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1.
  • Grey, Marie, et al. (author)
  • Acceleration of α-synuclein aggregation by exosomes
  • 2015
  • In: Journal of Biological Chemistry. - 1083-351X. ; 290:5, s. 2969-2982
  • Journal article (peer-reviewed)abstract
    • Exosomes are small vesicles released from cells into extra-cellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins flotillin-1 and alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effect of exosomes derived from naive cells and cells that over-express α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry we found several phospholipid classes in the exosomes, including phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.
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2.
  • Abouhmad, Adel, et al. (author)
  • Exploring the enzymatic and antibacterial activities of novel mycobacteriophage lysin b enzymes
  • 2020
  • In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:9
  • Journal article (peer-reviewed)abstract
    • Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37◦C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.
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3.
  • Andersson, Jenny Marie, et al. (author)
  • Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract
  • 2017
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 114:18, s. 3592-3601
  • Journal article (peer-reviewed)abstract
    • The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of 1H-13C solid-state nuclear magnetic spectroscopy, small-And wide-Angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, Lα(o), at physiological temperatures. This Lα(o) phase gradually changes into a liquid-disordered lamellar phase, Lα(d), when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one Lα(d) phase. Remarkably, it was possible to measure a large set of order parameter magnitudes /SCH/ from the liquiddisordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological selfassembly compositions for treatment of pathological states such as respiratory distress syndrome.
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4.
  • Berger, Karin, et al. (author)
  • Cereal Byproducts Have Prebiotic Potential in Mice Fed a High-Fat Diet
  • 2014
  • In: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 62:32, s. 8169-8178
  • Journal article (peer-reviewed)abstract
    • Barley husks, rye bran, and a fiber residue from oat milk production were processed by heat pretreatment, various separation steps, and treatment with an endoxylanase in order to improve the prebiotic potential of these cereal byproducts. Metabolic functions were intended to improve along with improved microbial activity. The products obtained were included in a high-fat mouse diet so that all diets contained 5% dietary fiber. In addition, high-fat and low-fat controls as well as partially hydrolyzed guar gum were included in the study. The soluble fiber product obtained from rye bran caused a significant increase in the bifidobacteria (log copies of 16S rRNA genes; median (25−75 percentile): 6.38 (6.04−6.66) and 7.47 (7.30−7.74), respectively; p < 0.001) in parallel with a tendency of increased production of propionic acid and indications of improved metabolic function compared with high-fat fed control mice. The oat-derived product caused an increase in the pool of cecal propionic (from 0.62 ± 0.12 to 0.94 ± 0.08) and butyric acid (from 0.38 ± 0.04 to 0.60 ± 0.04) compared with the high-fat control, and it caused a significant increase in lactobacilli (log copies of 16S rRNA genes; median (25−75 percentile): 6.83 (6.65−7.53) and 8.04 (7.86−8.33), respectively; p < 0.01) in the cecal mucosa. However, no changes in measured metabolic parameters were observed by either oat or barley products.
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5.
  • Börner, Tim, et al. (author)
  • A Process Concept for High-Purity Production of Amines by Transaminase-Catalyzed Asymmetric Synthesis: Combining Enzyme Cascade and Membrane-Assisted ISPR
  • 2015
  • In: Organic Process Research & Development. - : American Chemical Society (ACS). - 1083-6160 .- 1520-586X. ; 19:7, s. 793-799
  • Journal article (peer-reviewed)abstract
    • For the amine transaminase (ATA)-catalyzed synthesis of chiral amines, the choice of donor substrate is of high importance for reaction and process design. Alanine was investigated as an amine donor for the reductive amination of a poorly water-soluble ketone (4-phenyl-2-butanone) in a combined in situ product removal (ISPR) approach using liquid-membrane extraction together with an enzyme cascade. This ISPR strategy facilitates very high (>98%) product purity with an integrated enrichment step and eliminates product as well as coproduct inhibition. In the presented proof-of-concept alanine shows the following advantages over the other frequently employed amine donor isopropyl amine: (i) nonextractability of alanine affords high product purity without any additional downstream step and no losses via coextraction, (ii) higher maximum reaction rates, and (iii) broader acceptance among ATAs.
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6.
  • Börner, Tim, et al. (author)
  • Explaining Operational Instability of Amine Transaminases : Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability
  • 2017
  • In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 7:2, s. 1259-1269
  • Journal article (peer-reviewed)abstract
    • The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme-cofactor intermediate. Dissociation of the enzyme-PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure-function relationship suggesting that the cofactor-ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.
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7.
  • Börner, Tim, et al. (author)
  • Generic HPLC platform for automated enzyme reaction monitoring : Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
  • 2016
  • In: Biotechnology Journal. - : Wiley. - 1860-6768. ; 11:8, s. 1025-1036
  • Journal article (peer-reviewed)abstract
    • Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.
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8.
  • Börner, Tim, et al. (author)
  • Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase
  • 2017
  • In: ChemBioChem. - : Wiley. - 1439-4227. ; 18:15, s. 1482-1486
  • Journal article (peer-reviewed)abstract
    • Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.
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9.
  • Causevic, Ariana, et al. (author)
  • Effects of Lipase Immobilization Conditions and Support Materials for the Production of Structured Triacylglycerols
  • 2023
  • In: European Journal of Lipid Science and Technology. - : Wiley. - 1438-7697 .- 1438-9312. ; 125:2
  • Journal article (peer-reviewed)abstract
    • Structured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity-based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min−1 g−1 (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF “Amano” IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct. Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties.
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10.
  • Causevic, Ariana, et al. (author)
  • Impact of critical parameters influencing enzymatic production of structured lipids using response surface methodology with water activity control
  • 2022
  • In: Biochemical Engineering Journal. - : Elsevier BV. - 1369-703X. ; 187
  • Journal article (peer-reviewed)abstract
    • Structured lipids with desired properties can be produced by enzymatic transesterification. This is a multistep reaction with many factors influencing both the product yield and quality. In this study, the single and combined effects of water activity, temperature and substrate ratio were studied on the reaction between high oleic sunflower oil and ethyl stearate producing 1,2-stearin-3-olein (SOS) using immobilized lipase from Rhizopus oryzae (Lipase DF “Amano” IM). Product quality was assessed as the ratio between SOS and the unwanted isomer SSO. Response surface methodology was used to create models at several time points, for increased understanding and showing the importance of the factors studied, for efficient production of structured lipids. To set the water activity, a control system based on dry/humid nitrogen gas sparging was developed. The models were found to adequately describe the effect of the factors on product yield and quality as well as being robust. For the yield, it was found that the factors influencing the enzymatic activity were highly important initially whereas factors affecting the thermodynamic equilibrium were dominating later. Increased temperature and water activity had initial positive influence that shifted to substrate ratio and a negative influence of water activity as equilibrium was approached. Product quality was mostly affected by temperature negatively, as an increased temperature promoted acyl migration.
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11.
  • Causevic, Ariana, et al. (author)
  • Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy
  • 2021
  • In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1652
  • Journal article (peer-reviewed)abstract
    • There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.
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12.
  • Elovson Grey, Carl, et al. (author)
  • A mass spectrometric investigation of native and oxidatively inactivated chloroperoxidase
  • 2007
  • In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:9, s. 1055-1062
  • Journal article (peer-reviewed)abstract
    • The enzyme chloroperoxidase (CPO) found in Calclariomyces fumago is able to catalyze several stereoselective oxidation reaction by using a dean oxidant, usually hydrogen peroxide (H2O2), without the need for expensive cofactor generation. CPO's lack of operational stability however, is a major limitation for its commercial use. In the present study, a capillary-LC on-line trypsin- digestion system combined with reversed-phase chromatography and mass spectrometric detection was optimized for studying the primary sequence of CPO. Samples containing native CPO, CPO treated with H2O2, and CPO oxidatively inactivated by the use of indole and H2O2 were analyzed and compared. Three oxidized peptides were found in the samples treated with H2O2. Two additional oxidized peptides were found in the CPO samples that were completely inactivated, one of which contained an oxidized cysteine residue, Cys50, which is an essential amio acid due to its function as the axial ligand to the iron in the heme - the prosthetic group in CPO. In addition, the heme group was absent in the inactivated samples but was readily detected in other samples.
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13.
  • Falck, Peter, et al. (author)
  • Production of arabinoxylan-oligosaccharide mixtures of varying composition from rye bran by combination of process conditions and type of xylanase.
  • 2014
  • In: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 174, s. 118-125
  • Journal article (peer-reviewed)abstract
    • The aim was to study arabinoxylan-oligosaccharide production from rye bran using heat pretreatment and enzymatic hydrolysis. Due to the potential application in foods, the purity of arabinoxylan was also assessed. Rye bran was heat pretreated to improve xylanase-catalyzed hydrolysis of arabinoxylan into arabinoxylan-oligosaccharides. Enzymatic removal of starch and proteins before or after heat pretreatment increased the purity, although at lower yield. The most attractive process resulted in 62% (w/w) arabinoxylan content after ethanol precipitation. Using xylanases from two glycoside hydrolase families (RmXyn10A from GH10 and Pentopan Mono BG from GH11), different mixtures of unsubstituted and arabinose-substituted xylooligosaccharides were produced. GH10 gave a higher yield of short oligosaccharides (60% w/w) with xylobiose as the main product; xylobiose and xylotriose were the main products with GH11 (40% w/w). Thus, heat pretreatment combined with enzymatic hydrolysis can be used to produce arabinoxylan-oligosaccharides from rye bran that are potentially useful in functional foods.
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14.
  • Falck, Peter, et al. (author)
  • Xylooligosaccharides from Hardwood and Cereal Xylans Produced by a Thermostable Xylanase as Carbon Sources for Lactobacillus brevis and Bifidobacterium adolescentis.
  • 2013
  • In: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 61:30, s. 7333-7340
  • Journal article (peer-reviewed)abstract
    • To compare xylans from forestry with agricultural origins, hardwood xylan (birch) and cereal arabinoxylan (rye) were hydrolyzed using two variants of the xylanase RmXyn10A, full-length enzyme and catalytic module only, from Rhodothermus marinus . Cultivations of four selected bacterial species, using the xylooligosaccharide (XOS) containing hydrolysates as carbon source, showed selective growth of Lactobacillus brevis DSMZ 1264 and Bifidobacterium adolescentis ATCC 15703. Both strains were confirmed to utilize the XOS fraction (DP 2-5), whereas putative arabinoxylooligosaccharides from the rye arabinoxylan hydrolysate were utilized by only B. adolescentis. Escherichia coli did not grow, despite its capability to grow on the monosaccharides arabinose and xylose. It was also shown that Pediococcus parvulus strain 2.6 utilized neither xylose nor XOS for growth. In summary, RmXyn10A or its catalytic module proved suitable for high-temperature hydrolysis of hardwood xylan and cereal arabinoxylan, producing XOS that could qualify as prebiotics for use in functional food products.
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15.
  • Gil-Ramirez, Alicia, et al. (author)
  • Data on saponins, xylan and cellulose yield obtained from quinoa stalks after pressurized hot water extraction
  • 2018
  • In: Data in Brief. - : Elsevier BV. - 2352-3409. ; 20, s. 289-292
  • Journal article (peer-reviewed)abstract
    • The data we present below are linked to our research paper “Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)” (Gil-Ramírez et al., 2018) [1]. The objective is to provide supplementary information in order to facilitate the comprehension of the central composite experimental design (rotatable 22) used in the integrated process of extractions. Two factors, temperature and time of extraction are considered in the design. The responses are the yield of saponin, xylan and cellulose. First, the desirable linear regression obtained by the observed vs. predicted yields plot for each variable response confirm the validation of the model (Fig. 1). Second, the data presented here through Standardized Pareto Charts (Fig. 2), provides information about the effect of the time and temperature, as well as their interactions, in the yield of saponins, xylan and cellulose obtained in an integrated sequential extraction.
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16.
  • Grey, Carl, et al. (author)
  • Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102
  • 2003
  • In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis. - 1879-2871. ; 527:1-2, s. 27-36
  • Journal article (peer-reviewed)abstract
    • An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed. Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants). No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and -tocopherol. Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested. The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism. Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations. It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA. Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals.
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17.
  • Grey, Carl, et al. (author)
  • Antiproliferative effects of sea buckthorn (Hippophae rhamnoides L.) extracts on human colon and liver cancer cell lines
  • 2010
  • In: Food Chemistry. - : Elsevier BV. - 1873-7072 .- 0308-8146. ; 120:4, s. 1004-1010
  • Journal article (peer-reviewed)abstract
    • Sea buckthorn berries contain many bioactive compounds that have anticancer properties. To investigate whether the anti proliferative effects Could be associated with the presence of certain compounds. a sequential extraction was performed. The extraction started with heptane followed by ethyl acetate, ethanol, and water. A second protocol using ethanol:water (1:1) was also used. The contents of the extracts were determined and their effects on cell proliferation were investigated in both Caco-2 and Hep G2 cells. The ethyl acetate fraction was exclusively found to contain high levels of ursolic acid, together with low amounts of phenolics. The ethanol:water extracts contained high levels of phenolic compounds and proanthyocyanidin, but little ursolic acid. When the antiproliferative effects were examined, the strongest inhibitory effect was found in the ethyl acetate extract for the Caco-2 cells and in the ethanol:water extract for the Hep G2 cells. The antiproliferative effects were in both cases dose-dependent and were in the case of the ethyl acetate extract associated with an increase in apoptosis. The results obtained show that the choice of extraction solvent is of considerable importance and that ursolic acid might be more important than the polyphenols in inhibiting the cancer cell proliferation. (C) 2009 Elsevier Ltd. All rights reserved.
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18.
  • Grey, Carl, et al. (author)
  • Development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides
  • 2009
  • In: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 877:20-21, s. 1827-1832
  • Journal article (peer-reviewed)abstract
    • In the present study a HPAEC-PAD method is described that was developed for monitoring the consistency of N-glycosylation during the production and purification of recombinant proteins and monoclonal antibodies. The method successfully separated 18 neutral and sialylated oligosaccharides. Results obtained were compared with MALDI-TOF MS and it was shown that both methods gave similar results. in addition, a method validation was performed showing that the HPAEC-PAD analysis was well suited for the mapping and characterization of oligosaccharides. The method was found to be robust and additionally the precision was significantly better compared to the MALDI-TOF MS method. 
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19.
  • Grey, Carl, et al. (author)
  • Evaluation of multiple oxidation products for monitoring effects of antioxidants in Fenton oxidation of 2 '-deoxyguanosine
  • 2006
  • In: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 54:6, s. 2350-2358
  • Journal article (peer-reviewed)abstract
    • The objective of this study was to investigate the influence of the two antioxidants, ascorbic acid and (+)catechin, on the oxidation of 2'-deoxyguanosine (dG), using an iron-mediated Fenton reaction. The oxidation products 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8,5'-cyclo-2'-deoxyguanosine, together with the secondary oxidation products guanidinohydantoin and dehydroguanidinohydantoin, were identified and quantified through the use of an LC-MS/MS system. The results obtained showed that catechin inhibited the oxidation better than ascorbic acid did, indicating that the chelating ability of catechin rather than the radical scavenging mechanism alone is vital for the observed antioxidative efficiency. The correlation between the different oxidation products was found to be quite low, primarily because of the instability of 8-oxodG, making it prone to further oxidation. This led to apparent anti- and pro-oxidative results being obtained, emphasizing the potential problems in evaluating oxidative stress, by use of a single marker.
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20.
  • Grey, Carl, et al. (author)
  • Improved operational stability of chloroperoxidase through use of antioxidants.
  • 2008
  • In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 135:2, s. 196-201
  • Journal article (peer-reviewed)abstract
    • Chloroperoxidase (CPO) from Caldariomyces fumago is a potentially very useful enzyme due to its ability to catalyze a large variety of stereoselective oxidation reactions, but poor operational stability is a main limitation for commercial use. In the present study, the possibility of increasing the operational stability by use of antioxidants was investigated using the oxidation of indole as model reaction. Caffeic acid was the antioxidant showing the strongest positive effects, reaching a total turnover number (TTN) of 135,000 at pH 4 and 4mM hydrogen peroxide, compared to 28,700 in the absence of antioxidant. Portion-wise addition of hydrogen peroxide in the presence of caffeic acid caused a further increase in TTN to 171,000. An alternative way to reach high TTN was to use tert-butyl hydroperoxide as oxidant instead of hydrogen peroxide: a TTN of 600,000 was achieved although the reaction was quite slow. In this case, antioxidants did not have any positive effect. Possible mechanisms for the observed inactivation of CPO are discussed.
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21.
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22.
  • Grey, Carl, et al. (author)
  • Process development of oxygen-demanding reactions utilizing a simple design with parallel glass tube reactors - Evaluated using Gluconobacter oxydans (DSM 24525)
  • 2012
  • In: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 30:5-6, s. 441-445
  • Journal article (peer-reviewed)abstract
    • To investigate the possibility of using simple glass tubes as reactors for oxygen-demanding reactions, a setup was assembled to study the initial rate of conversion of glycerol to dihydroxyacetone (DHA) using Gluconobacter oxydans. Several parallel 10 mL glass tubes were incubated in a temperature-controlled shaker. The concentration of DHA was determined using a fast spectrophotometric HPLC-based method that could process 3 samples/min. It was shown that the obtained results were reproducible and the reaction rates remained constant throughout the reaction. Further, the system reached a high volumetric activity of 15.48 g DHA L-1 h(-1) consuming 86 mmol L-1 h(-1) oxygen before the system became mass-transfer limited, indicating a high diffusion of oxygen. It was concluded that the reactor system is well suited for process development where the requirement for oxygen is high and that the assay developed can be used to determine the initial rate of DHA production.
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23.
  • Grey, Carl (author)
  • Protection of Biomolecules by Antioxidants - Mechanisms and Applications
  • 2006
  • Doctoral thesis (other academic/artistic)abstract
    • Reactive oxygen species (ROS) consisting of various oxygen-based free radicals as well as other reactive non-radical species produced in O2-related metabolism or through other processes are involved in the oxidation of such vital biomolecules as DNA, proteins and lipids. The types of oxidation represented here are known to cause many different diseases and disorders in human beings, such as cancer, Alzheimer's disease and ageing. In addition, oxidation by ROS causes deterioration of food and is sometimes involved in the deactivation of enzymes. A particularly important source of ROS is believed to be the reaction of hydrogen peroxide with transition metal ions, mostly iron and copper, resulting in the formation of the highly deleterious hydroxyl radical, which is able to basically oxidize any organic molecule present. Several antioxidative defence mechanisms have evolved, serving to keep the biomolecules intact and prevent them from being damaged by ROS. The action of small-molecule antioxidants having the function of reacting with free radicals so as to prevent more vital molecules from being oxidized are an important part of this defence. The thesis is based on papers dealing with research on naturally occurring small-molecule antioxidants and the oxidation of DNA and proteins. In the case of DNA, a study was performed of the mechanisms involved, in which reaction intermediates and the formation of adducts were examined, using the iron-mediated Fenton reaction to induce oxidation of the nucleoside dG. Several oxidation products were identified and quantified by use of various reaction conditions. It was found that 8-oxodG, frequently used as a marker for oxidative DNA-damage, was highly susceptible to secondary oxidation. The measured level of this adduct being highly dependent upon both the reaction time and Fenton reagent concentrations involved. Two antioxidants, the iron-chelating antioxidant catechin and the strong radical-scavenger ascorbic acid, were evaluated by use of the same reaction system. It was concluded that catechin was more effective. Again, problems regarding 8-oxodG were observed, its being clearly shown that more reliable results could be obtained when a marker of more than one type was used to evaluate the effects of the antioxidants. In another study the effects of antioxidants were evaluated using a cellular test developed based on the Ames-tester strain Salmonella typhimurium TA102. Oxidation was induced by use of either hydrogen peroxide or the organic peroxide tert-butyl hydroperoxide (tBHP). Similar to the study just referred to, chelating antioxidants, in this case phenolic quercetin and caffeic acid, were the antioxidants found to be most effective. In the protein oxidation study carried out, use was made of the commercially interesting enzyme chloroperoxidase (CPO) from Caldariomyces fumago. This enzyme is able to catalyze various stereoselective oxygen-insertion reactions useful in the synthesis of drugs, for example, a clean source of hydrogen peroxide being used as the external oxidant. The main limitation of CPO is its poor stability, caused by its oxidative inactivation by hydrogen peroxide during the catalytic cycle. It was found that the inactivation was correlated with the oxidation of a key amino acid, a cysteine residue acting as the axial ligand to the prosthetic heme group of the enzyme, along with the disappearance of the heme. Furthermore, the addition of antioxidants significantly improved its operational stability, leading in the best case to 10 times as many turnovers of CPO being observed prior to its inactivation. Again it was found that a chelating antioxidant, caffeic acid in this case, was the antioxidant inhibiting unwanted oxidation the most. One thing these rather differing oxidation studies had in common was that the chelating antioxidants were clearly superior to the antioxidants that only possessed a radical scavenging mechanism. The results indicate two things in particular, first that the Fenton reaction is highly relevant in vivo and is thus a good way of producing radicals in in vitro assays, and secondly that the chelating mechanism is an important antioxidative mechanism, one which involves the action of small-molecule antioxidants both in vitro and in vivo.
  •  
24.
  • Grey, Carl, et al. (author)
  • Time and concentration dependence of Fenton-induced oxidation of dG
  • 2006
  • In: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 25:3, s. 259-278
  • Journal article (peer-reviewed)abstract
    • The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2'-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8,5'- cyclo-2'-deoxyguanosine (8,5'cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5'cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodC a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5'cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG atone.
  •  
25.
  • Hedström, Martin, et al. (author)
  • Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes
  • 2007
  • In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1146:1, s. 17-22
  • Journal article (peer-reviewed)abstract
    • A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150 mu m) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600 nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500 nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3 mu l, salt concentration 0.2-1 M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10 min.
  •  
26.
  • Heintz, Søren, et al. (author)
  • Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations
  • 2017
  • In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592. ; 114:3, s. 600-609
  • Journal article (peer-reviewed)abstract
    • An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid–liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L−1, a purity up to 70% gMPPA · gtot −1 and a recovery in the range of 80% mol · mol−1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600–609.
  •  
27.
  • Hoffmann, Christian, et al. (author)
  • Improved Alkyl Glycoside Synthesis by trans-Glycosylation through Tailored Microenvironments of Immobilized β-Glucosidase
  • 2020
  • In: ChemPlusChem. - : Wiley. - 2192-6506. ; 85:1, s. 137-141
  • Journal article (peer-reviewed)abstract
    • We present how the microenvironment can directly improve biocatalytic selectivity of immobilized β-glucosidase. β-Glucosidase from Thermotoga neapolitana was immobilized on a variety of functionalized off-stoichiometric thiol-ene (OSTE) particles, where highest activities were observed for thiol and imidazole functional particles. Compared to the soluble enzyme, the selectivity (rs/rh) between trans-glycosylation of p-nitrophenyl β-D-glucopyranoside (pNPG) with 1-propanol over hydrolysis was increased by a factor of 2–3 using particles containing imidazole (rs/rh of 6.7) and carboxylic acid moieties (rs/rh of 9.2), respectively. These results demonstrate clearly that enzyme selectivity depends directly on the local environment of the enzyme with the support.
  •  
28.
  • Lyttkens, Carl Hampus, et al. (author)
  • Understanding the politics of Pericles around 450 BC : The benefits of an economic perspective
  • 2018
  • In: Ancient History and Contemporary Social Science. - 9781474421775 ; 9, s. 269-292
  • Conference paper (peer-reviewed)abstract
    • Pericles is usually viewed as a great statesman and clever leader of the Athenians. In the mid-fifth century BC, however, he seems to have been in serious political trouble and may well have been in danger of losing a political struggle against his opponent Kimon. The fact that his incentives changed considerably at this point in time seems to have escaped attention in the literature. In contrast, we see this fierce competition as a motivation for several important policy measures that Pericles introduced at this particular time: the pay to jurors, the new law on citizenship (which has been a puzzle to many historians), and the building projects on the Acropolis and elsewhere. An economic rational-actor approach thus provides a diachronic analytical benefit by focusing on the way in which incentives change over time and a synchronic benefit by considering various decisions in a common framework.
  •  
29.
  • Mathew, Sindhu, et al. (author)
  • Analysis of carbonyl compounds in sea buckthorn for the evaluation of triglyceride oxidation, by enzymatic hydrolysis and derivatisation methodology
  • 2011
  • In: Food Chemistry. - : Elsevier BV. - 1873-7072 .- 0308-8146. ; 126:3, s. 1399-1405
  • Journal article (peer-reviewed)abstract
    • Carbonyl compounds formed in sea buckthorn berry (Hippophae rhamnoides) and oil samples as a result of lipid oxidation were determined by enzymatic hydrolysis followed by derivatisation with 2,4-dinitrophenylhydrazine and analysed by LC-UV and electrospray ionisation (ESI) tandem mass spectrometry (MS/MS). Several classes of carbonyl compounds such as saturated, unsaturated, linear and branched aldehydes and ketones, aromatic aldehyde, dicarbonyls and carboxy aldehydes were identified based on fragmentation pattern, molecular weight and retention time. The lower carbonyls such as formaldehyde, acetaldehyde and acetone were found to be predominant in the berry samples and acetaldehyde was found to be the most abundant carbonyl. In the sea buckthorn pulp oil sample, longer aldehydes and carboxy aldehydes dominated, thus clearly demonstrating the benefit of the enzymatic step when analysing oxidation products originating from triglycerides. (C) 2010 Elsevier Ltd. All rights reserved.
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30.
  • Mladenoska, Irina, et al. (author)
  • Competition between transglycosylation and hydrolysis in almond beta-glucosidase-catalyzed conversion of p-nitrophenyl-beta-D-glucoside in monophasic water/alcohol mixtures
  • 2007
  • In: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 25:5, s. 382-385
  • Journal article (peer-reviewed)abstract
    • Almond beta-glucosidase was used to catalyze the transglycosylation of p-nitrophenyl-beta-D-glucoside to alkyl glucosides, with hydrolysis to glucose as a side reaction. The conversions were carried out in alcohols with varying water contents below water saturation. Both the total reaction rate and the ratio between the transglycosylation and hydrolysis increased with increasing water activity, and at a fixed water activity in the different alcohols, rate and transglycosylation/hydrolysis ratio increased in the following order: 1-octanol < 1-hexanol < 1-butanol. Synthesis of alkyl glucosides by transglycosylation in monophasic alcohol media is thus most favorable for short chain alcohols, and should be carried out at high water content.
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31.
  • Mohammadi, Milad, et al. (author)
  • Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base
  • 2024
  • In: Food Chemistry. - 0308-8146. ; 442
  • Journal article (peer-reviewed)abstract
    • The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.
  •  
32.
  • Mojumdar, Enamul Haque, et al. (author)
  • Self-assembly in ganglioside-phospholipid systems : The co-existence of vesicles, micelles, and discs
  • 2020
  • In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:1
  • Journal article (peer-reviewed)abstract
    • Ganglioside lipids have been associated with several physiological processes, including cell signaling. They have also been associated with amyloid aggregation in Parkinson’s and Alzheimer’s disease. In biological systems, gangliosides are present in a mix with other lipid species, and the structure and properties of these mixtures strongly depend on the proportions of the different components. Here, we study self-assembly in model mixtures composed of ganglioside GM1 and a zwitterionic phospholipid, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). We characterize the structure and molecular dynamics using a range of complementary techniques, including cryo-TEM, polarization transfer solid state NMR, diffusion NMR, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and calorimetry. The main findings are: (1) The lipid acyl chains are more rigid in mixtures containing both lipid species compared to systems that only contain one of the lipids. (2) The system containing DOPC with 10 mol % GM1 contains both vesicles and micelles. (3) At higher GM1 concentrations, the sample is more heterogenous and also contains small disc-like or rod-like structures. Such a co-existence of structures can have a strong impact on the overall properties of the lipid system, including transport, solubilization, and partitioning, which can be crucial to the understanding of the role of gangliosides in biological systems.
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33.
  • Muratovska, Nina, et al. (author)
  • Engineering Saccharomyces cerevisiae for production of the capsaicinoid nonivamide
  • 2022
  • In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21
  • Journal article (peer-reviewed)abstract
    • BackgroundCapsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities.Results Saccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L−1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time.ConclusionsBaker’s yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.
  •  
34.
  • Muratovska, Nina, et al. (author)
  • Towards engineered yeast as production platform for capsaicinoids
  • 2022
  • In: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 59
  • Research review (peer-reviewed)abstract
    • Capsaicinoids are bioactive alkaloids produced by the chili pepper fruit and are known to be the most potent agonists of the human pain receptor TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1). They are currently produced by extraction from chili pepper fruit or by chemical synthesis. Transfer of the biosynthetic route to a microbial host could enable more efficient capsaicinoid production by fermentation and may also enable the use of synthetic biology to create a diversity of new compounds with potentially improved properties. This review summarises the current state of the art on the biosynthesis of capsaicinoid precursors in baker's yeast, Saccharomyces cerevisiae, and discusses bioengineering strategies for achieving total synthesis from sugar.
  •  
35.
  • NGO, NGOC, et al. (author)
  • Chemoenzymatic synthesis of the pH responsive surfactant octyl β-D-glucopyranoside uronic acid
  • 2020
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 104, s. 1055-1062
  • Journal article (peer-reviewed)abstract
    • Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl β-D-glucopyranoside (OG) was oxidized to the corresponding uronic acid (octyl β-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product and even at a substrate concentration of 60 mM, 85 % conversion was reached within 24 hours. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5-10.0).
  •  
36.
  • Ngo, Ngoc T.N., et al. (author)
  • Efficient laccase/TEMPO oxidation of alkyl glycosides : Effects of carbohydrate group and alkyl chain length
  • 2020
  • In: Journal of Biotechnology: X. - : Elsevier BV. - 2590-1559 .- 0168-1656. ; 8
  • Journal article (peer-reviewed)abstract
    • Alkyl glycosides with long hydrophobic chains have attractive surfactant properties, but their wider application is hampered by their low solubility in water. Here, a route to increased solubility by introduction of carboxyl groups via laccase/TEMPO oxidation is presented. The oxidation pathways for dodecyl β-maltoside and hexadecyl β-maltoside were studied in detail. Close to full conversion was achieved for both substrates and conditions were found under which the diacid products dominated, with only minor amounts of under-oxidized and over-oxidized products. Dodecyl β-maltoside oxidation was improved to give a yield of 85 % of the diacid derivative. Interestingly, in spite of low substrate solubility the oxidation of hexadecyl β-maltoside was very efficient in aqueous medium, due to the higher solubility of the products. Addition of organic cosolvents did not provide additional advantages. The method is promising for producing soluble anionic derivatives of alkyl glycosides in an environmentally friendly and efficient way.
  •  
37.
  • Ngo, Ngoc T. N., et al. (author)
  • Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)‐catalysed transglycosylation
  • 2021
  • In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 118:7, s. 2548-2558
  • Journal article (peer-reviewed)abstract
    • Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/TEMPO oxidation of a long‐carbohydrate‐chain alkyl glycoside, and cyclodextrin glucanotransferase (CGTase)‐catalysed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β‐D‐maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase‐catalysed coupling/disproportionation reactions with α‐cyclodextrin and dodecyl β‐D‐maltoside diuronic acid (DDM‐2COOH) or octyl β‐D‐glucuronic acid (OG‐COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non‐ionic substrates (dodecyl β‐D‐maltoside and octyl β‐D‐glucoside), Toruzyme exhibited high catalytic activity at pH 5‐6, but for the acidic substrates (DDM‐2COOH and OG‐COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM‐2COOH and OG‐COOH, which exist at lower pH (pKa about 3).
  •  
38.
  • Norlander, Siri, et al. (author)
  • Effect of kilning on the macronutrient composition profile of three Swedish oat varieties
  • In: Cereal Chemistry. - 0009-0352.
  • Journal article (peer-reviewed)abstract
    • Background and ObjectivesKilning is crucial in oat processing, to prevent rancidity and extend shelf-life. This study examines kilning effects on the macronutrient composition in Swedish oat varieties Galant, Fatima, and Belinda. We compared two kilning methods: one mimicking industrial practice and a simplified version. We analyzed dietary fibers (arabinoxylan, β-glucan), protein and amino acids, lipid profile, lipase activity, and antioxidant capacity in these oat samples.FindingsDistinct compositional differences were found: Galant had low lipid content, Fatima had elevated lipid and protein levels with fewer carbohydrates, and Belinda was rich in β-glucan and dietary fibers. Both kilning procedures had similar impacts on all varieties, causing no major changes in dietary fiber or total protein content, but resulting in a 20% decrease in soluble proteins. Kilning decreased levels of several amino acids in Belinda, while the l-glutamate/glutamine ratio increased across all varieties. Lipid analysis showed minimal kilning-induced changes; yet, antioxidative capacity diminished. Both kilning methods effectively inactivated lipases.ConclusionsThese findings emphasize macronutrient variations among oat varieties and the effect of kilning on soluble proteins, amino acids, and antioxidative capacity.Significance and NoveltyThis study underscores the need for precise control in oat processing, crucial in plant-based protein and novel food development for optimal quality and yield.
  •  
39.
  • Norlander, Siri, et al. (author)
  • Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6, displays activity on oat fibre xylan for prebiotic production
  • 2023
  • In: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 33:6, s. 490-502
  • Journal article (peer-reviewed)abstract
    • Carbohydrate active enzymes are valuable tools in cereal processing to valorise underutilized side streams. By solubilizing hemicellulose and modifying the fibre structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibres; arabinoxylo-oligosaccharides (AXOS). The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61 °C and optimum reaction conditions were determined to 55 °C and pH 6.5 on wheat arabinoxylan (WAX). HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, while the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homologue CtXyn5A, HhXyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using WAX and alkali extracted oat bran fibres as substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modelling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX, and X5, concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer AXOS ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to lack of important ligand interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.
  •  
40.
  • Paul, Catherine, et al. (author)
  • A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.
  • 2015
  • In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 99:17, s. 7101-7113
  • Journal article (peer-reviewed)abstract
    • 4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-β-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.
  •  
41.
  • Pawar, Sudhanshu, et al. (author)
  • Biofilm formation by designed co-cultures of Caldicellulosiruptor species as a means to improve hydrogen productivity
  • 2015
  • In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 8
  • Journal article (peer-reviewed)abstract
    • Background: Caldicellulosiruptor species have gained a reputation as being among the best microorganisms to produce hydrogen (H2) due to possession of a combination of appropriate features. However, due to their low volumetric H2 productivities (QH2), Caldicellulosiruptor species cannot be considered for any viable biohydrogen production process yet. In this study, we evaluate biofilm forming potential of pure and co-cultures of Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor owensensis in continuously stirred tank reactors (CSTR) and up-flow anaerobic (UA) reactors. We also evaluate biofilms as a means to retain biomass in the reactor and its influence on QH2. Moreover, we explore the factors influencing the formation of biofilm. Results: Co-cultures of C. saccharolyticus and C. owensensis form substantially more biofilm than formed by C. owensensis alone. Biofilms improved substrate conversion in both of the reactor systems, but improved the QH2 only in the UA reactor. When grown in the presence of each other’s culture supernatant, both C. saccharolyticus and C. owensensis were positively influenced on their individual growth and H2 production. Unlike the CSTR, UA reactors allowed retention of C. saccharolyticus and C. owensensis when subjected to very high substrate loading rates. In the UA reactor, maximum QH2 (approximately 20 mmol · L−1 · h−1) was obtained only with granular sludge as the carrier material. In the CSTR, stirring negatively affected biofilm formation. Whereas, a clear correlation was observed between elevated (>40 μM) intracellular levels of the secondary messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and biofilm formation. Conclusions: In co-cultures C. saccharolyticus fortified the trade of biofilm formation by C. owensensis, which was mediated by elevated levels of c-di-GMP in C. owensensis. These biofilms were effective in retaining biomass of both species in the reactor and improving QH2 in a UA reactor using granular sludge as the carrier material. This concept forms a basis for further optimizing the QH2 at laboratory scale and beyond.
  •  
42.
  • Ramirez, Alicia Gil, et al. (author)
  • Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)
  • 2018
  • In: Industrial Crops and Products. - : Elsevier BV. - 0926-6690. ; 121, s. 54-65
  • Journal article (peer-reviewed)abstract
    • World quinoa production is increasing due its high nutritional value. As a consequence, large quantities of stalks accumulate as unused byproducts. Here, we verify the presence of saponins in the stalks and present a biorefinery approach with quinoa stalks as feedstock, using an integrated processing scheme to separate saponins, xylan and cellulose. Saponins were extracted using pressurized hot water extraction (PHWE), optimized by a central composite experimental design (rotatable 22) with temperature and extraction time as factors. Xylan was extracted from the residual solid material after PHWE by an alkaline method using 0.5 M NaOH at 80 °C. Cellulose was purified from the remaining residuals using acetic and nitric acid at 120 °C, which resulted in recovery of white cotton-like cellulose, showing no need of further bleaching. The saponin yield was significantly increased at temperatures exceeding 110 °C, with highest amounts obtained at 195 °C (15.4 mg/g raw material). The yield in the following xylan extraction (maximum 120 mg/g raw material) was however significantly reduced when preceded by PHWE above 110 °C, indicating degradation of the polymer. Cellulose recovery (maximum 296 mg/g raw material) was less affected by variations in temperature and time in the preceding PHWE. The results obtained shows that tuning between saponin and xylan extraction is critical. This approach is foreseen to be applicable to the valorisation of residual fiber-rich biomass from various types of crops, besides quinoa.
  •  
43.
  • Rehn, Gustav, et al. (author)
  • Activity and stability of different immobilized preparations of recombinant E. coli cells containing omega-transaminase
  • 2012
  • In: Process Biochemistry. - : Elsevier BV. - 1873-3298 .- 1359-5113. ; 47:7, s. 1129-1134
  • Journal article (peer-reviewed)abstract
    • Production of chiral amines using omega-transaminases has been thoroughly studied in recent years. Immobilized w-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide. Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coil cells. The enzyme expressed was a modified Arthrobacter citreus omega-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone. The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2 g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4'-cyano-(alpha)-methylbenzylamine in three repeated bathes (cycle time >20 h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale. (c) 2012 Elsevier Ltd. All rights reserved.
  •  
44.
  • Rehn, Gustav, et al. (author)
  • An improved process for biocatalytic asymmetric amine synthesis by in situ product removal using a supported liquid membrane
  • 2016
  • In: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1381-1177. ; 123, s. 1-7
  • Journal article (peer-reviewed)abstract
    • Chiral amines are important building blocks in the pharmaceutical industry, and the biocatalytic synthesis of these compounds using ω-transaminases has been increasingly studied in recent years. In principal, asymmetric synthesis of chiral amines from a prochiral ketone is the preferable route, but it is often hampered by an unfavourable equilibrium position and product inhibition. An effective method for product removal is therefore necessary to drive the reaction towards product formation. In a recent study (Rehn et al., 2014) [29] we reported on the successful use of a supported liquid membrane (SLM) for the in situ product removal (ISPR) of (S)-α-methylbenzylamine (MBA) produced by Arthrobacter citreus ω-transaminase present in immobilized Escherichia coli cells. In the present work, we thoroughly discuss the factors influencing the performance of the SLM system and considerations for its successful use. Moreover, the system is further improved by implementing continuous control of the reactor pH using the amine donor substrate, and regeneration of the SLM unit at regular intervals to maintain the extraction performance, allowing the accumulation of 1.0 M (121 g/l) product in the stripping phase during operation for 91 h.
  •  
45.
  • Rehn, Gustav, et al. (author)
  • Chitosan flocculation: An effective method for immobilization of E. coli for biocatalytic processes.
  • 2013
  • In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 165:2, s. 138-144
  • Journal article (peer-reviewed)abstract
    • Immobilization of Escherichia coli cells containing a ω-transaminase was carried out by flocculation with chitosan and the preparation was used in asymmetric synthesis of (S)-4'-cyano-α-methylbenzylamine, recycled in five consecutive batches. Chitosans with different molecular weights and degrees of acetylation were compared and effects of varying the chitosan properties, cell concentration and ratio of cells to chitosan were studied. Immobilization was achieved by increasing the pH to slightly alkaline, which induced the formation of large fast sedimenting flocs. Although an effective immobilization was obtained using most types of chitosan, high molecular weight and low degree of acetylation were considered favourable properties, resulting in good floc stability and quick sedimentation. It was found that it was possible to affect the floc characteristics, by changing the ratio of cells to chitosan in such a way that preparations resembling either entrapped or cross-linked cells could be obtained. The volume of the sedimented preparation decreased approximately 50% when increasing the cell to chitosan ratio from 2g/g to 10g/g at a constant amount of cells. Despite very high concentrations of cells (10-100g cells/g chitosan) in the flocculated preparations, diffusion limitations were minimal. Flocculation with chitosan was considered a simple and effective method for immobilization of E. coli cells for biocatalytic processes.
  •  
46.
  • Rehn, Gustav, et al. (author)
  • Supported liquid membrane as a novel tool for driving the equilibrium of ω-transaminase catalyzed asymmetric synthesis.
  • 2014
  • In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 179, s. 50-55
  • Journal article (peer-reviewed)abstract
    • An attractive option to produce chiral amines of industrial importance is through asymmetric synthesis using ω-transaminase. However, reaching high yields often requires a strategy for shifting the equilibrium position. This paper describes a novel strategy for handling this problem. It involves the use of a supported liquid membrane (SLM) together with a packed bed reactor. The reactor contains Escherichia coli cells with ω-transaminase from Arthrobacter citreus, immobilized by flocculation with chitosan. The SLM consists of a hollow fibre membrane contactor in which the pores contain undecane. The system enables continuous extraction of the amine product and was used to successfully shift the equilibrium in asymmetric synthesis of (S)-α-methylbenzylamine (MBA). A conversion of 98% was reached, compared to 50% without product extraction. Moreover, a selective extraction of the produced MBA was realized. A high product concentration of 55g/l was reached after 80h, and the system showed promising potential for continuous operation.
  •  
47.
  • Sajib, Mursalin, 1987, et al. (author)
  • Valorization of Brewer's spent grain to prebiotic oligosaccharide: Production, xylanase catalyzed hydrolysis, in-vitro evaluation with probiotic strains and in a batch human fecal fermentation model
  • 2018
  • In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 268, s. 61-70
  • Journal article (peer-reviewed)abstract
    • Brewer's spent grain (BSG) accounts for around 85% of the solid by-products from beer production. BSG was first extracted to obtain water-soluble arabinoxylan (AX). Using subsequent alkali extraction (0.5 M KOH) it was possible to dissolve additional AX. In total, about 57% of the AX in BSG was extracted with the purity of 45–55%. After comparison of nine xylanases, Pentopan mono BG, a GH11 enzyme, was selected for hydrolysis of the extracts to oligosaccharides with minimal formation of monosaccharides. Growth of Bifidobacterium adolescentis (ATCC 15703) was promoted by the enzymatic hydrolysis to arabinoxylooligosaccharides, while Lactobacillus brevis (DSMZ 1264) utilized only unsubstituted xylooligosaccharides. Furthermore, utilization of the hydrolysates by human gut microbiota was also assessed in a batch human fecal fermentation model. Results revealed that the rates of fermentation of the BSG hydrolysates by human gut microbiota were similar to that of commercial prebiotic fructooligosaccharides, while inulin was fermented at a slower rate. In summary, a sustainable process to valorize BSG to functional food ingredients has been proposed.
  •  
48.
  • Shanker, Shiva, et al. (author)
  • Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and α-lipoic acid.
  • 2012
  • In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 157:2, s. 344-349
  • Journal article (peer-reviewed)abstract
    • Chemo-enzymatic synthesis of six esters of natural phenolics and α-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alpha-tocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants.
  •  
49.
  • Van Daele, Timothy, et al. (author)
  • Application of iterative robust model-based optimal experimental design for the calibration of biocatalytic models
  • 2017
  • In: Biotechnology Progress. - : Wiley. - 8756-7938. ; 33:5, s. 1278-1293
  • Journal article (peer-reviewed)abstract
    • The aim of model calibration is to estimate unique parameter values from available experimental data, here applied to a biocatalytic process. The traditional approach of first gathering data followed by performing a model calibration is inefficient, since the information gathered during experimentation is not actively used to optimize the experimental design. By applying an iterative robust model-based optimal experimental design, the limited amount of data collected is used to design additional informative experiments. The algorithm is used here to calibrate the initial reaction rate of an ω-transaminase catalyzed reaction in a more accurate way. The parameter confidence region estimated from the Fisher Information Matrix is compared with the likelihood confidence region, which is not only more accurate but also a computationally more expensive method. As a result, an important deviation between both approaches is found, confirming that linearization methods should be applied with care for nonlinear models.
  •  
50.
  • Vongkampang, Thitiwut, et al. (author)
  • Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production
  • 2021
  • In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14
  • Journal article (peer-reviewed)abstract
    • Background: Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose).Results: Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax=0.57 h−1) as on glucose (μmax=0.28 h−1). A study of the sugar uptake was made with diferent glucose–xylose ratios to fnd a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu=0.01 cmol L−1 and KI,xyl=0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar.Conclusions: C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specifc survival edge in thermophilic lignocellulosic degradation communities.
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