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  • 1.
    Ahlén, Gustaf
    et al.
    Recopharma AB.
    Strindelius, Lena
    Recopharma AB.
    Johansson, Tomas
    Recopharma AB.
    Nilsson, Anki
    Rrecopharma AB.
    Chatzissavidou, Nathalie
    Recopharma AB.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Holsgersson, Jan
    Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy.
    Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10Article in journal (Refereed)
    Abstract [en]

    Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented.OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays.Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect.Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.

  • 2. Andersson, Christian
    et al.
    Helmerius, Jonas
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Effects of neutralising agent, organic acids, and osmolarity on succinic acid production by Escherichia coli AFP1842008Conference paper (Other academic)
    Abstract [en]

    Using a low-cost medium Escherichia coli AFP184 has previously been reported to produce succinic acid with volumetric productivities close to 3 g L-1 h-1. At a total organic acid concentration of 30 g L-1 the productivity decreased drastically resulting in final succinate concentrations of 40 g L-1. The economical viability of biochemical succinic acid production would benefit from higher final succinic acid concentrations and volumetric productivities maintained at >2.5 g L-1 h-1 for an extended period of time. In the present work the effects of osmolarity and neutralising agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on succinic acid production by AFP184 were investigated. Highest concentration of succinic acid was obtained with Na2CO3, 75 g L-1. It was also found that the osmolarity resulting from succinate production and subsequent base addition, only marginally affected the productivity per viable cell. Organic acid inhibition due to the produced succinic acid on the other hand significantly reduced succinic acid productivity per viable cell. When using NH4OH productivity completely ceased at approximately 40 g L-1. Volumetric productivities remained at 2.5 g L-1 h-1 for 5 to 10 hours longer when using K- or Na-bases than when using NH4OH. However, loss of cell viability occurred, and together with the acid inhibition decreased the volumetric productivities. In this study it was demonstrated that by altering the neutralising agent it was possible to increase the period of high volumetric productivity in the anaerobic phase and improve the final succinic acid concentration by almost 100 %

  • 3. Andersson, Christian
    et al.
    Helmerius, Jonas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Berglund, Kris
    Rova, Ulrika
    Inhibition of succinic acid production in metabolically engineered Escherichia Coli by neutralizing agent, organic acids, and osmolarity2009In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 25, no 1, p. 116-123Article in journal (Refereed)
    Abstract [en]

    The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities >2.5 g L-1 h(-1) is important if production of succinic acid from. renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L-1. was obtained with Na2O3. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of similar to 40 g L-1. Volumetric productivities remained at 2.5 g L-1 h(-1) for tip to 10 h longer when K- or Na-bases where used instead of NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity.

  • 4. Andersson, Christian
    et al.
    Hodge, David
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli2007In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 23, no 2, p. 381-388Article in journal (Refereed)
    Abstract [en]

    Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.

  • 5. Andersson, Christian
    et al.
    Petrova, Ekaterina
    Luleå tekniska universitet.
    Berglund, Kris
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Maintaining high anaerobic succinic acid productivity by product removal2010In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 33, no 6, p. 711-718Article in journal (Refereed)
    Abstract [en]

    During dual-phase fermentations using Escherichia coli engineered for succinic acid production, the productivity and viable cell concentration decrease as the concentration of succinic acid increases. The effects of succinic acid on the fermentation kinetics, yield, and cell viability were investigated by resuspending cells in fresh media after selected fermentation times. The cellular succinic acid productivity could be restored, but cell viability continuously decreased throughout the fermentations by up to 80% and subsequently the volumetric productivity was reduced. Omitting complex nutrients in the resuspension media had no significant effect on cellular succinate productivity and yield, although the viable cell concentration and thus the volumetric productivity was reduced by approximately 20%. By resuspending the cells, the amount of succinate produced during a 100-h fermentation was increased by more than 60%. The results demonstrate that by product removal succinic acid productivity can be maintained at high levels for extended periods of time.

  • 6.
    Andersson, Christian
    et al.
    Luleå University of Technology.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Process for producing succinic acid from sucrosePatent (Other (popular science, discussion, etc.))
    Abstract [en]

    A process for hydrolyzing sucrose to glucose and fructose using succinic acid is described. The hydrolysate can be used to produce purified glucose and/or fructose or can be used as a carbon source for fermentations to produce various chemicals including succinic acid.

  • 7.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Dilokpimol, Adiphol
    Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University.
    Iancu, Laura
    Dupont Industrial Biosciences.
    Mäkelä, Miia R.
    Department of Microbiology, University of Helsink.
    Varriale, Simona
    Department of Chemical Sciences, University of Naples “Federico II”.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples “Federico II”.
    Hüttner, Silvia
    Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology.
    Uthoff, Stefan
    Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG.
    Steinbüchel, Alexander
    nstitut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster.
    Olsson, Lisbeth
    Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples “Federico II”.
    Hildén, Kristiina
    Department of Microbiology, University of Helsinki.
    de Vries, Ronald
    Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties2018In: Catalysts, ISSN 2073-4344, Vol. 8, no 6, article id 242Article in journal (Refereed)
    Abstract [en]

    Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

  • 8.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hunt, Cameron
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Varriale, Simona
    Department of Chemical Sciences, University of Naples “Federico II”.
    Gerogianni, Alexandra
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tailoring the specificity of the type C feruloyl esterase FoFaeC from Fusarium oxysporum towards methyl sinapate by rational redesign based on small molecule docking simulations2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 5, article id e0198127Article in journal (Refereed)
    Abstract [en]

    The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme’ active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

  • 9.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    Dupont Industrial Biosciences, Wageningen, the Netherlands.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG, Dortmund, Germany.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG, Dortmund, Germany.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimized Enzymatic Synthesis of Feruloyl Derivatives Catalyzed by Three Novel Feruloyl Esterases from Talaromyces wortmannii in Detergentless Microemulsions2018In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, p. 361-369Article in journal (Refereed)
    Abstract [en]

    Three novel feruloyl esterases (Fae125, Fae7262 and Fae68) from Talaromyces wortmanniioverexpressed in the C1 platform were evaluated for the transesterification of vinyl ferulatewith two acceptors of different size and lipophilicity (prenol and L-arabinose) in detergentless microemulsions. The effect of reaction conditions such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation were investigated. The type A Fae125 belonging to the subfamily 5 (SF5) of phylogenetic classification showed highest yields for the synthesis of both products after optimization of reaction conditions: 81.8% for prenyl ferulate and 33.0% for L-arabinose ferulate. After optimization, an 8-fold increase in the yield and a 12-fold increase in selectivity were achieved for the synthesis of prenyl ferulate.

