Influence of operation conditions on laccase-mediator removal of sterols from eucalypt pulp.

*Textile and Paper Engineering Department, ETSEIAT, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain. **Instituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080 Seville, Spain ***Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; E-mail address: [email protected] (M.B. Roncero)The way how sterols, the main lipophilic compounds present in eucalypt kraft pulp, are eliminated by an enzymatic stage using the laccase-mediator system was evaluated. With this purpose laccase-mediator stage (L) was applied on an Eucalyptus globulus pulp under different operation conditions following a three-variable (laccase dose, mediator dose and reaction time) sequential statistical plan, to optimise the removal of sterols. The decrease in pulp sterol content during the enzymatic treatment was related to the decrease in kappa number and to brightness increase, as well as with the increase in some oxidation products of sitosterol (namely 7-oxositosterol and stigmasta-3,5-dien-7-one). The increase in reaction time from 1 to 5 h strongly reduced the sterol content, while no more sterols were eliminated during the 5-7 h period. Increasing the laccase dose from 1 to 20 U g-1 of pulp produced a high reduction in pulp sterols, whereas the increase in mediator (1-hydroxybenzotriazole) dose (from 0.5 to 2.5 % of pulp weight) had only a slight influence in removing sterols. Therefore, at 16 U g-1 laccase dose, 0.5% mediator dose, 4 h of reaction, practically all the sterols were removed. Finally, it was demonstrated that sterols were more sensitive to a L stage (practically 100% of sterols were eliminated) than to a chlorine dioxide stage (54% of sterols eliminated).This work was funded by the Spanish MEC (BIOFIBER project, CTQ2007-68003-C02-01; and project BIO2007-28719-E), MICINN (ELLE project, AGL2008-00709) and the European Commission (project BIORENEW, NMP2-CT-2006-026456). Torraspapel S.A. (Zaragoza, Spain) and Novozymes (Bagsvaerd, Denmark) are gratefully acknowledged for supplying the pulp and enzymes used, respectively.Peer reviewe

Differential activity of lytic polysaccharide monooxygenases on celluloses of different crystallinity. Effectiveness in the sustainable production of cellulose nanofibrils

A series of cellulosic substrates has been produced, treated with lytic polysaccharide monooxygenase (LPMO) from Streptomyces ambofaciens (SamLPMO10C), and analyzed by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The activity of the bacterial LPMO showed high variability depending on the origin and degree of crystallinity of the substrate. Additionally, we tested the effectiveness of SamLPMO10C in the nanofibrillation of flax, a high crystalline agricultural fiber, as a single pretreatment or in combination with cellulases. All pretreatments were followed by a mechanical defibrillation by high-pressure homogenization (HPH) to obtain cellulose nanofibrils (NFC). The combined LPMO-cellulase treatment showed higher fibrillation yield, optical transmittance and carboxylate content than control reactions. Therefore, it could be explored as a promising green alternative to reduce the energy consumption in the production of NFC. To our knowledge, this is the first study reporting the effect of a bacterial LPMO in nanocellulose productionPostprint (author's final draft

Bacterial cellulose matrices to develop enzymatically active paper

This work studies the suitability of bacterial cellulose (BC) matrices to prepare enzymatically active nanocomposites, in a framework of more environmentally friendly methodologies. After BC production and purification, two kind of matrices were obtained: BC in aqueous suspension and BC paper. A lipase was immobilised onto the BC matrices by physical adsorption, obtaining Lipase/BC nanocomposites. Neither morphology nor crystallinity, measured by scanning electron microscopy (SEM) and X-Ray diffractometry (XRD) respectively, of the BC were affected by the binding of the protein. The activity of Lipase/BC suspension and Lipase/BC paper was tested under different conditions, and the operational properties of the enzyme were evaluated. A shift towards higher temperatures, a broader pH activity range, and slight differences in the substrate preference were observed in the immobilised lipase, compared with the free enzyme. Specific activity was higher for Lipase/BC suspension (4.2 U/mg) than for Lipase/BC paper (1.7 U/mg) nanocomposites. However, Lipase/BC paper nanocomposites showed improved thermal stability, reusability, and durability. Enzyme immobilised onto BC paper retained 60% of its activity after 48 h at 60 ºC. It maintained 100% of the original activity after being recycled 10 times at pH 7 at 60 ºC and it remained active after being stored for more than a month at room temperature. The results suggested that lipase/BC nanocomposites are promising biomaterials for the development of green biotechnological devices with potential application in industrials bioprocesses of detergents and food industry and biomedicine. Lipase/BC paper nanocomposite might be a key component of bioactive paper for developing simple, handheld, and disposable devices

Development of an antimicrobial bioactive paper made from bacterial cellulose

Bacterial cellulose (BC) has emerged as an attractive adsorptive material for antimicrobial agents due to its fine network structure, its large surface area, and its high porosity. In the present study, BC paper was first produced and then lysozymewas immobilized onto it by physical adsorption, obtaining a composite of lysozyme-BC paper. The morphology and the crystalline structure of the composite were similar to that of BC paper as examined by scanning electron microscopy and X-ray diffraction, respectively. Regarding operational properties, specific activities of immobilized and free lysozymewere similar. Moreover, immobilized enzyme showed a broaderworking temperature and higher thermal stability. The composites maintained its activity for at least 80 dayswithout any special storage. Lysozyme-BC paper displayed antimicrobial activity against Gram-positive and Gram-negative bacteria, inhibiting their growth by 82% and 68%, respectively. Additionally, the presence of lysozyme increased the antioxidant activity of BC paper by 30%. The results indicated that BC is a suitable material to produce bioactive paper as it provides a biocompatible environment without compromising the activity of the immobilized protein. BC paper with antimicrobial and antioxidant properties may have application in the field of active packaging

Bacterial Cellulose-Chitosan paper with antimicrobial and antioxidant activities

The production of paper-based bacterial cellulose-chitosan (BC-Ch) nanocomposites was accomplished following two different approaches. In the first, BC paper sheets were produced and then immersed in an aqueous solution of chitosan (BC-ChI); in the second, BC pulp was impregnated with chitosan prior to the production of paper sheets (BC-ChM). BC-Ch nanocomposites were investigated in terms of physical characteristics, antimicrobial and antioxidant properties, and the ability to inhibit the formation of biofilms on their surface. The two types of BC-Ch nanocomposites maintained the hydrophobic character, the air barrier properties, and the high crystallinity of the BC paper. However, BC-ChI showed a surface with a denser fiber network and with smaller pores than those of BC-ChM. Only 5% of the chitosan leached from the BC-Ch nanocomposites after 96 h of incubation in an aqueous medium, indicating that it was well retained by the BC paper matrix. BC-Ch nanocomposites displayed antimicrobial activity, inhibiting growth of and having a killing effect against bacteria Staphylococcus aureus and Pseudomonas aeruginosa and yeast Candida albicans. Moreover, BC-Ch papers showed activity against the formation of a biofilm on their surface. The incorporation of chitosan increased the antioxidant activity of the BC paper. Paper-based BC-Ch nanocomposites combined the physical properties of BC paper and the antimicrobial, antibiofilm, and antioxidant activities of chitosan

Laccase/TEMPO-mediated bacterial cellulose functionalization: production of paper-silver nanoparticles composite with antimicrobial activity

Bacterial cellulose (BC) was functionalized applying the Laccase/TEMPO oxidative treatment, leading to a five-fold increase of the concentration of carboxyl groups. Paper produced with this cellulose showed improved mechanical properties while maintaining barrier function against water and greases as compared to paper produced with non-oxidized BC. Also, the negative charge provided by the carboxyl groups on functionalized BC was used to generate silver nanoparticles (AgNPs), obtaining a BC paper and Ag composite. The presence of AgNPs in the composites was validated by SEM, EDS and ICP analysis, showing spherical, uniformly sized particles stabilized in the BC nanofibers matrix. Additionally, antimicrobial property of composites containing AgNPs was tested. The results showed the strong antimicrobial activity of the composites against Gram-positive and Gram-negative bacteria and fungi. The generation of Ag nanoparticles in a matrix that combine the physical characteristics of the BC nanofibers with the stiffness and the mechanical properties of paper produced composites that may have applicability in technological and biomedical uses

Bacterial cellulose for increasing barrier properties of paper products

Bacterial cellulose was combined with wood cellulose papers in order to obtain biomaterials with increased barrier properties. For this purpose, different parameters were assessed: two producing bacterial strains (Komagataeibacter xylinus and Gluconacetobacter sucrofermentans), two paper supports to hold bacterial cellulose (filter paper and eucalyptus paper), two kinds of combined biomaterials (composite and bilayer) and two drying temperatures (90ºC and room temperature). Papers with increased barrier properties (100º of water contact angle, 1220s of water drop test and air permeability ˂1µm (Pa·s)-1) were obtained by the addition of bacterial cellulose to each paper support. However, due to the lower initial barrier properties of filter paper, higher improvements were produced with this paper. In addition, bacterial cellulose provided smoother surfaces with higher gloss without a detrimental effect on physical properties. Higher resistance to water absorption was obtained with K. xylinus possibly explained by its longer size fibers than G. sucrofermentans, as analysed by SEM. Smoothness and gloss were specially increased in the bilayer biomaterial although resistance to air and water were further improved in the composite. In this biomaterial drying at high temperature had a detrimental effect. SEM analysis of the products obtained showed the intimate contact among fibers of bacterial cellulose and wood paper. Results obtained show the contribution of bacterial cellulose to improve the properties of paper and its potential for the design of new added value paper products from biomass

Laccase/TEMPO-mediated bacterial cellulose functionalization: production of paper-silver nanoparticles composite with antimicrobial activity

“This is a post-peer-review, pre-copyedit version of an article published in Cellulose. The final authenticated version is available online at: https://doi.org/10.1007/s10570-019-02678-5Bacterial cellulose (BC) was functionalized applying the Laccase/TEMPO oxidative treatment, leading to a five-fold increase of the concentration of carboxyl groups. Paper produced with this cellulose showed improved mechanical properties while maintaining barrier function against water and greases as compared to paper produced with non-oxidized BC. Also, the negative charge provided by the carboxyl groups on functionalized BC was used to generate silver nanoparticles (AgNPs), obtaining a BC paper and Ag composite. The presence of AgNPs in the composites was validated by SEM, EDS and ICP analysis, showing spherical, uniformly sized particles stabilized in the BC nanofibers matrix. Additionally, antimicrobial property of composites containing AgNPs was tested. The results showed the strong antimicrobial activity of the composites against Gram-positive and Gram-negative bacteria and fungi. The generation of Ag nanoparticles in a matrix that combine the physical characteristics of the BC nanofibers with the stiffness and the mechanical properties of paper produced composites that may have applicability in technological and biomedical usesPeer ReviewedPostprint (author's final draft

Bacterial cellulose for increasing barrier properties of paper products

Bacterial cellulose was combined with wood cellulose papers in order to obtain biomaterials with increased barrier properties. For this purpose, different parameters were assessed: two producing bacterial strains (Komagataeibacter xylinus and Gluconacetobacter sucrofermentans), two paper supports to hold bacterial cellulose (filter paper and eucalyptus paper), two kinds of combined biomaterials (composite and bilayer) and two drying temperatures (90 °C and room temperature). Papers with increased barrier properties (100° of water contact angle, 1220 s of water drop test and air permeability < 1 µm (Pa s)-1) were obtained by the addition of bacterial cellulose to each paper support. However, due to the lower initial barrier properties of filter paper, higher improvements were produced with this paper. In addition, bacterial cellulose provided smoother surfaces with higher gloss without a detrimental effect on physical properties. Higher resistance to water absorption was obtained with K. xylinus possibly explained by its longer size fibers than G. sucrofermentans, as analysed by SEM. Smoothness and gloss were specially increased in the bilayer biomaterial although resistance to air and water were further improved in the composite. In this biomaterial drying at high temperature had a detrimental effect. SEM analysis of the products obtained showed the intimate contact among fibers of bacterial cellulose and wood paper. Results obtained show the contribution of bacterial cellulose to improve the properties of paper and its potential for the design of new added value paper products from biomassPostprint (author's final draft

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1–2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.This work was supported by the Ministry of Science,Innovation and Universities (MICIU) and AgenciaEstatal de Investigacion (AEI), Spain (co-funded byFEDER funds/Development Fund–a Way to BuildEurope): RTI2018-099098-B-100 to AS/BH andRTI2018-094052-B-100 to AMV; and the RamonAreces Foundation: 20th National Competition forScientific and Technical Research in Life and MatterScience (2020) to IF. NL and JGS were recipients ofresearch assistant contracts linked to grant SAF2015-69145-R and RTI2018-099098-B-100, respectively. CMR was the recipient of a researchcontract (PEJD-2019-POST/BMD-16090) from the Education, Universities, Research and Spokesperson Counseling of the Community of Madrid