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  • 101.
    Marx, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Rios-Lombardia, Nicolas
    Farnberger, Judith F.
    Kroutil, Wolfgang
    Benitez-Mateos, Ana I.
    Lopez-Gallego, Fernando
    Moris, Francisco
    Gonzalez-Sabin, Javier
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases2018In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 360, no 11, p. 2157-2165Article in journal (Refereed)
    Abstract [en]

    Abstract Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1‐acetonaphthone yielded an enantiopure (R)‐amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)‐alcohol, which was easily converted into the previous (R)‐amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co‐immobilized on the same porous surface. This self‐sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA‐based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield.

  • 102.
    Mirkhani, Jawid
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Removal of pharmaceuticals from polluted water by use of melanin covered Escherichia coli2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
  • 103.
    Nameer, Samer
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    One-pot enzyme-catalyzed synthesis of dual-functional polyester macromers towards surface active hydrophobic filmsManuscript (preprint) (Other academic)
    Abstract [en]

    Selective enzyme catalysis is a valuable tool for the processing of monomers into value-added materials. In the present study natural resources were used to retrieve an ω-hydroxy fatty acid monomer containing an epoxide functionality. A procedure was developed for the synthesis of dual-functional oligomers by utilizing lipase catalysis in a one-pot synthesis route. The chemoselectivity of the enzyme allowed addition of thiol monomers to the retrieved epoxy monomers, without harming the epoxides, achieving a thiol-epoxy functional polyester resin. The synthesis reached full conversion (>99 %) after 8 h. It was possible to selectively crosslink the resin, through UV-initiated cationic polymerization of the epoxides into thiol-functional thermosets. The curing performance was followed in situ by Real-Time FTIR. The thiol groups on the surface of the film were accessible for post-modification.

  • 104.
    Nameer, Samer
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    One-pot enzyme-catalyzed synthesis of dual-functional polyester macromers towards surface-active hydrophobic films2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 79, p. 50294-50299Article in journal (Refereed)
    Abstract [en]

    Selective enzyme catalysis is a valuable tool for the processing of monomers into value-added materials. In the present study natural resources were used to retrieve an omega-hydroxy fatty acid monomer containing an epoxide functionality. A procedure was developed for the synthesis of dual-functional oligomers by utilizing lipase catalysis in a one-pot synthesis route. The chemoselectivity of the enzyme allowed addition of thiol monomers to the retrieved epoxy monomers, without harming the epoxides, achieving a thiol-epoxy functional polyester resin. The synthesis reached full conversion (> 99%) after 8 h. It was possible to selectively crosslink the resin through UV-initiated cationic polymerization of the epoxides into thiol-functional thermosets. The curing performance was followed in situ by real-time FTIR. The thiol groups on the surface of the film were accessible for post-modification.

  • 105.
    Nilsson, Charlotte
    et al.
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Land and Water Resources Engineering.
    Lakshmanan, Ramnath
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Renman, Gunno
    KTH, School of Architecture and the Built Environment (ABE), Sustainable development, Environmental science and Engineering, Land and Water Resources Engineering.
    Kuttuva Rajarao, Gunaratna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Efficacy of reactive mineral-based sorbents for phosphate, bacteria, nitrogen and TOC removal - Column experiment in recirculation batch mode2013In: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 47, no 14, p. 5165-5175Article in journal (Refereed)
    Abstract [en]

    Two mineral-based materials (Polonite and Sorbulite) intended for filter wells in on-site wastewater treatment were compared in terms of removal of phosphate (PO4-P), total inorganic nitrogen (TIN), total organic carbon (TOC) and faecal indicator bacteria (Escherichia coli and Enterococci). Using an innovative, recirculating system, septic tank effluent was pumped at a hydraulic loading rate of 3000 L m(2) d(-1) into triplicate bench-scale columns of each material over a 90-day period. The results showed that Polonite performed better with respect to removal of PO4-P, retaining on average 80% compared with 75% in Sorbulite. This difference was attributed to higher CaO content in Polonite and its faster dissolution. Polonite also performed better in terms of removal of bacteria because of its higher pH value. The total average reduction in E. coli was 60% in Polonite and 45% in Sorbulite, while for Enterococci the corresponding value was 56% in Polonite and 34% in Sorbulite. Sorbulite removed TIN more effectively, with a removal rate of 23%, while Polonite removed 11% of TIN, as well as TOC. Organic matter (measured as TOC) was accumulated in the filter materials but was also released periodically. The results showed that Sorbulite could meet the demand in removing phosphate and nitrogen with reduced microbial release from the wastewater treatment process.

  • 106. Nobili, Alberto
    et al.
    Steffen-Munsberg, Fabian
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. University of Greifswald, Germany .
    Kohls, Hannes
    Trentin, Ivan
    Schulzke, Carola
    Höhne, Matthias
    Bornscheuer, Uwe T.
    Engineering the Active Site of the Amine Transaminase from Vibrio fluvialis for the Asymmetric Synthesis of Aryl-Alkyl Amines and Amino Alcohols2015In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 7, no 5, p. 757-760Article in journal (Refereed)
    Abstract [en]

    Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of a-hydroxy ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30-fold increased activity in the conversion of (S)-phenylbutylamine and (R)-phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98% ee.

  • 107.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    The History of Pyrosequencing®2015In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1315, p. 3-15Article in journal (Refereed)
    Abstract [en]

    One late afternoon in the beginning of January 1986, bicycling from the lab over the hill to the small village of Fulbourn, the idea for an alternative DNA sequencing technique came to my mind. The basic concept was to follow the activity of DNA polymerase during nucleotide incorporation into a DNA strand by analyzing the pyrophosphate released during the process. Today, the technique is used in multidisciplinary fields in academic, clinical, and industrial settings all over the word. This technique can be used for both single-base sequencing and whole-genome sequencing, depending on the format used.In this chapter, I give my personal account of the development of Pyrosequencing()-beginning on a winter day in 1986, when I first envisioned the method-until today, nearly 30 years later.

  • 108.
    Oddsdottir, Hildur Aesa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Robustness analysis of elementary flux modes generated by column generation2016In: Mathematical Biosciences, ISSN 0025-5564, E-ISSN 1879-3134, Vol. 273, p. 45-56Article in journal (Refereed)
    Abstract [en]

    Elementary flux modes (EFMs) are vectors defined from a metabolic reaction network, giving the connections between substrates and products. EFMs-based metabolic flux analysis (MFA) estimates the flux over each EFM from external flux measurements through least-squares data fitting. The measurements used in the data fitting are subject to errors. A robust optimization problem includes information on errors and gives a way to examine the sensitivity of the solution of the EFMs-based MFA to these errors. In general, formulating a robust optimization problem may make the problem significantly harder. We show that in the case of the EFMs-based MFA, when the errors are only in measurements and bounded by an interval, the robust problem can be stated as a convex quadratic programming (QP) problem. We have previously shown how the data fitting problem may be solved in a column-generation framework. In this paper, we show how column generation may be applied also to the robust problem, thereby avoiding explicit enumeration of EFMs. Furthermore, the option to indicate intervals on metabolites that are not measured is introduced in this column generation framework. The robustness of the data is evaluated in a case-study, which indicates that the solutions of our non-robust problems are in fact near-optimal also when robustness is considered, implying that the errors in measurement do not have a large impact on the optimal solution. Furthermore, we showed that the addition of intervals on unmeasured metabolites resulted in a change in the optimal solution.

  • 109.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On dynamically identifying elementary flux modes for a poly-pathway model of metabolic reaction networksManuscript (preprint) (Other academic)
    Abstract [en]

    he aim with poly-pathway models is to model variations in the metabolic behavior of cells in response to changes in their external environment.By considering the elementary flux modes of a metabolic network, the network can be reduced to a set of macroscopic reactions. The macroscopic reactions connect external substrates to products, where each reaction is associated with a kinetic equation.Since enumerating all elementary flux modes is prohibitive for complex networks, these types of models are usually limited to simple networks. In this work we consider an algorithm for identifying elementary flux modes for a poly-pathway model. First we consider a dynamic identification of elementary flux modes and model parameters using column generation. However, due to non-linearity in one optimization problem involved in column generation, elementary flux mode identification can not be guaranteed with that column generation approach. In order to still be able to identify elementary flux modes, an approximation algorithm is derived and tested for the model identification. In a case study, the algorithm is shown to work well in practice and obtains a near-optimal solution compared to a method in which all elementary flux modes are enumerated beforehand.

  • 110.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On the Robustness of Elementary-Flux-Modes-based Metabolic Flux AnalysisManuscript (preprint) (Other academic)
    Abstract [en]

    Elementary flux modes (EFMs) are vectors defined from a metabolic reaction network, giving the connections between substrates and products. EFMs-based metabolic flux analysis (MFA) estimates the flux over each EFM from external flux measurements through least-squares data fitting. The measurements used in the data fitting are subject to errors. A robust optimization problem includes information on errors and gives a way to examine the sensitivity of the solution of the EFMs-based MFA to these errors. In general, formulating a robust optimization problem may make the problem significantly harder. We show that in the case of the EFMs-based MFA the robust problem can be stated as a convex quadratic programming problem. We have previously shown how the data fitting problem may be solved in a column-generation framework. In this paper, we show how column generation may be applied also to the robust problem. Furthermore, the option to indicate intervals on metabolites that are not measured is introduced in this column generation framework. The robustness of the data is evaluated in a case-study, which indicated that the solutions of our non-robust problems are in fact near-optimal also when robustness is considered, implying that the errors in measurement do not have a large impact on the optimal solution. Furthermore, we showed that the addition of intervals on unmeasured metabolites resulted in a change in the optimal solution.

  • 111.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On dynamically generating relevant elementary flux modes in a metabolic network using optimization2014In: Journal of Mathematical Biology, ISSN 0303-6812, E-ISSN 1432-1416Article in journal (Refereed)
    Abstract [en]

    Elementary flux modes (EFMs) are pathways through a metabolic reaction network that connect external substrates to products. Using EFMs, a metabolic network can be transformed into its macroscopic counterpart, in which the internal metabolites have been eliminated and only external metabolites remain. In EFMs-based metabolic flux analysis (MFA) experimentally determined external fluxes are used to estimate the flux of each EFM. It is in general prohibitive to enumerate all EFMs for complex networks, since the number of EFMs increases rapidly with network complexity. In this work we present an optimization-based method that dynamically generates a subset of EFMs and solves the EFMs-based MFA problem simultaneously. The obtained subset contains EFMs that contribute to the optimal solution of the EFMs-based MFA problem. The usefulness of our method was examined in a case-study using data from a Chinese hamster ovary cell culture and two networks of varied complexity. It was demonstrated that the EFMs-based MFA problem could be solved at a low computational cost, even for the more complex network. Additionally, only a fraction of the total number of EFMs was needed to compute the optimal solution.

  • 112.
    Okoli, Chuka
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology. KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Development of Protein-Functionalized Magnetic Iron Oxide Nanoparticles: Potential Application in Water Treatment2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The treatment of water to make it safe for human consumption is a problem of immense concern, both in developing and developed countries. However, the production of clean water with chemicals as coagulants has several drawbacks associated with cost, health risks and complexity in sludge management. The application of nanotechnology in water treatment is a fast growing discipline proposed as an efficient alternative that will combat these hurdles. The aim of this thesis is to develop new water treatment strategies in a more eco-friendly manner based on a bottom-up approach using: (i) a natural coagulant protein from Moringa oleifera purified with nanoscale magnetic iron oxide nanoparticles for in situ treatment; and (ii) a protein-functionalized nanoparticle (MOCP-MNPs) system by means of binding the coagulant protein onto the nanoparticles in order to develop a potential reusable water treatment process.

    Magnetic iron oxide nanoparticles with different surface chemistry have been prepared from co-precipitation in aqueous solution and (water-in-oil and oil-in-water) microemulsion methods.  

    The prepared nanoparticles were studied in terms of size, morphology, magnetic behavior, structure, surface area including surface chemical structure and charges using different techniques such as TEM, VSM/SQUID, XRD, BET, FT-IR and zeta potential. The prepared nanoparticles exhibited a size ranging from 2-30 nm with superparamagnetic properties. The Moringa oleifera  coagulant protein (MOCP) with known molecular mass (6.5 kDa) was purified from the crude Moringa oleifera (MO) seed extracts using nanoparticles prepared from both methods. The obtained MOCP exhibits comparable coagulation activity with alum in terms of water turbidity removal, implying alternative replacement to chemical coagulants. This technique can be easily applied where natural materials are available locally.

    Studies on the interaction between MOCP and surface modified nanoparticles were essential to understand the binding mechanism for the development of a protein-functionalized nanoparticle. Based on in silico investigation, the overall molecular docking studies reveal the interactions between protein-ligand complexes by electrostatic, van der Waals and hydrogen-bonding; which imply, that there are at least two binding sites is i.e. one located at the core binding site (TEOS and APTES ligand) while the other located at the side chain residues (TSC and Si60-OH).

    This work underscores advancement in the development and use of MOCP-MNPs for potential water treatment. About 70% turbidity removal was achieved gravimetrically using MOCP-MNPs (60 min) in high and low turbid waters, whereas alum requires 180 min to reduce the turbidity especially in low turbid waters. The turbidity removal efficiency was enhanced by the use of MOCP-MNPs under the influence of an external magnetic field. More than 95% turbidity removal was achieved within 12 min in high and low turbid waters when MOCP-MNPs were used. The combination of natural coagulant protein and magnetic nanoparticles as well as the use of applied magnetic field enhanced the performance coagulating/flocculating properties in the water samples.

    These results suggest a successful development of MOCP-MNPs as demonstrated in the regeneration study. The data shown in this work represent novel potential water treatment strategies that could be cost-effective, simple, robust and environmentally friendly whilst utilizing biocompatible materials. 

  • 113.
    Okoli, Chuka
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology.
    Boutonnet, Magali
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology.
    Järås, Sven
    KTH, School of Chemical Science and Engineering (CHE), Chemical Engineering and Technology, Chemical Technology.
    Rajarao-Kuttuva, Gunaratna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Albanova University Center, Sweden.
    Protein-functionalized magnetic iron oxide nanoparticles: Time efficient potential-water treatment2014In: Nanotechnology for Sustainable Development, First Edition, Springer, 2014, p. 127-136Chapter in book (Other academic)
    Abstract [en]

    Recent advances in nanoscience suggest that the existing issues involving water quality could be resolved or greatly improved using nanomaterials, especially magnetic iron oxide nanoparticles. Magnetic nanoparticles have been synthesized for the development and use, in association with natural coagulant protein for water treatment. The nanoparticles size, morphology, structure, and magnetic properties were characterized by transmission electron microscope, X-ray diffraction, and superconducting quantum interference device magnetometry. Purified Moringa oleifera protein was attached onto microemulsions-prepared magnetic iron oxide nanoparticles (ME-MION) to form stable protein-functionalized magnetic nanoparticles (PMO+ME-MION). The turbidity removal efficiency in both synthetic and surface water samples were investigated and compared with the commonly used synthetic coagulant (alum) as well as PMO. More than 90 % turbidity could be removed from the surface waters within 12 min by magnetic separation of PMO+ME-MION; whereas gravimetrically, 70 % removal in high and low turbid waters can be achieved within 60 min. In contrast, alum requires 180 min to reduce the turbidity of low turbid water sample. These data support the advantage of separation with external magnetic field (magnetophoresis) over gravitational force. Time kinetics studies show a significant enhancement in ME-MION efficiency after binding with PMO implying the availability of large surface of the MEMION. The coagulated particles (impurities) can be removed from PMO+ME-MION by washing withmild detergent or cleaning solution. To our knowledge, this is the first report on surface water turbidity removal using protein-functionalized magnetic nanoparticle.

  • 114.
    Okoli, Chuka
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sengottiyan, Selvaraj
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Natarajan Arul, Murugan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Pavankumar, Asalapuram Ramachand
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Kuttuva Rajarao, Gunaratna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    In silico modeling and experimental evidence of coagulant protein interaction with precursors for nanoparticle functionalization2013In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 31, no 10, p. 1182-1190Article in journal (Refereed)
    Abstract [en]

    The design of novel protein-nanoparticle hybrid systems has applications in many fields of science ranging from biomedicine, catalysis, water treatment, etc. The main barrier in devising such tool is lack of adequate information or poor understanding of protein-ligand chemistry. Here, we establish a new strategy based on computational modeling for protein and precursor linkers that can decorate the nanoparticles. Moringa oleifera (MO2.1) seed protein that has coagulation and antimicrobial properties was used. Superparamagnetic nanoparticles (SPION) with precursor ligands were used for the protein-ligand interaction studies. The molecular docking studies reveal that there are two binding sites, one is located at the core binding site; tetraethoxysilane (TEOS) or 3-aminopropyl trimethoxysilane (APTES) binds to this site while the other one is located at the side chain residues where trisodium citrate (TSC) or Si-60 binds to this site. The protein-ligand distance profile analysis explains the differences in functional activity of the decorated SPION. Experimentally, TSC-coated nanoparticles showed higher coagulation activity as compared to TEOS- and APTES-coated SPION. To our knowledge, this is the first report on in vitro experimental data, which endorses the computational modeling studies as a powerful tool to design novel precursors for functionalization of nanomaterials; and develop interface hybrid systems for various applications.

  • 115.
    Palo-Nieto, Carlos
    et al.
    Mid Sweden University.
    Afewerki, Samson
    Anderson, Mattias
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tai, Cheuk-Wai
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Córdova, Armando
    Integrated Heterogeneous Metal/Enzymatic Multiple Relay Catalysis for Eco-Friendly and Asymmetric Synthesis2016In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, p. 3932-3940Article in journal (Refereed)
    Abstract [en]

    Organic synthesis is in general performed using stepwise transformations where isolation and purification of key intermediates is often required prior to further reactions. Herein we disclose the concept of integrated heterogeneous metal/enzymatic multiple relay catalysis for eco-friendly and asymmetric synthesis of valuable molecules (e.g., amines and amides) in one-pot using a combination of heterogeneous metal and enzyme catalysts. Here reagents, catalysts, and different conditions can be introduced throughout the one-pot procedure involving multistep catalytic tandem operations. Several novel cocatalytic relay sequences (reductive amination/amidation, aerobic oxidation/reductive amination/amidation, reductive amination/kinetic resolution and reductive amination/dynamic kinetic resolution) were developed. They were next applied to the direct synthesis of various biologically and optically active amines or amides in one-pot from simple aldehydes, ketones, or alcohols, respectively.

  • 116. Pandya, J.
    et al.
    Venalis, P.
    Al-Khalili, Lubna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hossain, M.
    Stache, V.
    Lundberg, I. E.
    Malmström, V.
    Fasth, A.
    CD28null T cells are myotoxic to autologous muscle cells from patients with polymyositis2014In: Scandinavian Journal of Rheumatology, ISSN 0300-9742, E-ISSN 1502-7732, Vol. 43, p. 9-10Article in journal (Other academic)
  • 117. Pandya, Jayesh
    et al.
    Venalis, Paulius
    Al-Khalili, Lubna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hossain, Mohammad Shahadat
    Lundberg, Ingrid E.
    Malmström, Vivianne
    Fasth, Andreas E. R.
    CD28null T Cells from Polymyositis Patients Are Cytotoxic to Autologous Muscle Cells in Vitro Via Perforin-Dependent Mechanisms2014In: Arthritis & Rheumatology (Hoboken), ISSN 2326-5191, Vol. 66, p. S1193-S1193Article in journal (Other academic)
  • 118. Papapetridis, I
    et al.
    Pronk, J. T.
    van Maris, Antonius
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Klaassen, P.
    Eukaryotic cell with increased production of fermentation product2017Other (Other academic)
  • 119. Papapetridis, Ioannis
    et al.
    van Dijk, Marlous
    van Maris, Antonius J. A.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Delft University Technology, Netherlands.
    Pronk, Jack T.
    Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae2017In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 10, no 1, article id 107Article in journal (Refereed)
    Abstract [en]

    Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD(+)-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP(+)-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1 Delta gpd2 Delta strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP(+)-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.

  • 120.
    Pavankumar, Asalapuram R.
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Kayathri, Rajarathinam
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Murugan, Natarajan Arul
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Zhang, Qiong
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Okoli, Chuka
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Rajarao, Gunaratna K.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Dimerization of a flocculent protein from Moringa oleifera: experimental evidence and in silico interpretation2014In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 32, no 3, p. 406-415Article in journal (Refereed)
    Abstract [en]

    Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein-protein docking studies and binding free energy calculations. The structure of MO2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.

  • 121.
    Pavankumar, Asalapuram Ramachandran
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Norén, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Singh, Lakhvinder
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gowda, Naveen Kumar Chandappa
    Scaling-up the production of recombinant Moringa oleifera coagulant protein for large-scale water treatment applications2014In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 4, no 14, p. 7136-7141Article in journal (Refereed)
    Abstract [en]

    Provision of safe drinking water, devoid of aetiologies is an all-time challenge due to the usage of unsafe chemicals in most of the water treatment processes. In this regard, proteins of Moringa oleifera (MO) provide most promising solutions, as its crude seed extracts are being used as natural 'water clarifiers' in drinking and wastewater treatments. Among MO seed proteins, a 6.5 kDa coagulant protein (MO coagulant protein; MOCP) has gained importance because of its antimicrobial and water clarification properties. Considering the biomedical and commercial values of the MO plant, in this study, the cDNA coding for MOCP was cloned and over expressed in E. coli BL21 (DE3). After bench-scale experiments, for the first time, the recombinant protein production was scaled up to 7 L bioreactors in Luria Broth supplemented with 1% glucose. About 42 mg of Moringa oleifera coagulating recombinant protein (MOCRP; 6.5 kDa) was purified using superparamagnetic iron oxide nanoparticles (SPION) from 1 litre of the over expressed E. coli cell cultures. The purified MOCRP revealed an efficient coagulation activity (similar to 66%) even with 0.1 mg L-1; while very good antimicrobial response was recorded against a set of Gram-negative and Gram-positive microorganisms at as low as 10 mg L-1. Thus, we report the over expression and purification of this dual-function protein (MOCRP) from moringa for the first time, using bioreactors with an economical and user-friendly approach for large-scale applications.

  • 122.
    Pavankumar, Asalapuram Ramachandran
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Singh, Lakhvinder
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Identification of Moringa oleifera protein responsible for the decolorization and pesticide removal from drinking water and industrial effluent - an in silico and in situ evaluation2015In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 90, no 8, p. 1521-1526Article in journal (Refereed)
    Abstract [en]

    Natural products are always in demand, especially in the food and water treatment industry, to reduce health hazards caused by the prolonged use of chemicals. Though crude seed extract of Moringa oleifera (MOCE) is used for decolouration, the protein responsible for such activity is not fully known. In this study, in silico analysis of Moringa oleifera coagulant protein (MOCP; a predominant oligomeric protein in MOCE) was undertaken to check its molecular interactions with water and soil pollutants, in order to identify the protein accountable for such activities. The molecular docking studies of MOCP with azo dyes like congo red, tartrazine) and a pesticide (dichlorodiphenyltrichloroethane) revealed a strong binding affinity (-5.66, -5.33 and -5.04, respectively, kJ mol(-1)) between the protein and the pollutants through hydrogen bonding and electrostatic interactions. Further, these results were verified in situ with MOCP, a recombinant form of MOCP (MOCRP) and MOCE against congo red (100 mg L-1) and revealed the dye removal efficiency of 63.8%, 65.7%, and 72.3%, respectively. While the jar test results of synthetic coloured water and industrial textile effluent containing congo red showed 51.6% and 58.3%. Hence, we believe that the MOCP is responsible for multiple activities of MOCE and suggest its prospective use for large-scale treatment of drinking water and industrial effluents.

  • 123. Pawar, Prashant Mohan-Anupama
    et al.
    Ratke, Christine
    Balasubramanian, Vimal K.
    Chong, Sun-Li
    Gandla, Madhavi Latha
    Adriasola, Mathilda
    KTH, School of Biotechnology (BIO).
    Sparrman, Tobias
    Hedenström, Mattias
    Szwaj, Klaudia
    Derba-Maceluch, Marta
    Gaertner, Cyril
    Mouille, Gregory
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Tenkanen, Maija
    Jönsson, Leif J.
    Mellerowicz, Ewa J.
    Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification2017In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 214, no 4, p. 1491-1505Article in journal (Refereed)
    Abstract [en]

    High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

  • 124.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO).
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara, Monica
    KTH, School of Biotechnology (BIO).
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Design and flux modelling for recombinant production of 3-Hydroxybutyrate in Escherichia coli2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, p. S167-S167Article in journal (Other academic)
  • 125.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara-Martinez, Monica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply2016In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, article id 91Article in journal (Refereed)
    Abstract [en]

    Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

  • 126. Poborilova, Zuzana
    et al.
    Ohlsson, Anna B.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Torkel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Vildova, Anna
    Babula, Petr
    DNA methylation changes caused by lawsone2014In: International Journal of Agricultural and Biosystems Engineering, ISSN 2319-3913, Vol. 8, no 7, p. 654-657Article in journal (Refereed)
  • 127. Poborilova, Zuzana
    et al.
    Ohlsson, Anna B.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Torkel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Vildova, Anna
    Provaznik, Ivo
    Babula, Petr
    DNA hypomethylation concomitant with the overproduction of ROS induced by naphthoquinone juglone on tobacco BY-2 suspension cells2015In: Environmental and Experimental Botany, ISSN 0098-8472, E-ISSN 1873-7307, Vol. 113, p. 28-39Article in journal (Refereed)
    Abstract [en]

    Juglone, 5-hydroxy-1,4-naphthoquinone, is a naturally occurring plant secondary metabolite with allelopathic and cytotoxic properties. The cytotoxic effect of juglone and changes at the level of DNA methylation were investigated on a plant cell model, tobacco BY-2 cell suspension culture. We found that juglone in a concentration-dependent manner inhibits cell growth and causes creation of reactive oxygen species (ROS) that were visible mainly in nucleus, nucleolus, cytoplasm, and plasma membrane. Analysis of the changes in DNA methylation level using Luminometric Methylation Assay showed that juglone at the concentrations of 60 and 80 mu m causes hypomethylation of DNA. Decrease in DNA methylation was confirmed by the more sensitive technique, confocal immunofluorescence microscopy, already at 30 mu m, and was accompanied by significant overproduction of hydrogen peroxide, hydroxyl radicals, and superoxide anion radicals. The highest percentage of the signs of programed cell death, especially apoptic-like bodies that indicate DNA fragmentation was observed at 30 mu m of juglone. On the basis of results we suggest that overproduction of ROS is the main mechanism that causes hypomethylation of DNA, which may contribute to the initiation of the programmed cell death.

  • 128. Ratke, Christine
    et al.
    Pawar, Prashant Mohan-Anupama
    Balasubramanian, Vimal K.
    Naumann, Marcel
    Duncranz, Mathilda Lönnäs
    Derba-Maceluch, Marta
    Gorzsas, Andras
    Endo, Satoshi
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mellerowicz, Ewa J.
    Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification2015In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 13, no 1, p. 26-37Article in journal (Refereed)
    Abstract [en]

    The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

  • 129. Ravichandran, R.
    et al.
    Åstrand, Carolina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Patra, H. K.
    Turner, Anthony P. F.
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Phopase, J.
    Intelligent ECM mimetic injectable scaffolds based on functional collagen building blocks for tissue engineering and biomedical applications2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 34, p. 21068-21078Article in journal (Refereed)
    Abstract [en]

    Hydrogels comprising natural extracellular matrix (ECM) components are very attractive as scaffolds for regenerative medicine applications due to their inherent biointeractive properties. Responsive materials that adapt to their surrounding environments and regulate transport of ions and bioactive molecules manifest significant advantages for biomedical applications. Although there are many exciting challenges, the opportunity to design, fabricate and engineer stimuli-responsive polymeric systems based on ECM components is particularly attractive for regenerative medicine. Here we describe a one-pot approach to fabricate in situ fast gellable intelligent ECM mimetic scaffolds, based on methacrylated collagen building blocks with mechanical properties that can be modulated in the kPa-MPa range and that are suitable for both soft and hard tissues. Physiochemical characterizations demonstrate their temperature and pH responsiveness, together with the structural and enzymatic resistance that make them suitable scaffolds for long-term use in regenerative medicine and biomedical applications. The multifunctionality of these hydrogels has been demonstrated as an in situ depot-forming delivery platform for the adjustable controlled release of proteins and small drug molecules under physiological conditions and as a structural support for adhesion, proliferation and metabolic activities of human cells. The results presented herein should be useful to the design and fabrication of tailor-made scaffolds with tunable properties that retain and exhibit sustained release of growth factors for promoting tissue regeneration.

  • 130. Rex, E.
    et al.
    Rosander, Erica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Røyne, F.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ulmanen, J.
    A systems perspective on chemical production from mixed food waste: The case of bio-succinate in Sweden2017In: Resources, Conservation and Recycling, ISSN 0921-3449, E-ISSN 1879-0658, Vol. 125, p. 86-97Article in journal (Refereed)
    Abstract [en]

    The option of producing the chemical succinic acid from bio-based resources is well in line with current political and industrial ambitions for a bio-based economy. A little explored but intriguing biomass feedstock opportunity is food waste. Mixed food waste is especially appealing as it represents less resource competition than more homogenous food waste fractions. The feasibility of producing succinic acid from mixed food waste depends on both technical and societal system structures. Therefore, to assess the production prospect, it is important to investigate all relevant system components. This study explores from such multiple perspectives the feasibility of chemical production as a viable added pathway for mixed food waste, using microbial production of succinic acid from municipal solid waste in Sweden as an example. The perspectives explored are: 1) feedstock feasibility, 2) societal drivers and barriers for technology progress, and 3) resource availability. Findings show that even though, from a technical feasibility and resource availability perspective, production seems possible, it lacks institutional support and actor commitment and alignment for development in Sweden. Findings also show that a holistic and interdisciplinary systems perspective contributes valuable insight when assessing prospects for bio-based chemicals.

  • 131. Rodrigo, Gustav
    et al.
    Gruvegard, Mats
    Van Alstine, James M.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. JMVA Biotech, Sweden.
    Antibody Fragments and Their Purification by Protein L Affinity Chromatography2015In: ANTIBODIES, ISSN 2073-4468, Vol. 4, no 3, p. 259-277Article, review/survey (Refereed)
    Abstract [en]

    Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteriasuggesting its consideration as a key unit operation in antibody fragment processing.

  • 132.
    Rüdiger, Arne
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hedfors, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Trey, Stacy
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. SP Trätek, SP Technical Research Institute of Sweden.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Chemoenzymatic Route to Renewable Thermosets Based on a Suberin Monomer2013In: Journal of renewable materials, ISSN 2164-6341, Vol. 1, no 2, p. 124-140Article in journal (Refereed)
    Abstract [en]

    The present study describes the use of an epoxy functional fatty acid, 9,10-epoxy-18-hydroxyoctadecanoic acid (EFA), extracted from birch (Betula pendula) outer bark to produce thermosets. The purified epoxy fatty acid was polymerized by enzyme-catalyzed polycondensation utilizing Candida antarctica lipase B (CalB) to form oligomers with targeted degrees of polymerization (DP) of 3, 6, and 9 and obtained DPs of 2.3, 5.9 and 7.3, respectively. It was determined that it is possible to first enzymatically polymerize and aliphatically endcap the epoxy functional fatty acid resulting in controlled oligomer lengths while also maintaining the epoxy functionality for further reaction by main-chain homo-epoxy cationic photopolymerization. The enzymatic polymerized oligomers were characterized in terms of conversion of the residual epoxy groups (FT-IR), the thermal properties (DSC, TGA) and the purity by MALDI-TOF and 1H-NMR. The amorphous thermoset films with varying degrees of crosslinking resulting from the cationically photopolymerized oligomers, were characterized in terms of their thermal properties and residual epoxy content (FT-IR ATR). The crosslinked polyesters formed insoluble, amorphous, and transparent films. This work demonstrates that thermoset films with designed properties can be effectively made with the use of forest products to reduce the petroleum-based plastics market.

  • 133.
    Sandersjöö, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Kostallas, George
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 1, p. 155-162Article in journal (Refereed)
    Abstract [en]

    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  • 134. Scheidt, Thomas
    et al.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Anderson, Mattias
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chen, Yujie
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Yi, Dong
    Fessner, Wolf-Dieter
    Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases2015In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, p. 1721-1731Article in journal (Refereed)
    Abstract [en]

    omega-Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)- or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify omega-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify omega-transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase-catalyzed transacylation to furnish enantiomerically pure (R)and (S)-configured amines. The latter are instrumental for the rapid enantioselectivity typing of omega-transaminases. This method was used to characterize two novel (S)-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

  • 135. Sehl, Torsten
    et al.
    Bock, Saskia
    Marx, Lisa
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Maugeri, Zaira
    Walter, Lydia
    Westphal, Robert
    Vogel, Constantin
    Menyes, Ulf
    Erhardt, Martin
    Mueller, Michael
    Pohl, Martina
    Rother, Doerte
    Asymmetric synthesis of (S)-phenylacetylcarbinol - closing a gap in C-C bond formation2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 2, p. 380-384Article in journal (Refereed)
    Abstract [en]

    (S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging. As no highly selective enzymes or chemical catalysts were available, we used semi-rational enzyme engineering to tailor a potent biocatalyst to be >97% stereoselective for the synthesis of (S)-PAC. By optimizing the reaction and process used, industrially relevant product concentrations of >48 g L-1 (up to 320 mM) were achieved. In addition, the best enzyme variant gave access to a broad range of ring-substituted (S)-PAC derivatives with high stereoselectivity, especially for meta-substituted products.

  • 136. Sehl, Torsten
    et al.
    Bock, Saskia
    Marx, Lisa
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Maugeri, Zaira
    Walter, Lydia
    Westphal, Robert
    Vogel, Constantin
    Menyes, Ulf
    Erhardt, Martin
    Mueller, Michael
    Pohl, Martina
    Rother, Doerte
    Asymmetric synthesis of (S)-phenylacetylcarbinol - closing a gap in C-C bond formation2017In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 19, no 2, p. 380-384Article in journal (Refereed)
    Abstract [en]

    (S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging. As no highly selective enzymes or chemical catalysts were available, we used semi-rational enzyme engineering to tailor a potent biocatalyst to be >97% stereoselective for the synthesis of (S)-PAC. By optimizing the reaction and process used, industrially relevant product concentrations of >48 g L-1 (up to 320 mM) were achieved. In addition, the best enzyme variant gave access to a broad range of ring-substituted (S)-PAC derivatives with high stereoselectivity, especially for meta-substituted products.