  • 10.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    Dupont Industrial Biosciences.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Screening of novel feruloyl esterases from Talaromyces wortmannii for the development of efficient and sustainable syntheses of feruloyl derivatives2019In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 120, p. 124-135Article in journal (Refereed)
    Abstract [en]

    The feruloyl esterases Fae125, Fae7262 and Fae68 from Talaromyces wortmannii were screened in 10 different solvent: buffer systems in terms of residual hydrolytic activity and of the ability for the transesterification of vinyl ferulate with prenol or L-arabinose. Among the tested enzymes, the acetyl xylan-related Fae125 belonging to the phylogenetic subfamily 5 showed highest yield and selectivity for both products in alkane: buffer systems (n-hexane or n-octane). Response surface methodology, based on a 5-level and 6-factor central composite design, revealed that the substrate molar ratio and the water content were the most significant variables for the bioconversion yield and selectivity. The effect of agitation, the possibility of DMSO addition and the increase of donor concentration were investigated. After optimization, competitive transesterification yields were obtained for prenyl ferulate (87.5-92.6%) and L-arabinose ferulate (56.2-61.7%) at reduced reaction times (≤ 24 h) resulting in good productivities (> 1 g/L/h, >300 kg product/kg FAE). The enzyme could be recycled for six consecutive cycles retaining 66.6% of the synthetic activity and 100% of the selectivity.

  • 11.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Leonov, Laura
    DuPont Industrial Biosciences.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Papadopoulou, Adamantia
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Ralli, Marianna
    Korres Natural Products.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions2018In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 1, p. 511-511Article in journal (Refereed)
  • 12.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Leonov, Laura
    DuPont Industrial Biosciences.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Papadopoulou, Adamantia
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Ralli, Marianna
    Korres Natural Products.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions2017In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, no 8, p. 3213-3226Article in journal (Refereed)
    Abstract [en]

    Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

  • 13.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Papadopoulou, Adamantia
    Laboratory of Cell Proliferation & Ageing, Institute of Biosciences & Applications NCSR “Demokritos”, T. Patriarchou Grigoriou & Neapoleos.
    Iancu, Laura
    DuPont Industrial Biosciences.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Ralli, Marianna
    Korres Natural Products.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Piechot, Alexander
    Taros Chemicals GmbH & Co.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimization of enzymatic synthesis of l-arabinose ferulate catalyzed by feruloyl esterases from Myceliophthora thermophila in detergentless microemulsions and assessment of its antioxidant and cytotoxicity activities2018In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 65, p. 100-108Article in journal (Refereed)
    Abstract [en]

    The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferulate (VFA) with l-arabinose in detergentless microemulsions. The effect of parameters such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation was investigated. FaeA1 offered the highest transesterification yield (52.2 ± 4.3%) after 8 h of incubation at 50 °C using 80 mM VFA, 55 mM l-arabinose and 0.02 mg FAE mL−1 in a mixture comprising of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. The ability of l-arabinose ferulate (AFA) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was significant (IC50 386.5 μM). AFA was not cytotoxic even at high concentrations (1 mM) however was found to be pro-oxidant at concentrations higher than 20 μM when the antioxidant activity was determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in human skin fibroblasts.

  • 14.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Varriale, Simona
    Department of Chemical Sciences, University of Naples "Federico II".
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Faraco, Voncenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 15, p. 6519-6543Article in journal (Refereed)
    Abstract [en]

    Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry

  • 15.
    Bauer, Fredric
    et al.
    Lund University.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hulteberg, Christian
    Lund University.
    Lundgren, Joakim
    Luleå University of Technology, Department of Engineering Sciences and Mathematics, Energy Science.
    Mesfun, Sennai
    Luleå University of Technology, Department of Engineering Sciences and Mathematics, Energy Science.
    Nilsson, Robert
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Wännström, Sune
    SP Technical Research Institute of Sweden.
    Comparative system analysis of carbon preserving fermentations for biofuels production2013Report (Refereed)
  • 16.
    Behravan, Gity
    et al.
    Umeå universitet.
    Sen, Srikanta
    Stockholms Universitet.
    Rova, Ulrika
    Thelander, Lars
    Umeå universitet.
    Eckstein, Fritz
    Max-Planck-Institut fur Experimentelle Medizin.
    Gräslund, Astrid
    Stockholms Universitet.
    Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate1995In: Biochimica et Biophysica Acta, Gene Structure and Expression, ISSN 0167-4781, E-ISSN 1879-2634, Vol. 1264, no 3, p. 323-329Article in journal (Refereed)
    Abstract [en]

    Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2′-azido-2′-deoxycytidine 5′-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that ‘normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W 103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type

  • 17.
    Berglund, Kris
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Andersson, Christian
    Luleå University of Technology.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Process for the production of succinic acidPatent (Other (popular science, discussion, etc.))
    Abstract [en]

    A process for the production of succinic acid can comprise supplying a media with E. coli AFP 184 and a high sugar concentration under aerobic conditions, then converting the media to aerobic conditions. Such a process can be useful when performed in conjunction with the production of ethanol in a biorefmery .

  • 18.
    Berglund, Kris
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Fermentative Upgrading of Xylose2009In: NWBC-2009: The 2nd Nordic wood biorefinery conference : Finlandia Hall, Helsinki, Finland, September 2-4, 2009 : Proceedings-Posters / [ed] Annemari Kuokka-Ihalainen., Helsinki: KCL Re-inventing paper , 2009Conference paper (Other academic)
    Abstract [en]

    Chemical pulp mills such as Kraft, soda, or sulfite mills are current examples of biorefineries that can convert lignocellulosic biomass into energy, pulp or cellulose derivatives, and tall oil.  While existing viscose pulps use a hemicellulose extraction to generate soluble sugars for ethanol production, in general there still exists a large potential for other more profitable applications of the biomass (Fig. 1), i.e. the mill needs to present a widespread product portfolio.  The biofuels under development from fermentation that will be discussed are the diesel fuel oxygenates dibutyl succinate and diethyl succinate to be used for reduced particulate emissions and fossil fuel replacement for diesel engines and butanol for Otto engines.  It's important to stress that succinic acid, butanol and ethanol, needed for the production of the diesel additives and gasoline replacement will be produced from renewable resources and hence replacing products currently produced from non-renewable fossil sources.  Since wood will be used, there will be no issue of competing with raw material used for food production.  Besides biofuel production, succinic acid and butanol, can be used directly or further refined into numerous different products classified as green chemicals.

