction time. Coupling of protein-based hydrophobins or bovine serum albumin using the EDAC/NHS system led to the desired increase in hydrophobicity and detection of protein on cellulose. Coupling was investigated using contact angle measurements and SEM microscopy paired with elemental analysis for oxygen and nitrogen. Hence, the coupled hydrophobins led to a significant increase of the initial contact angle by 33% with water drop stability of over 200 s. In contrary, pure cellulose obtained no visible water drop and for surfaces with uncoupled hydrophobins no stable contact angle, with a soaking time dropping to 55 s was achieved. As a result, the thorough study revealed that the new combinatorial approach of surface functionalization and protein coupling led to the successful increase of hydrophobicity

Cultivation of heterotrophic algae on paper waste material and digestate

This study investigates the cellulases-catalyzed hydrolysis of newspaper waste and paper slurries as carbon source in combination with digestate from anaerobic digestion as nitrogen source for the heterotrophic cultivation of Chlorella sorokiniana. The enzymatic hydrolysis of paper slurries resulted in a hydrolysate containing 27.2 g l 121 glucose reflecting a 78% cellulose conversion. A maximum Chlorella sorokiniana biomass yield of 3.64 g l 121 was achieved within 91 h of cultivation under consumption of 9.7 g l 121 glucose. The most efficient cultivation converted 1 g l 121 glucose into 0.42 g l 121 biomass. A strong decrease in pH of the batch cultures due to ammonium consumption and CO2 production by algae cells inhibited full glucose consumption for high concentrations of 17 g l 121 glucose. Characterizing the amino acid content and pattern of the biomass revealed an amino acid fraction of 32.9% with Alanine as the most frequent amino acid

Biorefining: the role of endoglucanases in refining of cellulose fibers

With an annual production of more than 400 million tons, paper is the main product of the largest biorefinery process industrially implemented. Enzymes have been used for pulp refining to dramatically reduce energy consumption. However, exact mechanisms related to the individual enzymes are hardly understood. Yet, this knowledge would be important to predict enzyme performance in industrial processes. Three commercial refining enzyme formulations showed different endoglucanase (1.25 nkat mg 121\u201313.7 nkat mg 121), \u3b2-glucosidase (0.57 nkat mg 121\u20131.34 nkat mg 121) and xylanase activities (1.78 nkat ml 121\u201362.1 nkat mg 121) on model substrates. Additionally, distinct amounts of reducing sugars from hardwood sulfate pulp were released. Endoglucases were purified from each formulation by using hydrophobic interaction and anion exchange chromatography and showed molecular weights from 20 to 55 kDa and specific activities ranging between 3.11 and 26.3 nkat mg 121 according to endoglucanase specific derivatized cellopentaose (CellG5). Refining trials of hardwood sulfate pulp were conducted using a PFI laboratory mill and fiber properties such as degree of refining or fiber length and properties of formed sheets like tensile index were monitored. Thereby, enzymes were dosed based on identical endoglucanase activity on CellG5. Enzyme formulations and purified endoglucanases led to an increase of the degree of refining of up to 47.9 [\ub0SR] at 6000 PFI revolutions while the tensile index was improved by up to 76.0 Nm g 121. In summary, refining effects can be primarily attributed to endoglucanases indicating activity on CellG5 being a suitable parameter for enzyme dosing

Effects of enzymes on the refining of different pulps

Comparative studies of the effects of two commercial enzyme formulations on fiber refining were conducted. Extensive basic characterisation of the enzymes involved, assessment of their hydrolytic activities on different model substrates as well as on different pulps (softwood sulfate, softwood sulfite, hardwood sulfate) were evaluated. Both enzyme formulations showed endoglucanase as well as some xylanase and \u3b2-glucosidase activity. In addition, Enzyme A reached a CMC end viscosity of 19.5\u2009mPa compared to 11.1\u2009mPa for Enzyme B. Reducing sugar release almost doubled from 695\u2009\u3bcmol\u2009mL 121 for hardwood sulfate pulp to 1300\u2009\u3bcmol\u2009mL 121 for softwood sulfite pulp with Enzyme B under the same conditions. Enzyme A increased the degree of refining even under non-ideal conditions from 23\u2009\ub0SR to up to 50\u2009\ub0SR. Further characterization of hand sheets, made from enzyme pre-treated and refined cellulose fibers with Enzyme A and B, showed that Enzyme A had the best effects leading to hand sheets with increased tensile strength and low air permeability. In summary, the increase in the degree of refining seen for Enzyme A correlated to higher xylanase and \u3b2-glucosidase activity and lower endoglucanase activity

Enzymatic systems for cellulose acetate degradation

Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable

Enzymatic Systems for Cellulose Acetate Degradation

Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable