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  • 1.
    Ayan, Hilal
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Identify synthetic polymers used in cosmetics and further test their biodegradation in aqueous setup in order to assess their impact on the environment2017Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
    Abstract [en]

    Plastics have a wide application field, where cosmetic products are one of them. Polymers which are building blocks to plastics exists in many variants, overall they can be categorized into two groups; microplastics and water soluble polymers. Both polymer types are important to study and understand since polymers in general are not covered by any legislation. To gain a more profound understanding of their impact on environment this study was conducted. In collaboration with SSNC (Naturskyddsföreningen), a database containing hundreds cosmetic products was processed. The most occurring polymers were quantified and prevalent ingredients having “poly” in their name were selected for further investigation namely Nylon 12-20 (microplast) and Acrylates C/10-30 Alkyl-crosspolymer (water soluble). A standardized analysis method OECD 301 F was performed to test the polymers biodegrading ability. Results from biodegradation method showed that, neither of the two polymers is readily biodegradable in aqueous environment, despite their different properties. In connection with the obtained results, a filtration analysis was performed, with the purpose to determine the possibility to capture the polymers using microfilters. Results mainly showed flowthrough of both polymers. Relating the results to reality implies that these polymers are not captured in waste water treatment plant due to inefficient filtration and thereby spread to the environment. In addition more research should be devoted to water-soluble polymers and their impact on nature. Based on all compiled results, it is proposed that legislation addressing microplastics should be edited and revised in such a way that water soluble polymers are included in future prohibitions (against microplastics).

  • 2.
    Azeem, Muhammad
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi. Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan.
    Barba Aliaga, Marina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Borg-Karlson, Anna-Karin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Organisk kemi. Division of Organic Chemistry, Institute of Technology, Tartu University, Tartu 50411, Estonia.
    Terenius, O.
    Broberg, A.
    Rajarao, Gunaratna Kuttuva
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides2019Inngår i: Microbiology Research, ISSN 0944-5013, E-ISSN 1618-0623, Vol. 223-225, s. 129-136Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

  • 3.
    Björlenius, Berndt
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Pharmaceuticals – improved removal from municipal wastewater and their occurrence in the Baltic Sea2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Pharmaceutical residues are found in the environment due to extensive use in human and veterinary medicine. The active pharmaceutical ingredients (APIs) have a potential impact in non-target organisms. Municipal wastewater treatment plants (WWTPs) are not designed to remove APIs.

    In this thesis, two related matters are addressed 1) evaluation of advanced treatment to remove APIs from municipal wastewater and 2) the prevalence and degradation of APIs in the Baltic Sea.

    A stationary pilot plant with nanofiltration (NF) and a mobile pilot plant with activated carbon and ozonation were designed to study the removal of APIs at four WWTPs. By NF, removal reached 90%, but the retentate needed further treatment. A predictive model of the rejection of APIs by NF was developed based on the variables: polarizability, globularity, ratio hydrophobic to polar water accessible surface and charge. The pilot plants with granular and powdered activated carbon (GAC) and (PAC) removed more than 95% of the APIs. Screening of activated carbon products was essential, because of a broad variation in adsorption capacity. Recirculation of PAC or longer contact time, increased the removal of APIs. Ozonation with 5-7 g/m3 ozone resulted in 87-95% removal of APIs. Elevated activity and transcription of biomarkers indicated presence of xenobiotics in regular effluent. Chemical analysis of APIs, together with analysis of biomarkers, were valuable and showed that GAC-filtration and ozonation can be implemented to remove APIs in WWTPs, with decreased biomarker responses.

    Sampling of the Baltic Sea showed presence of APIs in 41 out of 43 locations. A developed grey box model predicted concentration and half-life of carbamazepine in the Baltic Sea to be 1.8 ng/L and 1300 d respectively.

    In conclusion, APIs were removed to 95% by GAC or PAC treatment. The additional treatment resulted in lower biomarker responses than today and some APIs were shown to be widespread in the aquatic environment.

  • 4.
    Björlenius, Berndt
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Ripszám, M.
    Haglund, P.
    Lindberg, R. H.
    Tysklind, M.
    Fick, J.
    Pharmaceutical residues are widespread in Baltic Sea coastal and offshore waters – Screening for pharmaceuticals and modelling of environmental concentrations of carbamazepine2018Inngår i: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 633, s. 1496-1509Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The consumption of pharmaceuticals worldwide coupled with modest removal efficiencies of sewage treatment plants have resulted in the presence of pharmaceuticals in aquatic systems globally. In this study, we investigated the environmental concentrations of a selection of 93 pharmaceuticals in 43 locations in the Baltic Sea and Skagerrak. The Baltic Sea is vulnerable to anthropogenic activities due to a long turnover time and a sensitive ecosystem in the brackish water. Thirty-nine of 93 pharmaceuticals were detected in at least one sample, with concentrations ranging between 0.01 and 80 ng/L. One of the pharmaceuticals investigated, the anti-epileptic drug carbamazepine, was widespread in coastal and offshore seawaters (present in 37 of 43 samples). In order to predict concentrations of pharmaceuticals in the sub-basins of the Baltic Sea, a mass balance-based grey box model was set up and the persistent, widely used carbamazepine was selected as the model substance. The model was based on hydrological and meteorological sub-basin characteristics, removal data from smaller watersheds and wastewater treatment plants, and statistics relating to population, consumption and excretion rate of carbamazepine in humans. The grey box model predicted average environmental concentrations of carbamazepine in sub-basins with no significant difference from the measured concentrations, amounting to 0.57-3.2 ng/L depending on sub-basin location. In the Baltic Sea, the removal rate of carbamazepine in seawater was estimated to be 6.2 10(-9) s(-1) based on a calculated half-life time of 3.5 years at 10 degrees C, which demonstrates the long response time of the environment to measures phasing out persistent or slowly degradable substances such as carbamazepine. Sampling, analysis and grey box modelling were all valuable in describing the presence and removal of carbamazepine in the Baltic Sea.

  • 5. Bracher, J. M.
    et al.
    Verhoeven, M. D.
    Wisselink, H. W.
    Crimi, B.
    Nijland, J. G.
    Driessen, A. J. M.
    Klaassen, P.
    Van Maris, Antonius J.A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. Delft University of Technology, Netherlands.
    Daran, J. -MG.
    Pronk, J. T.
    The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae2018Inngår i: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, nr 1, artikkel-id 63Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

  • 6. Bracher, Jasmine M.
    et al.
    Martinez-Rodriguez, Oscar A.
    Dekker, Wijb JC
    Verhoeven, Maarten D.
    van Maris, Antonius
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Pronk, Jack T.
    Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains2019Inngår i: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 19, nr 1, artikkel-id foy104Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N-2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.

  • 7.
    Brechmann, Nils Arnold
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Eriksson, Per-Olov
    Eriksson, Kristofer
    Oscarsson, Sven
    Buijs, Jos
    Shokri, Atefeh
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Hjälm, Göran
    Chotteau, Véronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture2019Inngår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development

    of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified

    CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding

    capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an

    IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step

    from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were

    obtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scale

    purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,

    where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein

    A capture step, the mAb purity was similar to the one obtained by column chromatography, while

    the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead

    mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient

    single step, which directly connected the culture to the downstream process without cell clarification.

    Purification of mAb directly from non-clarified cell broth without cell separation can provide

    significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

  • 8.
    Brännström, Sara
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Finnveden, Maja
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Johansson, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malmström, Eva
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Itaconate based polyesters: Selectivity and performance of esterification catalysts2018Inngår i: European Polymer Journal, ISSN 0014-3057, E-ISSN 1873-1945, Vol. 103, s. 370-377Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The performance of different esterification catalysts was studied for the use in synthesis of renewable polyesters from dimethyl itaconate (DMI), dimethyl succinate (DMS) and 1,4-butanediol (BD). Itaconic acid and derivatives such as DMI are interesting monomers because of their multiple functionalities and previous work has shown great potential. However, the multiple functionalities also pose challenges to avoid side reactions such as thermally initiated, premature, radical crosslinking and/or isomerization of the 1,1-disubstituted unsaturation. Additionally, the two carboxylic acids have inherently different reactivity. One key factor to control reactions with IA is to understand the performance of different catalysts. In this study, six esterification catalysts were investigated; immobilized Candida antarctica lipase B (CalB), titanium(IV)butoxide (Ti(OBu)4), p-toluenesulfonic acid (pTSA), sulfuric acid (H2SO4), 1,8-diazabicycloundec-7-ene (DBU), and 1,5,7-triazabicyclodec-5-ene (TBD). CalB and Ti(OBu)4 were selected for further characterization with appreciable differences in catalytic activity and selectivity towards DMI. CalB was the most effective catalysts and was applied at 60 °C while Ti(OBu)4 required 160 °C for a reasonable reaction rate. CalB was selective towards DMS and the non-conjugated side of DMI, resulting in polyesters with itaconate-residues mainly located at the chain ends, while Ti(OBu)4 showed low selectivity, resulting in polyesters with more randomly incorporated itaconate units. Thermal analysis of the polyesters showed that the CalB-catalyzed polyesters were semi-crystalline, whereas the Ti(OBu)4-catalyzed polyesters were amorphous, affirming the difference in monomer sequence. The polyester resins were crosslinked by UV-initiated free radical polymerization and the material properties were evaluated and showed that the crosslinked materials had similar material properties. The films from the polyester resins catalyzed by CalB were furthermore completely free from discoloration whereas the film made from the polyester resins catalyzed with Ti(OBu)4 had a yellow color, caused by the catalyst. Thus, it has been shown that CalB can be used to attain sustainable unsaturated polyesters resins for coating applications, exhibiting equally good properties as resins obtained from traditional metal-catalysis.

  • 9.
    Brännström, Sara
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Finnveden, Maja
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Razza, Nicolo
    Politecn Torino, Dept Appl Sci & Technol, Corso Duca Abruzzi 24, I-10129 Turin, Italy..
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malmström, Eva
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Sangermano, Marco
    Politecn Torino, Dept Appl Sci & Technol, Corso Duca Abruzzi 24, I-10129 Turin, Italy..
    Johansson, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Tailoring Thermo-Mechanical Properties of Cationically UV-Cured Systems by a Rational Design of Vinyl Ether Ester Oligomers using Enzyme Catalysis2018Inngår i: Macromolecular Chemistry and Physics, ISSN 1022-1352, E-ISSN 1521-3935, Vol. 219, nr 21, artikkel-id 1800335Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There is a demand for new sustainable polymeric materials. Vinyl ethers are, in this context, attractive oligomers since they polymerize fast, are non-toxic, and can be polymerized under ambient conditions. The availability of vinyl ether oligomers is, however, currently limited due to difficulties in synthesizing them without using tedious synthesis routes. This work presents the synthesis of a series of vinyl ether ester oligomers using enzyme catalysis under solvent-free conditions and the subsequent photoinduced cationic polymerization to form polymer thermosets with T(g)s ranging from -10 to 100 degrees C. The whole process is very efficient as the synthesis takes less than 1 h with no need for purification and the crosslinking is complete within 2 min.

  • 10.
    Chen, Shan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Stability and inactivation mechanisms of two transaminases2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In the past decades, more and more enzymes are employed as biocatalysts in industrial processes because of their advantages, such as high efficiency, substrate selectivity and stereoselectivity. Among them, amine transaminases (ATAs) are pyridoxal 5’-phosphate (PLP) dependent enzymes. ATAs have gained attention for their excellent performance in chiral amine synthesis, and their broad substrate acceptance. However, the low operational stability of amine transaminases still limits their application in industry.

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) has been selected for further investigation for its relatively low operational stability. Co-solvents and various additives have been added to the enzyme storage solution to improve its storage stability at various temperatures. Co-lyophilization of Cv-ATA with surfactants has been applied to improve its enzymatic activity in neat organic solvents.

    As a PLP-dependent dimeric enzyme, the Cv-ATA is not primarily inactivated due to tertiary structural changes. Instead, both dimer dissociation and PLP release may affect the enzyme stability. Therefore, the inactivation pathway of the Cv-ATA during operational conditions was explored. The unfolding of the enzyme was detected by several methods, and the detection of fluorescence intensity spectrum of tryptophan is extensively applied for its high sensitivity. The phosphate group of PLP can be coordinated into the phosphate group binding cup, which may influence the enzyme structural stability. Therefore, the effect of both PLP and inorganic phosphate ions (present in phosphate buffer) on the enzyme stability was explored.

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is another amine transaminase, which catalyses the same biocatalytic reaction and has a similar substrate scope as Cv-ATA. However, there is still a lack of data on the stability of Vf-ATA. Consequently, the operational stability of Vf-ATA in various environments was studied.

  • 11.
    Chen, Shan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Svedendahl Humble, Maria
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum2018Inngår i: Molecular Catalysis, ISSN 2468-8231, Vol. 446, s. 115-123Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5’-phosphate (PLP)dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer.The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

  • 12.
    Chen, Shan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Svedendahl, Maria
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Characterization of the operational stability of a transaminase from Vibrio fluvialisManuskript (preprint) (Annet vitenskapelig)
  • 13.
    Chen, Shan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Svedendahl, Maria
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Inactivation pathway underlying the operational instability of an amine transaminase from Chromobacterium violaceumManuskript (preprint) (Annet vitenskapelig)
  • 14.
    Chen, Shan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Campillo-Brocal, Jonatan C.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Svedendahl Humble, Maria
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Characterization of the stability of Vibrio fluvialis JS17 amine transaminase2018Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 282, s. 10-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.

  • 15.
    Dorau, Robin
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Gorbe, Tamas
    Humble, Maria Svedendahl
    Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering2018Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, nr 4, s. 338-346Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36%), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

  • 16.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO).
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009Inngår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, nr 4, s. 1029-1036Artikkel i tidsskrift (Fagfellevurdert)
  • 17.
    Feng, Zhaoxuan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Odelius, Karin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Rajarao, Gunaratna Kuttuva
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hakkarainen, Minna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Polymerteknologi.
    Microwave carbonized cellulose for trace pharmaceutical adsorption2018Inngår i: Chemical Engineering Journal, ISSN 1385-8947, E-ISSN 1873-3212, Vol. 346, s. 557-566Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A promising sustainable strategy to valorize cellulose to high-value adsorbents for trace pharmaceuticals, like diclofenac sodium (DCF), in the water is demonstrated. Carbon nanospheres (CN) as the DCF adsorbent were derived from cellulose through a one-pot microwave-assisted hydrothermal carbonization method. CN exhibited efficient DCF removal (100% removal of 0.001 mg/mL DCF in 30 s and 59% removal of 0.01 mg/mL DCF in 1 h). The adsorption kinetics and isotherm data were well-fitted with the pseudo-second-order kinetic model and Langmuir model, respectively. The adsorption process was endothermic and spontaneous as confirmed by the thermodynamic parameters. Multiple characterization techniques including SEM/EDS, FTIR, FTIR-imaging and zeta potential were applied to qualitatively investigate the adsorption process. π-π stacking and hydrogen bonding were proposed as the dominant adsorption interactions. CN also demonstrated effective adsorption capacity towards three other commonly-detected contaminants in the wastewater including ketoprofen (KP), benzophenone (BZP), and diphenylamine (DPA), each bearing partial structural similarity with DCF. The affinity of the contaminants towards CN followed the order DPA > BZP > DCF > KP, which could be explained by the different configurations and chemical units. It was speculated that for DCF and KP, the steric hindrance and electrostatic repulsion produced by dissociated carboxyl groups can impede the adsorption process as compared to DPA and BZP. This methodology could offer further insights into the drug adsorption on the cellulose-derived carbon adsorbents and the use of bioderived carbons for treatment of wastewaters contaminated with pharmaceuticals.

  • 18. Fernández-Niño, Miguel
    et al.
    Pulido, Sergio
    Stefanoska, Despina
    Pérez, Camilo
    González-Ramos, Daniel
    van Maris, Antonius JA
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Marchal, Kathleen
    Nevoigt, Elke
    Swinnen, Steve
    Identification of novel genes involved in acetic acid tolerance of Saccharomyces cerevisiae using pooled-segregant RNA sequencing2018Inngår i: FEMS yeast research, Vol. 18, nr 8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN. PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.

  • 19.
    Finnveden, Maja
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Enzyme catalysis towards bio-based UV-curable buildingblocks2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Polymeric materials are found in virtually all areas of daily life; they are found in everything from packages keeping our food safe to the buildings where we spend our days, and the production is a worldwide industry. Although polymeric materials play a big part in sustainable solution’s, a lot can be done to develop more environmental methods for producing them. Both the process conditions and the resources that go in are important to consider. As more people understand that we need to manage our planet’s resources and ecosystem differently the demand for sustainable materials is increasing.

    Catalysis is a key for designing chemistry for the environment and an interesting alternative is enzyme catalysis. Enzymes are proteins working as catalysts in biochemical reactions. One of the most prominent features of enzymes’ is their selectivity, which means that they have preferences towards forming one product over others. Using enzymes’ as catalysts in synthetic chemical reactions the selectivity can be used to produce a wide range of products without side reaction occurring. Further benefits of using enzyme catalysis include high rate acceleration and working under mild reaction conditions.

    In the work presented here the selectivity and efficiency of enzymes have been combined with photochemistry in new efficient methods for the synthesis ofpolymeric materials. The enzymes used were the well-known lipase B form Candida antarctica and an esterase/acyltransferase from Mycobacterium smegmatis.

    The thesis divides into three parts in which three kinds of components were synthesized by enzyme catalysis: (i) unsaturated polyesters; (ii) vinyl ether building-blocks; and (iii) bio-based polyamides. In the first two parts the efficiency and selectivity of enzyme catalysis at low temperatures were utilized to synthesize building-blocks that can be further used for photopolymerization. By using enzyme catalysis structures that can be difficult or even impossible to access with conventional chemistry have been made. In part (iii) photochemistry was used to synthesize a monomer that was polymerized by enzyme catalysis to produce polyamides.

    All three parts presented in this thesis show the potential of the combination of enzymes and photochemistry to give access to polymeric materials under benign conditions. The work thus advances the capacity to manufacture building-blocks to create new sustainable polymeric materials.

  • 20.
    Finnveden, Maja
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Brännström, Sara
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Johansson, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Malmström, Eva
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Novel sustainable synthesis of vinyl ether ester building blocks, directly from carboxylic acids and the corresponding hydroxyl vinyl ether, and their photopolymerization2018Inngår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 8, nr 44, s. 24716-24723Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Increased environmental awareness has led to a demand for sustainable, bio-based materials. Consequently, the development of new benign synthesis pathways utilizing a minimum of reaction steps and available bio-based building blocks is needed. In the present study, vinyl ether alcohols and functional carboxylic acids were used to synthesize bifunctional vinyl ether esters using the immobilized enzyme Candida antarctica lipase B as a catalyst. Vinyl ethers are attractive alternatives to (meth)acrylates due to low allergenic hazards, low toxicity, and fast polymerization; however, difficult synthesis limits the monomer availability. The synthesis was performed in one-pot and the described method was successful within a broad temperature range (22-90 degrees C) and in various organic solvents as well as in the bulk. The synthesis of different vinyl ether esters reached high conversions (above 90%) after less than 1 h and products were purified by removing the enzyme by filtration using only small amounts of acetone. This approach is a straightforward route to reach monomers with multiple types of functionalities that can be used as different photo-curable thermoset resins. In this work, this was demonstrated by polymerizing the monomers with cationic and radical UV-polymerization. By changing the functional carboxylic acids, the architecture of the final polymer can be tailored, herein demonstrated by two examples. In the developed versatile method, carboxylic acids can be used directly as acyl donors, constituting a more sustainable alternative to the carboxylic acid derivatives used today.

  • 21.
    Finnveden, Maja
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. KTH Royal Institute of Technology.
    Hendil-Forssell, Peter
    Claudino, Mauro
    Johansson, Mats
    KTH, Tidigare Institutioner (före 2005), Fiber- och polymerteknologi.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Lipase Catalyzed Synthesis of renewable plant oil-based polyamidesManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Enzyme catalyzed synthesis towards renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality was homopolymerized at 80 and 140°C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1200 times lower showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB type monomer was copolymerized with 1,12-diaminododecan to synthesize oligoamides of two different lengths.

  • 22.
    Finnveden, Maja
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hendil-Forssell, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Claudino, Mauro
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Johansson, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Lipase-Catalyzed Synthesis of Renewable Plant Oil-Based Polyamides.2019Inngår i: Polymers, ISSN 2073-4360, E-ISSN 2073-4360, Vol. 11, nr 11, artikkel-id E1730Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Enzyme catalyzed synthesis of renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality, was homopolymerized at 80 and 140 °C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as a catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1.2 × 103 times lower, showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB-type monomer was copolymerized with 1,12-diaminododecane to synthesize oligoamides of two different lengths.

  • 23.
    Finnveden, Maja
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Semlitsch, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    He, Oscar
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Mono-substitution of symmetric diesters: selectivity of Mycobacterium smegmatis acyltransferase variants2019Inngår i: Catalysis Science & Technology, ISSN 2044-4753, E-ISSN 2044-4761Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A method for selectively reacting one, out of two identical carboxylic esters in a symmetric diester has been developed. An esterase from Mycobacterium smegmatis (MsAcT) has a restricted active site resulting in a narrow acyl donor specificity. This constraint was used to develop a selective synthesis route from divinyl adipate (a symmetric diester) towards mixed vinyl adipate esters. To find a suitable catalyst, the wild type (wt) MsAcT and two MsAcT variants: a single point mutant (L12A) and a double point mutant (T93A/F154A), were immobilized and studied under solvent-free conditions. Out of the tested catalysts, MsAcT L12A was the most selective for mono-transesterification of divinyl adipate. When divinyl adipate was reacted with 1.5 equivalents of a hydroxyl vinyl ether full conversion of DVA was observed yielding over 95% mixed diester. Furthermore, the limitations for longer dicarboxylic esters were studied, showing that MsAcT T93A/F154A tolerated up to at least dimethyl sebacate.

  • 24.
    Fons, Joaquin Gomis
    et al.
    Lund Univ, Chem Engn, Lund, Sweden..
    Andersson, Niklas
    Lund Univ, Chem Engn, Lund, Sweden..
    Nilsson, Bernt
    Lund Univ, Chem Engn, Lund, Sweden..
    Schwarz, Hubert
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Chotteau, Véronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Small scale end-to-end mAb platform with a continuous, integrated and compact process2019Inngår i: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 257Artikkel i tidsskrift (Annet vitenskapelig)
  • 25.
    Guevara-Martínez, Mónica
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Perez-Zabaleta, Mariel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Gustavsson, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Quillaguamán, Jorge
    Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Larsson, Gen
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli2019Inngår i: Applied Microbiology and Biotechnology, s. 1-12Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

  • 26.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010Inngår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, nr 3, s. 785-789Artikkel i tidsskrift (Fagfellevurdert)
  • 27.
    Hagrot, Erika
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Macroscopic models of Chinese hamster ovary cell cultures2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Biopharmaceuticals treat a range of diseases, and is a growing sector within the pharmaceutical industry. A majority of these complex molecules are produced by genetically modified mammalian cells in large-scale cell cultures. Biopharmaceutical process development is costly and labor intensive, and has often been based on time-consuming empirical methods and trial-and-error. Mathematical modeling has great potential to speed up this work. A central question however, engaging researchers from various fields, is how to translate these complex biological systems into feasible and useful models.

    For biopharmaceutical production, macroscopic kinetic flux modeling has been proposed. This model type is derived from typical data obtained in the industry, and has been able to simulate cell growth and the uptake/secretion of important metabolites. Often, however, their scope is limited to specific culture conditions due to e.g. the lack of information on reaction kinetics, limited data sets, and simplifications to achieve calculability.

    In this thesis, the macroscopic kinetic model type is the starting point, but the goal is to capture a variety of culture conditions, as will be necessary for future applications in process optimization. The effects of varied availability of amino acids in the culture medium on cell growth, uptake/secretion of metabolites, and product secretion were studied in cell cultures.

    In Paper I, the established methodology of Metatool was tested: (i) a simplified metabolic network of approx. 30 reactions was defined; (ii) all possible so-called elementary flux modes (EFMs) through the network were identified using an established mathematical algorithm; and (iii) the effect on each flux was modelled by a simplified generalized kinetic equation. A limitation was identified; the Metatool algorithm could only handle simple networks, and therefore several reactions had to be discarded. In this paper, a new strategy for the kinetics was developed. A pool of alternative kinetic equations was created, from which a smaller set could be given higher weight as determined via data-fitting. This improved the simulations.

    The identification of EFMs was further studied in papers II–IV. In Paper II, a new algorithm was developed based on the column generation optimization technique, that in addition to the network also accounts for the data from one of the parallel cultures. The method identifies a subset of the EFMs that can optimally fit the data, even in more complex metabolic networks.

    In Paper III, a kinetic model based on EFM subsets in a 100 reaction network was generated, which further improved the simulations. Finally, in Paper IV, the algorithm was extended to EFM identification in a genome-scale network. Despite the high complexity, small subsets of EFMs relevant to the experimental data could be e ciently identified.

  • 28.
    Hagrot, Erika
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Oddsdóttir, Hildur Aesa
    KTH, Skolan för teknikvetenskap (SCI), Matematik (Inst.), Optimeringslära och systemteori.
    Forsgren, Anders
    KTH, Skolan för teknikvetenskap (SCI), Matematik (Inst.), Optimeringslära och systemteori.
    Chotteau, Véronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Identification of experimentally relevant elementary flux mode subsets in a genome-scale metabolic network of CHO cell metabolism using column generationManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    An elementary flux mode (EFM) is a stoichiometrically balanced pathway through a metabolic network that links extracellular substrates to products. For large and complex networks, finding all such pathways is a computational challenge due to the combinatorial explosion of possible modes. Herein, we show how a new algorithm based on the column generation optimization technique can be applied to efficiently identify small sets of pathways in a genome-scale metabolic network. We examine the metabolism of Chinese hamster ovary (CHO) cells by identifying pathways that are relevant to data obtained in a pseudo-perfusion cell culture experiment. Based on the identified pathways, we examine the intracellular metabolism behind the uptake and utilization of extracellular medium components for the biomass and mAb synthesis, and the generation of extracellular metabolic by-products.

  • 29.
    Hagrot, Erika
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Sweden.
    Oddsdóttir, Hildur Æsa
    KTH, Skolan för teknikvetenskap (SCI), Matematik (Inst.), Optimeringslära och systemteori.
    Mäkinen, Meeri
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Sweden.
    Forsgren, Anders
    KTH, Skolan för teknikvetenskap (SCI), Matematik (Inst.), Optimeringslära och systemteori.
    Chotteau, Véronique
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Sweden; WCPR, Wallenberg Centre for Protein Research, Sweden.
    Novel column generation-based optimization approach for poly-pathway kinetic model applied to CHO cell culture2019Inngår i: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 8, artikkel-id e00083Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mathematical modelling can provide precious tools for bioprocess simulation, prediction, control and optimization of mammalian cell-based cultures. In this paper we present a novel method to generate kinetic models of such cultures, rendering complex metabolic networks in a poly-pathway kinetic model. The model is based on subsets of elementary flux modes (EFMs) to generate macro-reactions. Thanks to our column generation-based optimization algorithm, the experimental data are used to identify the EFMs, which are relevant to the data. Here the systematic enumeration of all the EFMs is eliminated and a network including a large number of reactions can be considered. In particular, the poly-pathway model can simulate multiple metabolic behaviors in response to changes in the culture conditions. We apply the method to a network of 126 metabolic reactions describing cultures of antibody-producing Chinese hamster ovary cells, and generate a poly-pathway model that simulates multiple experimental conditions obtained in response to variations in amino acid availability. A good fit between simulated and experimental data is obtained, rendering the variations in the growth, product, and metabolite uptake/secretion rates. The intracellular reaction fluxes simulated by the model are explored, linking variations in metabolic behavior to adaptations of the intracellular metabolism.

  • 30. Hakkaart, Xavier DV
    et al.
    Pronk, Jack T
    van Maris, Antonius JA
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    A Simulator-Assisted Workshop for Teaching Chemostat Cultivation in Academic Classes on Microbial Physiology2017Inngår i: Journal of Microbiology & Biology Education, Vol. 18, nr 3Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state. In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/or chemostat cultivation.

  • 31.
    Hallberg, Martin
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Leitner, C.
    Haltrich, D.
    Divne, Christina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase2004Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 341, nr 3, s. 781-796Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.

  • 32. Holmquist, M.
    et al.
    Martinelle, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Berglund, P.
    Mid Sweden University.
    Clausen, I. G.
    Patkar, S.
    Svendsen, A.
    Hult, K.
    Lipases from Rhizomucor miehei and Humicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity1993Inngår i: Journal of Protein Chemistry, ISSN 02778033 (ISSN), Vol. 12, nr 6, s. 749-757Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S- enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: E(S) = 8.5; H. lanuginosa: E(S) = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (E(R) = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E(R) = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E(S) = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E(S) = 1.9) and increased the enantioselectivity with the aromatic ester (E(R) = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E(S) = 17.4) and a decreased enantioselectivity with the phenyl ester (E(R) = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

  • 33.
    Hörnström, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Engineering and applications of surface displayed tyrosinase on Escherichia coli2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The rise of biotechnology has provided a toolbox to deal with major challenges related to pollution and health. Microbial enzymes constitute powerful macromolecules with applications in environmental technology, and industrial and medical production. The display of enzymes on cellular surfaces promotes external access to reactants, thereby simplifying production and cost-effectiveness of bioprocesses. To this end, a system for the surface display of the oxidative enzyme tyrosinase was developed, optimized and implemented. The first part of the thesis focused on developing tyrosinase surface-display via autotransport-based secretion in Escherichia coli. Initially, the presence of active surface-displayed tyrosinase, catalyzing the oxidation, of L-tyrosine was verified. Next, the components of the surface expression system were systematically engineered to yield an optimized tyrosinase-displaying strain with five times higher biomass-specific tyrosinase activity. The second half of the thesis applied the surface-displayed tyrosinase for wastewater treatment and biosensor development. It was found that the catalyzed oxidation of L- tyrosine resulted in the deposition of melanin at the E. coli cell surface. The resulting melanized cells were used in a membrane bioreactor for adsorption of the pharmaceutical chloroquine from an aqueous solution, with a specific binding capacity of 140 mg/g cells and allowed simple cell regeneration by lowering the pH. In a second application, the tyrosinase- display system was integrated into a genetic circuit with regulated oxidation and production of L-tyrosine in response to specific toxins. By employing the resulting cells in an electrochemical cell, the circuit generated a means to directly and selectively link biological information to an electrical output. Overall, the results in this thesis highlight the functionality of the surface expression methodology and demonstrates its versatile applicability.

  • 34.
    Hörnström, David
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Larsson, Gen
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gustavsson, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Molecular optimization of autotransporter-based tyrosinase surface display2019Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, nr 2, s. 486-494Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.

  • 35.
    Jahic, Mehmedalija
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Gustavsson, Malin
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Jansen, Ann-Katrin
    Martinelle, Mats
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi. KTH, Tidigare Institutioner (före 2005), Bioteknologi. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes2003Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 102, nr 1, s. 45-53Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

  • 36. Juergens, H.
    et al.
    Niemeijer, M.
    Jennings-Antipov, L. D.
    Mans, R.
    Morel, J.
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Pronk, J. T.
    Gardner, T. S.
    Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics2018Inngår i: Scientific Data, E-ISSN 2052-4463, Vol. 5, artikkel-id 180195Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous ‘Materials and Methods’ sections in scientific journals, thus promoting reproducibility and reusability of published studies.

  • 37.
    Kjellmer, Ingemar
    et al.
    Univ Gothenburg, Sahlgrens Acad, Inst Clin Sci, Dept Pediat, Gothenburg, Sweden..
    Lindecrantz, Kaj
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. Karolinska Inst, Dept Clin Sci Intervent & Technol, Stockholm, Sweden..
    Rosen, Karl G.
    Univ Gothenburg, Sahlgrens Acad, Dept Physiol, Gothenburg, Sweden..
    ST analysis of the fetal electrocardiogram - Comments on recent experimental data2019Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, nr 8, artikkel-id e0221210Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In their paper, Andriessen at al present a validation of fetal ECG analysis and the clinical STAN device in midgestation fetal lambs exposed to 25 minutes of umbilical cord occlusion. The study presents results that contrast remarkably from previously published experimental data which raises a number of questions and comments. The most striking finding of Andriessen et al is the recording of an extremely high number of alarms from the STAN equipment during control conditions when no alarms at all are expected. These patterns have never been seen, neither in the clinical situation nor in our own fetal sheep studies. The reason for this becomes apparent when their way of recording the FECG is scrutinized. In their assessment of STAN, Andriessen at al use an assumed negative aVF lead with the assumption that it will reflect the FECG in the same way as the unipolar scalp lead used clinically. The signal used for disqualification of STAN is itself not qualified to properly represent the fetal scalp lead signal that STAN is designed for. To question a proven technology is fully accepted but those attempting would be asked to argue along fully validated data and related analysis including questioning of their own data.

  • 38.
    Land, Henrik
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Campillo-Brocal, Jonatan C.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Svedendahl Humble, Maria
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization2019Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, nr 10, s. 1297-1304Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.

  • 39.
    Land, Henrik
    et al.
    Uppsala university.
    Ruggieri, Federica
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Szekrenyi, Anna
    Technishe Universität Darmstadt.
    Fessner, Wolf-Dieter
    Technishe Universität Darmstadt.
    Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary AminesManuskript (preprint) (Annet vitenskapelig)
  • 40.
    Land, Henrik
    et al.
    Uppsala University.
    Ruggieri, Federica
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Szekrenyi, Anna
    Fessner, Wolf-Dieter
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines2019Inngår i: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. n/a, nr n/aArtikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A protein engineering approach for expanding the substrate scope of the (S)-selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl- and benzyl-substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2-diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)-1,2-diphenylethylamine with ee >99 % under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20 % (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzyl-substituted primary amines.

  • 41.
    Lu, Ke
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Yang, Liyun
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem, Ergonomi.
    Abtahi, F.
    Lindecrantz, Kaj
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Rödby, K.
    Seoane, F.
    Wearable cardiorespiratory monitoring system for unobtrusive free-living energy expenditure tracking2019Inngår i: IFMBE Proceedings, Springer, 2019, nr 1, s. 433-437Konferansepaper (Fagfellevurdert)
    Abstract [en]

    In this work, we want to introduce combined heart rate and respiration monitoring for more accurate energy expenditure tracking on free-living subjects. We have developed a wearable cardiorespiratory monitoring system with unobtrusive heart rate measurement and ventilation estimation function for this purpose. The system is based on a garment with integrated textile electrodes for one-lead electrocardiogram and impedance pneumography measurements. A pilot experiment has been performed to prove the concept and to evaluate the characteristics of heart rate and ventilation estimated by our system in relation to energy expenditure. In the experiment, ventilation shows a better linearity in relation to the energy expenditure at the low intensity region than heart rate. Based on these characteristics, a model combining heart rate and ventilation for energy expenditure estimation is proposed which shows a significantly lower estimation error than the heart rate only model.

  • 42. Löfgren, J.
    et al.
    Görbe, T.
    Oschmann, M.
    Svedendahl, Maria
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Bäckvall, J. -E
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.

  • 43. Marques, W. L.
    et al.
    Mans, R.
    Henderson, R. K.
    Marella, E. R.
    Horst, J. T.
    Hulster, E. D.
    Poolman, B.
    Daran, J. -M
    Pronk, J. T.
    Gombert, A. K.
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae2018Inngår i: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, s. 121-133Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.

  • 44. Marques, Wesley Leoricy
    et al.
    van der Woude, Lara Ninon
    Luttik, Marijke AH
    van den Broek, Marcel
    Nijenhuis, Janine Margriet
    Pronk, Jack T
    van Maris, Antonius JA
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi. Department of Biotechnology, Delft University of Technology, Delft, Netherlands.
    Mans, Robert
    Gombert, Andreas K
    Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter2018Inngår i: Yeast, Vol. 35, nr 12, s. 639-652Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (mu = 0.19 h(-1) and mu = 0.08 h(-1), respectively). All five mutants displayed sucrose-induced proton uptake (13-50 mu mol H+ (g biomass)(-1) min(-1)). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1(I209F C265F G326C)) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h(-1)) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.

  • 45.
    Marx, Lisa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Chemo-enzymatic cascades for the synthesis of chiral high-value chemicals2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Chiral amines are frequent in today´s top selling pharmaceuticals. Classical organic synthesis of pharmaceuticals is often work intensive involving many synthesis steps, the use of protection group chemistry, heavy metal catalysts and chiral crystallization techniques. In recent years biocatalysts have proven their outstanding ability to synthesize chiral compounds. In this work the possibility of employing biocatalysts as alternative catalysts for API (active pharmaceutical ingredient) synthesis was explored. Three compounds currently on the market were selected as viable case studies: Cinacalcet (a hyperparathyroidism drug), Vyvanse (an ADHD-drug) and Sertraline (an antidepressant). Two enzyme classes were investigated to directly provide the chiral amines - transaminases and imine reductases. Ketoreductases were also investigated to provide the chiral amine via the chiral alcohol. Laccases and hydrolases were employed to complete the synthesis pathways to the final API. In the case of Vyvanse a true one-pot, two-step enzymatic cascade was achieved by a transaminase and hydrolase. For Cinacalcet a chemo-enzymatic cascade could be demonstrated. Both transaminase and ketoreductase gave excellent enantioselectivities and high yield for the key intermediates, which could then be chemically converted into the final API with good yield. For Sertraline the best yield of one diastereomer precursor could be achieved by a ketoreductase, followed by further enzymatic and chemical steps to the final API. Transaminases and imine reductases both have potential in synthesizing the key amine precursors or the APIs themselves. But to date selectivity and yield are insufficient for industrial application in a lot of cases. This work demonstrates the potential of enzymes to serve as viable alternatives to organo-metallic synthesis. Furthermore enzymes have the potential to simplify work-up because of their excellent enantioselectivity. Finally, a scale-up of a one-step transamination to the key chiral precursor of Cinacalcet demonstrated the enzyme´s applicability in larger volume and at higher substrate concentration.

  • 46.
    Marx, Lisa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Ríos-Lombardía, Nicolás
    EntreChem SL.
    Süss, Philipp
    Enzymicals AG.
    Höhne, Matthias
    University of Greifswald.
    Morís, Francisco
    EntreChem SL.
    González-Sabín, Javier
    EntreChem SL.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Chemoenzymatic Synthesis of SertralineManuskript (preprint) (Annet vitenskapelig)
  • 47.
    Marx, Lisa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Süss, Philipp
    Enzymicals AG.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Fractional Factorial Experimental Design and Scale-Up of a Transaminase ProcessManuskript (preprint) (Annet vitenskapelig)
  • 48.
    Marx, Lisa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Süss, Philipp
    Enzymicals AG.
    Morís, Francisco
    EntreChem SL.
    González-Sabín, Javier
    EntreChem SL.
    Berglund, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Enzymatic one-pot two-step cascade for the synthesis of VyvanseManuskript (preprint) (Annet vitenskapelig)
  • 49.
    Master, Emma
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Rudsander, Ulla
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Zhou, Welin
    Henriksson, Hongbin
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Divne, Christina
    KTH, Tidigare Institutioner (före 2005), Bioteknologi. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Denman, Stuart
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Wilson, David
    Teeri, Tuula
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 31, s. 10080-10089Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

  • 50.
    Neubauer, P.
    et al.
    Tech Univ Berlin, Dept Biotechnol, Berlin, Germany..
    Golson, R.
    BioSilta Oy, Oulu, Finland..
    Neubauer, A.
    BioSilta Oy, Oulu, Finland..
    Ukkonen, K.
    BioSilta Oy, Oulu, Finland..
    Krause, M.
    BioSilta Oy, Oulu, Finland..
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO).
    Ottosson, J.
    KTH, Skolan för bioteknologi (BIO).
    Larsen, M. Wittrup
    KTH, Skolan för bioteknologi (BIO).
    Hult, Karl
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Vasala, A.
    BioSilta Oy, Oulu, Finland..
    Using EnBase (TM) to enhance recombinant protein production2009Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 25, s. S190-S190Artikkel i tidsskrift (Annet vitenskapelig)
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