  • 19.
    Berglund, Kris
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Fermentation-Based Building Blocks for Renewable Resource-Based Surfactants2010In: Surfactants from renewable resources, Chichester: John Wiley & Sons Ltd , 2010, p. 127-141Chapter in book (Refereed)
    Abstract [en]

    'new' top-ranked building blocks; Citric acid recovery from fermentation broths and CaCO3 precipitation; Citric, acetic and lactic acid - top three industrial carboxylic acids; Fermentation-based building blocks for renewable resource-based surfactants; Fermentation-based building blocks for surfactants; Filamentous fungi, Aspergillus niger and Candida yeast strains; New fermentation-based building blocks; Organic acid metabolites - as hydrophilic moiety; Sulfonates - largest market share of anionic surfactants; Sulfosuccinate class of surfactants

  • 20.
    Bonturi, Nemailla
    et al.
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Nilsson, Robert
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Miranda, Everson Alves
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Single Cell Oil Producing Yeasts Lipomyces starkeyi and Rhodosporidium toruloides: Selection of Extraction Strategies and Biodiesel Property Prediction2015In: Energies, ISSN 1996-1073, E-ISSN 1996-1073, Vol. 8, no 6, p. 5040-5052Article in journal (Refereed)
    Abstract [en]

    Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

  • 21.
    Cerullo, Gabriella
    et al.
    University of Naples “Federico II”, Naples, Italy.
    Varriale, Simona
    University of Naples “Federico II”, Naples, Italy.
    Bozonnet, Sophie
    Université de Toulouse, Toulouse, France.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Gherbovet, Olga
    Université de Toulouse, Toulouse, France.
    Fauré, Régis
    Université de Toulouse, Toulouse, France.
    Piechot, Alexander
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Jütten, Peter
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Brás, Joana L.A.
    NzyTech LDA, Lisbon, Portugal.
    Fontes, Carlos M.G.A.
    NzyTech LDA, Lisbon, Portugal.
    Faraco, Vincenza
    University of Naples “Federico II”, Naples, Italy.
    Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants2019In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 51, p. 14-20Article in journal (Refereed)
    Abstract [en]

    The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

  • 22.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Organosolv Biomass Pretreatment for Flexible Fuel Production (Swedish Energy Agency)2016Other (Other (popular science, discussion, etc.))
  • 23.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: BIOcatalytic Carbon Capture and Conversion of steel flue gas to liquid hydrocarbons (FORMAS)2016Other (Other (popular science, discussion, etc.))
  • 24.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes2012In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 87, no 7, p. 903-907Article in journal (Refereed)
    Abstract [en]

    Background: Mycelium of the medicinal mushroom shiitake, Lentinus edodes, is a potential source for production of the blood cholesterol reducing compound eritadenine. To increase the mycelial biomass and in turn the production of eritadenine, a potential growth promoting substance in the form of a water extract of distillers dried grains with solubles (DDGS) was added to the culture media. Results: The hot water extract of DDGS was shown to considerably increase the growth of shiitake mycelia in bioreactor cultivations; the mycelial yield was 2-3 times higher than in the control, and the highest final biomass concentration obtained was 3.4 g L -1. Further, by using shake flask cultures as inoculums the bioreactor cultivation time could be reduced by 1 week for some of the experiments. The highest final titer of eritadenine in the present study was 25.1 mg L -1, which was about 2 times higher than in the control, and was also obtained when a water extract of DDGS was added to the culture medium. Conclusion: It was demonstrated that a water extract of DDGS promoted the growth of shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production

  • 25.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hodge, David
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Production of the bioactive compound eritadenine by submerged cultivation of shiitake (Lentinus edodes) mycelia2008In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 56, no 8, p. 2609-2612Article in journal (Refereed)
    Abstract [en]

    Fruit bodies and mycelia of shiitake mushroom (Lentinus edodes) have been shown to contain the cholesterol-reducing compound eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)butyric acid. In the search for a production method for eritadenine, shiitake mycelia were investigated in the present study. The mycelia were cultivated both in shake flasks and in bioreactors, to investigate the effects of pH, stirring rate, and reactor type on the production and distribution of eritadenine. Both the biomass and the culture broth were examined for their eritadenine content. In the shake flasks, the final concentration of eritadenine was 1.76 mg/L and eritadenine was equally distributed between the mycelia and the growth media. In the bioreactors, the shiitake mycelia were found to contain eritadenine in relatively low levels, whereas the majority, 90.6-98.9%, was detected in the growth media. Applying a stirring rate of 250 rpm during bioreactor cultivation resulted in the highest eritadenine concentrations: 10.23 mg/L when the pH was uncontrolled and 9.59 mg/L when the pH was controlled at 5.7. Reducing the stirring rate to 50 rpm resulted in a decreased eritadenine concentration, both at pH 5.7 (5.25 mg/L) and when pH was not controlled (5.50 mg/L). The mycelia in the shake flask cultures appeared as macroscopic aggregates, whereas mycelia cultivated in bioreactors grew more as freely dispersed filaments. This study demonstrates for the first time the extra- and intracellular distribution of eritadenine produced by shiitake mycelial culture and the influence of reactor conditions on the mycelial morphology and eritadenine concentrations.

  • 26.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Patra, Anuttam
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Ramser, Kerstin
    Luleå University of Technology, Department of Computer Science, Electrical and Space Engineering, Signals and Systems.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Solid state characterization of sodium eritadenate2011In: American Journal of Analytical Chemistry, ISSN 2156-8251, E-ISSN 2156-8278, Vol. 2, no 2, p. 164-173Article in journal (Refereed)
    Abstract [en]

    Knowledge of the solid state is of great importance in the development of a new active pharmaceutical ingredient, since the solid form often dictates the properties and performance of the drug. In the present study, solid state characteristics of the sodium salt of the candidate cholesterol reducing compound eritadenine, 2(R), 3(R))-dihydroxy-4-(9-adenyl)-butanoic acid, were investigated. The compound was crystallized by slow cooling from water and various aqueous ethanol solutions, at different temperatures. Further, the compound solution was subjected to lyophilization and to high vacuum drying. The resulting solids were screened for polymorphism by micro Raman spectroscopy (λex = 830 nm) and the crystallinity was investigated by X-ray powder diffraction. Further, thermal analysis was applied to study possible occurrence of solvates or hydrates. Solids obtained from slow cooling showed crystallinity, whereas rapid cooling gave rise to more amorphous solids. Analysis of difference spectra of the Raman data for solids obtained from slow cooling of solution revealed subtle differences in the structures between crystals derived from pure water and crystals derived from aqueous ethanol solutions. Finally, from the thermal analysis it was deduced that crystals obtained from pure water were stoichiometrically dihydrates whereas crystals obtained from aqueous ethanol solutions were 2.5 hydrates; this formation of different hydrates were supported by the Raman difference analysis.

  • 27.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Ramser, Kerstin
    Luleå University of Technology, Department of Computer Science, Electrical and Space Engineering, Signals and Systems.
    Rova, Ulrika
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Raman analysis of synthetic eritadenine2008In: Journal of Raman Spectroscopy, ISSN 0377-0486, E-ISSN 1097-4555, Vol. 39, no 10, p. 1464-1468Article in journal (Refereed)
    Abstract [en]

    Eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid, is a cholesterol-reducing compound naturally occurring in the shitake mushroom (Lentinus edodes). To identify the unknown Raman spectrum of this compound, pure synthetic eritadenine was examined and the vibrational modes were assigned by following the synthesis pathway. This was accomplished by comparing the known spectra of the starting compounds adenine and D-ribose with the spectra of a synthesis intermediate, methyl 5-(6-Aminopurin-9H-9-yl)-2,3-O-isopropylidene-5-deoxy-β-D-ribofuranoside (MAIR) and eritadenine. In the Raman spectrum of eritadenine, a distinctive vibrational mode at 773 cm-1 was detected and ascribed to vibrations in the carbon chain, ν(C--C). A Raman line that arose at 1212 cm-1, both in the Raman spectrum of MAIR and eritadenine, was also assigned to ν(C--C). Additional Raman lines detected at 1526 and at 1583 cm-1 in the Raman spectrum of MAIR and eritadenine were assigned to ν(N--C) and a deformation of the purine ring structure. In these cases the vibrational modes are due to the linkage between adenine and the ribofuranoside moiety for MAIR, and between adenine and the carbon chain for eritadenine. This link is also the cause for the disappearance of adenine specific Raman lines in the spectrum of both MAIR and eritadenine. Several vibrations observed in the spectrum of D-ribose were not observed in the Raman spectrum of eritadenine due to the absence of the ribose ring structure. In the Raman spectrum of MAIR some of the D-ribose specific Raman lines disappeared due to the introduction of methyl and isopropylidene moieties to the ribose unit. With the approach presented in this study the so far unknown Raman spectrum of eritadenine could be successfully identified and is presented here for the first time.

  • 28.
    Enman, Josefine
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Berglund, Kris A.
    Quantification of the bioactive compound eritadenine in selected strains of shiitake mushroom (Lentinus edodes)2007In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 55, no 4, p. 1177-1180Article in journal (Refereed)
    Abstract [en]

    Cardiovascular disease is one of the most common causes of death in the Western world, and a high level of blood cholesterol is considered a risk factor. The edible fungus, shiitake mushroom (Lentinus edodes), contains the hypocholesterolemic agent eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid. This study was conducted to quantify the amount of the cholesterol reducing agent eritadenine in shiitake mushrooms, in search of a potential natural medicine against blood cholesterol. The amounts of eritadenine in the fruit bodies of four different shiitake mushrooms, Le-1, Le-2, Le-A, and Le-B, were investigated in this study. To achieve this goal, methanol extraction was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In addition, enzymes that degrade the fungal cell walls were also used to elucidate if the extraction could be further enhanced. To analyze the target compound, a reliable and reproducible HPLC method for separation, identification, and quantification of eritadenine was developed. The shiitake strains under investigation exhibit up to 10 times higher levels of eritadenine than previously reported for other shiitake strains. Further, pretreating the mushrooms with hydrolytic enzymes before methanol extraction resulted in an insignificant increase in the amount of eritadenine released. These results indicate the potential for delivery of therapeutic amounts of eritadenine from the ingestion of extracts or dried concentrates of shiitake mushroom strains.

  • 29.
    Faisal, Abrar
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zarebska, Agata
    Saremi, Pardis
    Korelskiy, Danil
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Ohlin, Lindsay
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Hedlund, Jonas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    Grahn, Mattias
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering.
    MFI zeolite as adsorbent for selective recovery of hydrocarbons from ABE fermentation broths2014In: Adsorption, ISSN 0929-5607, E-ISSN 1572-8757, Vol. 20, no 2-3, p. 465-470Article in journal (Refereed)
    Abstract [en]

    1-Butanol and butyric acid are two interesting compounds that may be produced by acetone, butanol, and ethanol fermentation using e.g. Clostridium acetobutylicum. The main drawback, restricting the commercialization potential of this process, is the toxicity of butanol for the cell culture resulting in low concentrations of this compound in the broth. To make this process economically viable, an efficient recovery process has to be developed. In this work, a hydrophobic MFI type zeolite with high silica to alumina ratio was evaluated as adsorbent for the recovery of butanol and butyric acid from model solutions. Dual component adsorption experiments revealed that both butanol and butyric acid showed a high affinity for the hydrophobic MFI zeolite when adsorbed from aqueous model solutions. Multicomponent adsorption experiments using model solutions, mimicking real fermentation broths, revealed that the adsorbent was very selective to the target compounds. Further, the adsorption of butyric and acetic acid was found to be pH dependent with high adsorption below, and low adsorption above, the respective pKa values of the acids. Thermal desorption of butanol from MFI type zeolite was also studied and a suitable desorption temperature was identified.

  • 30.
    Filatov, D.
    et al.
    Umeå universitet.
    Ingemarson, R.
    Umeå universitet.
    Johansson, E.
    Umeå universitet.
    Rova, Ulrika
    Thelander, L.
    Umeå universitet.
    Mouse ribonucleotide reductase: from genes to proteins1995In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 23, no 4, p. 903-905Article in journal (Refereed)
  • 31.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Belotserkovsky, Jaroslav
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Isaksson, Leif
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 29Article in journal (Refereed)
    Abstract [en]

    BackgroundArginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. ResultsFirstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. ConclusionsIn this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

  • 32.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rusanova-Naydenova, Daniela
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Simultaneous Quantification of L-Arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach2019In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 24, no 4, article id 802Article in journal (Refereed)
    Abstract [en]

    Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

  • 33.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rusanova-Naydenova, Daniela
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tuning of the Carbon-to-Nitrogen Ratio for the Production of L-Arginine by Escherichia coli2017In: Fermentation, ISSN 2311-5637, Vol. 3, no 4, article id 60Article in journal (Refereed)
    Abstract [en]

    L-arginine, an amino acid with a growing range of applications within the pharmaceutical, cosmetic, food, and agricultural industries, can be produced by microbial fermentation. Although it is the most nitrogen-rich amino acid, reports on the nitrogen supply for its fermentation are scarce. In this study, the nitrogen supply for the production of l-arginine by a genetically modified Escherichia coli strain was optimised in bioreactors. Different nitrogen sources were screened and ammonia solution, ammonium sulphate, ammonium phosphate dibasic, and ammonium chloride were the most favourable nitrogen sources for l-arginine synthesis. The key role of the C/N ratio for l-arginine production was demonstrated for the first time. The optimal C/N molar ratio to maximise l-arginine production while minimising nitrogen waste was found to be 6, yielding approximately 2.25 g/L of l-arginine from 15 g/L glucose with a productivity of around 0.11 g/L/h. Glucose and ammonium ion were simultaneously utilized, showing that this ratio provided a well-balanced equilibrium between carbon and nitrogen metabolisms.

  • 34.
    Gustafsson, Anki
    et al.
    Recopharma AB, Huddinge.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Strindelius, Lena
    Recopharma AB, Huddinge.
    Johansson, Thomas
    Recopharma AB, Huddinge.
    Fleckenstein, Tilly
    Recopharma AB, Huddinge.
    Chatzissavidou, Nathalie
    Recopharma AB, Huddinge.
    Lindberg, Linda
    Absorber AB, Stockholm.
    Ångström, Jonas
    Göteborgs universitet.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Holgersson, Jan
    Karolinska Institutet.
    Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 8, p. 1071-1086Article in journal (Refereed)
    Abstract [en]

    Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG2b) carrying mostly O-glycans and, as a control, the a1-acid glycoprotein (AGP/mIgG2b) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. P. pastoris-produced PSGL-1/mIgG2b was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by a-mannosidases including an a1,2- but not an a1,6-mannosidase. Electrospray ionization - ion trap mass spectrometry (ESI-MS) confirmed the presence of O-glycans containing up to 9 hexoses with the penta- and hexasaccharides being the predominant ones. a1,2- and a1,3-linked, but not a1,6-linked, mannose residues were detected by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG2b to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG2b expressed in P. pastoris carried O-glycans mainly comprised of a-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.

  • 35. Helmerius, Jonas
    et al.
    Walter, Jonas Vinblad von
    Smurfit Kappa Kraftliner AB.
    Rova, Ulrika
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Production of value added chemicals from xylan extraction in a Kraft pulp mill and the effect on pulp quality2008Conference paper (Other academic)
    Abstract [en]

    In the Kraft process hemicelluloses are lost in the cooking procedure to the black liquor stream, which is subsequently burnt in the recovery boiler to recover cooking chemicals and to produce steam and energy. Hemicelluloses have a low heating value compared to lignin and therefore recovery of hemicelluloses at an earlier stage of the Kraft process followed by biochemical conversionintohighvalue-conversion intohighvalue-into high value-added products might offer a muchbettereconomicopportunity.much better economic opportunityIn collaboration with the research and development department of Smurfit Kappa Kraftliner AB, Piteå, Sweden, alkali and water extractions of birch wood were performed under conditions compatible with the Kraft process, at different times, temperatures and alkali charges. The extraction conditions were set in a range suitable with the current pulp process at Smurfit Kappa Kraftliner. TherequirementsforprocessThe requirements for process configurations, based on either hot water or alkali extraction were also explored. ThexylanyieldsfromdifferentextractiontrialswereThe xylan yields from different extraction trials were measured and the chips from those extraction trials were cooked, refined and turned into sheets of paper. The effects on paper quality were compared with a reference pulp made from the same wooden chips. Recovered xylans from water extracted birch wood chips were subjected to secondary hydrolysis, enzymatic or sulphuricacid.sulphuric acidDetoxification of the hydrolysate with active carbon and regulation of pH were performed before fermentation. FermentationofthexyloseFermentation of the xylose hydrolysate to succinic acid was demonstrated by the use of thethe succinic acid producer Escherichia coli AFP184.

  • 36. Helmerius, Jonas
    et al.
    Walter, Jonas Vinblad von
    Smurfit Kappa Kraftliner AB.
    Rova, Ulrika
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Hodge, David
    Production of value added chemicals from xylan extraction in a Kraft pulp mill and the effect on pulp quality2008Conference paper (Other academic)
    Abstract [en]

    In the Kraft process hemicelluloses are lost in the cooking procedure to the black liquor stream, which is subsequently burnt in the recovery boiler to recover cooking chemicals and to produce steam and energy. Hemicelluloses have a low heating value compared to lignin and therefore recovery of hemicelluloses at an earlier stage of the Kraft process followed by biochemical conversion into high value-added products might offer a much better economic opportunity. In collaboration with the research and development department of Smurfit Kappa Kraftliner AB, Piteå, Sweden, alkali and water extractions of birch wood were performed under conditions compatible with the Kraft process, at different times, temperatures and alkali charges. The extraction conditions were set in a range suitable with the current pulp process at Smurfit Kappa Kraftliner. The requirements for process configurations, based on either hot water or alkali extraction were also explored. The xylan yields from different extraction trials were measured and the chips from those extraction trials were cooked, refined and turned into sheets of paper. The effects on paper quality were compared with a reference pulp made from the same wooden chips. Recovered xylans from water extracted birch wood chips were subjected to secondary hydrolysis, enzymatic or sulphuric acid. Detoxification of the hydrolysate with active carbon and regulation of pH were performed before fermentation. Fermentation of the xylose hydrolysate to succinic acid was demonstrated by the use of the succinic acid producer Escherichia coli AFP184.

  • 37.
    Helmerius, Jonas
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Walter, Jonas Vinblad von
    Luleå tekniska universitet.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Berglund, Kris
    Hodge, David B.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Impact of hemicellulose pre-extraction for bioconversion on birch Kraft pulp properties2010In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 101, no 15, p. 5996-6005Article in journal (Refereed)
    Abstract [en]

    The combination of hemicellulose extraction with chemical pulping processes is one approach to generate a sugar feedstock amenable to biochemical transformation to fuels and chemicals. Extractions of hemicellulose from silver birch (Betula pendula) wood chips using either water or Kraft white liquor (NaOH, Na2S, and Na2CO3) were performed under conditions compatible with Kraft pulping, using times ranging between 20 and 90 min, temperatures of 130-160 °C, and effective alkali (EA) charges of 0-7%. The chips from select extractions were subjected to subsequent Kraft pulping and the refined pulps were made into handsheets. Several metrics for handsheet strength properties were compared with a reference pulp made without an extraction step. This study demonstrated that white liquor can be utilized to extract xylan from birch wood chips prior to Kraft cooking without decreasing the pulp yield and paper strength properties, while simultaneously impregnating cooking alkali into the wood chips. However, for the alkaline conditions tested extractions above pH 10 resulted in low concentrations of xylan. Water extractions resulted in the highest final concentrations of xylan; yielding a liquor without the presence of toxic or inhibitory inorganics and minimal soluble aromatics that we demonstrate can be successfully enzymatically hydrolyzed to monomeric xylose and fermented to succinic acid. However, water extractions were found to negatively impact some pulp properties including decreases in compression strength, bursting strength, tensile strength, and tensile stiffness while exhibiting minimal impact on elongation and slight improvement in tearing strength index.

  • 38. Hodge, David
    et al.
    Andersson, Christian
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Detoxification requirements for bioconversion of softwood dilute acid hydrolyzates to succinic acid2009In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 44, no 5, p. 309-316Article in journal (Refereed)
    Abstract [en]

    In this work an Escherichia coli metabolically engineered to ferment lignocellulosic biomass sugars to succinic acid was tested for growth and fermentation of detoxified softwood dilute sulfuric acid hydrolyzates, and the minimum detoxification requirements were investigated with activated carbon and/or overliming treatments. Detoxified hydrolyzates supported fast growth and complete fermentation of all hydrolyzate sugars to succinate at yields comparable to pure sugar, while untreated hydrolyzates were unable to support either growth or fermentation. Activated carbon treatment was able to remove significantly more HMF and phenolics than overliming. However, in some cases, overliming treatment was capable of generating a fermentable hydrolyzate where activated carbon treatment was not. The implications of this are that in addition to the known organic inhibitors, the changes in the inorganic content and/or composition due to overliming are significant to the hydrolyzate toxicity. It was also found that any HMF remaining after detoxification was completely metabolized during aerobic cell growth on the hydrolyzates that were capable of supporting growth.

  • 39. Hodge, David
    et al.
    Andersson, Christian
    Helmerius, Jonas
    Walter, J. Vinblad von
    Rova, Ulrika
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Succinic acid production from forest based raw materials2008Conference paper (Other academic)
    Abstract [en]

    Lignocellulosic biomass and particularly hemicellulose from the forest products industry represents a large reservoir of sugars with the potential to be converted to higher value products through bioprocessing. This presentation will cover several projects regarding the fractionation and conversion of lignocellulose to succinic acid, a potentially important platform molecule in the synthesis of a number of commodity and specialty chemicals. The first of these investigates the feasibility of integrating a hardwood hemicellulose sugar extraction step into a Kraft pulping process with the intention of utilizing the hemicellulose as a fermentation feedstock. The requirements on processing configurations for hemicellulose extraction and recovery are compared, and a number of experimental parameters affecting the extraction (alkali, temperature, time) are investigated. Pulp quality is an important property and hemicellulose extraction can result in negatively affect the strength of the paper, which is also investigated. The second portion of the work deals with the fermentation requirements for microbial conversion of dilute acid hydrolyzed softwood to succinic acid. In particular, activated carbon and overliming detoxifications were tested for the ability to remove fermentation inhibitors and improve the fermentability of the hydrolyzates.

  • 40.
    Hodge, David
    et al.
    Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing.
    Helmerius, Jonas
    Walter, Jonas Vinblad von
    Sun Pine Biodiesel AB, Piteå.
    Lindström, Curt
    Smurfit Kappa Kraftliner AB, Piteå.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Impact of hemicellulose pre-extraction for bioconversion on birch kraft pulp properties2009Conference paper (Other academic)
    Abstract [en]

    The carbohydrate portion of lignocellulosic feedstocks are ideally suited to conversion via biochemical transformations because of their crucial role in cellular metabolism. The combination of hemicelluloses extraction with pulping processes could be one way to generate a sugar feedstock amenable to biochemical transformation to fuels and chemical intermediates. White liquor, green liquor, and water HC extractions of birch wood were performed under conditions compatible with the Kraft process, at different times, temperatures and alkali charges. The effective alkali charge was in extractions between 0%-7% and temperature between 130°C-160°C for 20-90 minutes. The xylan yields from different HC extractions were measured and the chips from select HC extractions were cooked, and the refined pulps were made into hand sheets. Several metrics for hand sheet quality were compared with a reference pulp made from the same wood chips. It is possible using white liquor to extract xylan from birch wood chips prior Kraft cooking without decreasing the pulp yield and paper strength properties, and at the same time achieve an impregnation of the wood chips. It is not possible in that extraction to attain extracted and hydrolyzed liquor containing a fermentable concentration of xylose, 2.63 g/L in this study. Increased extracted wood material, increased final acetic acid concentration and decreased final xylan concentration together with increased effective alkali charge at the same extraction temperature and time in white liquor extractions performed support that xylan degradation increases. Using white liquor or green liquor under the conditions investigated degrades xylan resulting in significant losses of xylose that could have been used as substrate in fermentation processes.

  • 41.
    Hu, Shu-Hong
    et al.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Gee, Christine L.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Latham, Catherine F.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Rowlinson, Scott W.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Rova, Ulrika
    Jones, Alun
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Halliday, Judy A.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Bryant, Nia J.
    Wellcome Laboratory of Cell Biology, Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, The University of Glasgow.
    James, David E.
    Garvan Institute of Medical Research, 384 Victoria Road, Darlinghurst, Sydney.
    Martin, Jennifer F.
    Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland.
    Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin42003In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 31, no 2, p. 305-310Article in journal (Refereed)
    Abstract [en]

    Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

  • 42.
    Hunt, Cameron J
    et al.
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia..
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tanksale, Akshat
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia..
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Haritos, Victoria S
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia.
    Insights into substrate binding of ferulic acid esterases by arabinose and methyl hydroxycinnamate esters and molecular docking2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 17315Article in journal (Refereed)
    Abstract [en]

    Ferulic acid esterases (FAE, EC 3.1.1.73) cleave the arabinose hydroxycinnamate ester in plant hemicellulose and other related substrates. FAE are commonly categorised as type A-D based on catalytic activities towards model, short alkyl chain esters of hydroxycinnamates. However, this system correlates poorly with sequence and structural features of the enzymes. In this study, we investigated the basis of the type A categorisation of an FAE from Aspergillus niger, AnFaeA, by comparing its activity toward methyl and arabinose hydroxycinnamate esters. kcat/Km ratios revealed that AnFaeA hydrolysed arabinose ferulate 1600-fold, and arabinose caffeate 6.5 times more efficiently than their methyl ester counterparts. Furthermore, small docking studies showed that while all substrates adopted a catalytic orientation with requisite proximity to the catalytic serine, methyl caffeate and methyl p-coumarate preferentially formed alternative non-catalytic conformations that were energetically favoured. Arabinose ferulate was unable to adopt the alternative conformation while arabinose caffeate preferred the catalytic orientation. This study demonstrates that use of short alkyl chain hydroxycinnnamate esters can result in activity misclassification. The findings of this study provide a basis for developing a robust classification system for FAE and form the basis of sequence-function relationships for this class.

  • 43.
    Häggström, Caroline
    et al.
    Wibax AB.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Brandberg, Tomas
    SEKAB E-Technology, School of Engineering, University of Borås.
    Hodge, David
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Integration of Ethanol Fermentation with Second Generation Biofuels Technologies2014In: Biorefineries: Integrated Biochemical Processes for Liquid Biofuels, Amsterdam: Elsevier, 2014, p. 161-187Chapter in book (Refereed)
    Abstract [en]

    This chapter presents an overview of the challenges associated with integrating yeast fermentation into cellulosic biofuel processes, as well as the approaches that might overcome these challenges. The chapter first introduces the design considerations for first-generation ethanol fermentation processes using sugar cane and corn as feedstocks, with an emphasis on process constraints and operation strategies. The chapter then explores methods for improving yield, titer, productivity, and economics. These processing methods illustrate the challenges posed by the fermentation of ethanol from lignocellulose hydrolyzates, especially the differences in process constraints for high-productivity, high-product titer operations. Finally, the chapter discusses an example of aerobic seed cultivation of yeast using a hydrolyzate of dilute acid-hydrolyzed softwood hemicellulose

  • 44. Häggström, Caroline
    et al.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Brandberg, Tomas
    SEKAB E-Technology.
    Hodge, David
    Chemical Engineering & Materials Science, Michigan State University, East Lansing, MI.
    Media requirements for aerobic cultivation of saccharomyces cerevisiae TMB 3400-F30-3 on undetoxified softwood dilute acid hydrolyzate2009Conference paper (Other academic)
    Abstract [en]

    The purpose of this study was to develop a low-cost industrial nutrient medium for the aerobic cultivation of a hydrolyzate-adapted Saccharomyces cerevisiae TMB3400-FT30-3 for subsequent fermentation of dilute acid hydrolyzed softwood. This xylose-utilizing yeast is a promising ethanologen for converting lignocellulosic biomass sugars to ethanol. The dilute acid pretreated softwood hydrolyzate used in the process is a readily available and economic option for a carbon source for cell growth in the fermentation seed tank and has the added advantage of contributing to cell adaptation before the fermentation. The focus of the work was to determine the minimal level of externally-supplied media supplementation in the form of beet molasses and nitrogen in the form of ammonium sulfate that will still result in near-maximal biomass yields during fed-batch aerobic growth. The experimental work showed that it is possible to supply only 10 % of the sugars in the form of beet molasses (or 6.7 g/L of concentrated molasses ) without any significant reduction of the biomass yield from its near maximum value of 0.45 g biomass / g sugar. It was not until the molasses addition was below 9 % that a statistically significant reduction in yield was observed. Reducing the nitrogen from 0.12 to 0.06 g N / g biomass resulted in a statistically significant decrease of the yield.

  • 45.
    Jaros, Adam
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Acetate adaptation of clostridia tyrobutyricum for improved fermentation production of butyrate2013In: SpringerPlus, E-ISSN 2193-1801, Vol. 2, no 1Article in journal (Refereed)
    Abstract [en]

    Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium capable of utilizing xylose for the fermentation production of butyrate. Hot water extraction of hardwood lingocellulose is an efficient method of producing xylose where autohydrolysis of xylan is catalysed by acetate originating from acetyl groups present in hemicellulose. The presence of acetic acid in the hydrolysate might have a severe impact on the subsequent fermentations. In this study the fermentation kinetics of C. tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate equivalents were studied. Analysis of xylose batch fermentations found that even in the presence of high levels of acetate, acetate adapted strains had similar fermentation kinetics as the parental strain cultivated without acetate. The parental strain exposed to acetate at inhibitory conditions demonstrated a pronounced lag phase (over 100 hours) in growth and butyrate production as compared to the adapted strain (25 hour lag) or non-inhibited controls (0 lag). Additional insight into the metabolic pathway of xylose consumption was gained by determining the specific activity of the acetate kinase (AK) enzyme in adapted versus control batches. AK activity was reduced by 63% in the presence of inhibitory levels of acetate, whether or not the culture had been adapted.

  • 46.
    Jaros, Adam
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Berglund, Kris
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Effect of acetate on fermentation production of butyrate2012In: Cellulose Chemistry and Technology, ISSN 0576-9787, Vol. 46, no 5-6, p. 341-347Article in journal (Refereed)
    Abstract [en]

    A carbon source for the fermentation production of butyrate is xylose extracted from ligno-cellulosic material by hot water extraction. Although this auto-hydrolysis of hemicellulose can provide a low-cost source of xylose, the process generates a high level of acetic acid that might inhibit subsequent fermentations. This study focuses on the effects of acetate on the production of butyrate from xylose by batch fermentations with a selected strain Clostridium tyrobutyricum.At initial acetate concentrations of 17.6 g L-1 and 26.3 g L-1 in the media, C. tyrobutyricum cultures exhibited a lag phase (45 and 118 hours, respectively) in terms of sugar consumption, butyrate production and cell biomass growth, lowering the overall production rate. Butyrate fermentations performed with high concentrations of acetate in the media demonstrated a re-uptake of acetate into the butyrate production pathway and after the lag phase, all cultures adapted to the inhibitory acetate, which increased the final butyrate yields by 12.6% (32.6 g L-1 compared to 28.5 g L-1).

  • 47.
    Kalogiannis, Konstantinos G
    et al.
    Chemical Process and Energy Resources Institute (CPERI), Centre for Research and Technology Hellas (CERTH), 6th km Harilaou-Thermi Rd, 57001 Thessaloniki, Greece.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Aspden, James
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Lappas, Angelos A
    Chemical Process and Energy Resources Institute (CPERI), Centre for Research and Technology Hellas (CERTH), 6th km Harilaou-Thermi Rd, 57001 Thessaloniki, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Acid Assisted Organosolv Delignification of Beechwood and Pulp Conversion towards High Concentrated Cellulosic Ethanol via High Gravity Enzymatic Hydrolysis and Fermentation2018In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 23, no 7, article id 1647Article in journal (Refereed)
    Abstract [en]

    Background: Future biorefineries will focus on converting low value waste streams to chemical products that are derived from petroleum or refined sugars. Feedstock pretreatment in a simple, cost effective, agnostic manner is a major challenge.

    Methods: In this work, beechwood sawdust was delignified via an organosolv process, assisted by homogeneous inorganic acid catalysis. Mixtures of water and several organic solvents were evaluated for their performance. Specifically, ethanol (EtOH), acetone (AC), and methyl- isobutyl- ketone (MIBK) were tested with or without the use of homogeneous acid catalysis employing sulfuric, phosphoric, and oxalic acids under relatively mild temperature of 175 °C for one hour.

    Results: Delignification degrees (DD) higher than 90% were achieved, where both AC and EtOH proved to be suitable solvents for this process. Both oxalic and especially phosphoric acid proved to be good alternative catalysts for replacing sulfuric acid. High gravity simultaneous saccharification and fermentation with an enzyme loading of 8.4 mg/gsolids at 20 wt.% initial solids content reached an ethanol yield of 8.0 w/v%.

    Conclusions: Efficient delignification combining common volatile solvents and mild acid catalysis allowed for the production of ethanol at high concentration in an efficient manner

  • 48.
    Kalogiannis, Konstantinos G.
    et al.
    Chemical Process and Energy Resources Institute (CPERI), Centre for Research and Technology Hellas (CERTH), Thessaloniki, Greece.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Lappas, Angelos A.
    Chemical Process and Energy Resources Institute (CPERI), Centre for Research and Technology Hellas (CERTH), Thessaloniki, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Aromatics from Beechwood Organosolv Lignin through Thermal and Catalytic Pyrolysis2019In: Energies, ISSN 1996-1073, E-ISSN 1996-1073, Vol. 12, no 9, article id 1606Article in journal (Refereed)
    Abstract [en]

    Biomass fractionation, as an alternative to biomass pretreatment, has gained increasing research attention over the past few years as it provides separate streams of cellulose, hemicellulose, and lignin. These streams can be used separately and can provide a solution for improving the economics of emerging biorefinery technologies. The sugar streams are commonly used in microbial conversions, whereas during recent years lignin has been recognized as a valuable compound as it is the only renewable and abundant source of aromatic chemicals. Successfully converting lignin into valuable chemicals and products is key in achieving both environmental and economic sustainability of future biorefineries. In this work, lignin retrieved from beechwood sawdust delignification pretreatment via an organosolv process was depolymerized with thermal and catalytic pyrolysis. ZSM-5 commercial catalyst was used in situ to upgrade the lignin bio-oil vapors. Lignins retrieved from different modes of organosolv pretreatment were tested in order to evaluate the effect that upstream pretreatment has on the lignin fraction. Both thermal and catalytic pyrolysis yielded oils rich in phenols and aromatic hydrocarbons. Use of ZSM-5 catalyst assisted in overall deoxygenation of the bio-oils and enhanced aromatic hydrocarbons production. The oxygen content of the bio-oils was reduced at the expense of their yield. Organosolv lignins were successfully depolymerized towards phenols and aromatic hydrocarbons via thermal and catalytic pyrolysis. Hence, lignin pyrolysis can be an effective manner for lignin upgrading towards high added value products

  • 49.
    Karnaouri, Anthi C.
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zerva, Anastasia
    Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Dimarogona, Maria
    Section of Process and Environmental Engineering, Department of Chemical Engineering, University of Patras, Patras, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts2019In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 279, p. 362-372Article in journal (Refereed)
    Abstract [en]

    Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.

  • 50.
    Karnaouri, Anthi C
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Department of Chemical Sciences and Technologies, Via della Ricerca Scientifica, University of Rome Tor Vergata.
    Lange, Heikko
    Department of Chemical Sciences and Technologies, Via della Ricerca Scientifica, University of Rome Tor Vergata.
    Crestini, Claudia
    Department of Chemical Sciences and Technologies, Via della Ricerca Scientifica, University of Rome Tor Vergata.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Chemoenzymatic Fractionation and Characterization of Pretreated Birch Outer Bark2016In: ACS Sustainable Chemistry and Engineering, ISSN 2168-0485, Vol. 4, no 10, p. 5289-5302Article in journal (Refereed)
    Abstract [en]

    In this study, the application of different chemical and enzymatic treatment methods for the fractionation of the birch outer bark components was evaluated. More specifically, untreated and steam exploded, hydrothermally and organosolv treated bark samples were incubated with enzyme mixtures that consisted of cellulases, hemicellulases and esterases, and the effect of enzymes was analyzed with 31P NMR and {13C-1H} HSQC. The biocatalysts performed the cleavage of ester bonds resulting in reduction of methoxy and aliphatic groups in the remaining solid fraction, whereas the aromatic fraction remained intact. Moreover, the suberin and lignin fraction were isolated chemically and their properties were characterized by gas chromatography (GC-MS), 31P NMR, {13C-1H} HSQC and gel permeation chromatography (GPC). It was demonstrated that the lignin fraction was enriched in guaiacyl phenolics but still contained some associated aliphatic acids and carbohydrates, whereas the suberin fraction presented a polymodal pattern of structures with different molecular weight distributions. This work will help in getting a deeper fundamental knowledge of the bark structure, the intermolecular connection between lignin and suberin fractions, as well as the potential use of enzymes in order to degrade the recalcitrant bark structure toward its valorization.

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