  • 137.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Building blocks for polymer synthesis by enzymatic catalysis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them.

    Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers.

    In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties.

    The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups.

  • 138.
    Semlitsch, Stefan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mixed vinyl adipate esters through selective synthesis using a designed esterase/acyltransferaseManuscript (preprint) (Other academic)
    Abstract [en]

    Methods for selective syntheses of mixed dicarboxylic esters from symmetrical diesters are of great interest. An esterase from Mycobacterium smegmatis, MsAcT, has shown to be an efficient catalyst for acyl transfer reactions using both alcohols and amines. It has a restricted active site resulting in a narrow acyl donor specificity. This limitation was used for the development of a selective synthesis of mixed vinyl adipate esters from divinyl adipate. The single mutant, L12A, achieved over 90% conversion of divinyl adipate with three different alcohols leading to the corresponding mixed vinyl adipate esters. This method allows the synthesis of vinyladipoyl esters at mild reaction conditions.

  • 139.
    Semlitsch, Stefan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Torron, Susana
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Enzymatic catalysis as a versatile tool for the synthesis of multifunctional, bio-based oligoester resins2016In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 18, no 7, p. 1923-1929Article in journal (Refereed)
    Abstract [en]

    The use of enzymes as selective catalysts for processing renewable monomers into added value polymers and materials has received increased attention during the last decade. In the present work Candida antarctica lipase B (CalB) was used as catalyst in one-pot routes to synthesise multifunctional oligoester resins based on an epoxy-functional omega-hydroxy-fatty acid (EFA) extracted from birch bark. The chemoselective enzymatic process resulted in three different EFA-based telechelic oligomers with targeted molecular weights; containing maleimide, methacrylate or oxetane as end-groups, respectively. The enzyme catalysed synthesis of the maleimide and the oxetane telechelic oligomers reached full conversion of monomers (>95%) after 2 h. In the case of methacrylate functional oligomer the EFA monomer reached full conversion (>98%) after 2 h but the integration of the methacrylate moiety took more than 10 h. This was due to a rate limiting reaction path using ethylene glycol dimethacrylate as substrate. The oligomer products were characterised by NMR, MALDI-TOF-MS and SEC.

  • 140. Singh, Lakhvinder
    et al.
    Sharma, Praveen
    Pavankumar, Asalapuram Ramachandran
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Lab4Life Biores Private Ltd, Bangalore, Karnataka, India.
    Chemical Oxygen Demand Elimination and Decolorization of Textile Industrial Effluent by an Indigenous Fungal Species Aspergillus foetidus2015In: CLEAN - Soil, Air, Water, ISSN 1863-0650, E-ISSN 1863-0669, Vol. 43, no 3, p. 456-461Article in journal (Refereed)
    Abstract [en]

    This study reports the biodegradation capability of an indigenous fungal species Aspergillus foetidus for decolorization and removal of chemical oxygen demand from textile effluent. Model designed experiments were performed to construct the quadratic model and high regression coefficients between variables and responses indicated suitability of the model. Further, the experimental conditions were optimized for fungal species A. foetidus 8796 to show its efficiency to remove color from textile effluents. Aspergillus foetidus 8796 revealed up to 91.3% of color removal activity and 82.58% of chemical oxygen demand removal capability under the optimal conditions of this study. The extracellular enzymatic activities of this fungal species evidenced an excellent evaluation of experimental data to propose A. foetidus, as a cost effective solution to treat the effluents from textile industry.

  • 141. Spinka, M.
    et al.
    Seiferheld, S.
    Zimmermann, P.
    Bergner, E.
    Blume, A. -K
    Schierhorn, A.
    Reichenbach, Tom
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Pertermann, R.
    Ehrt, C.
    König, S.
    Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 9, p. 1285-1298Article in journal (Refereed)
    Abstract [en]

    The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

  • 142.
    Steffen-Munsberg, Fabian
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Greifswald University.
    Matzel, Philipp
    Sowa, Miriam A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Bornscheuer, Uwe T.
    Höhne, Matthias
    Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, p. 4511-4521Article in journal (Refereed)
    Abstract [en]

    Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme’s function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved ‘flipping’ arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

  • 143.
    Steffen-Munsberg, Fabian
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Greifswald University, Germany.
    Vickers, Clare
    Kohls, Hannes
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Mallin, Hendrik
    Nobili, Alberto
    Skalden, Lilly
    van den Bergh, Tom
    Joosten, Henk-Jan
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Höhne, Matthias
    Bornscheuer, Uwe T.
    Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications2015In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 5, p. 566-604Article in journal (Refereed)
    Abstract [en]

    In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

  • 144.
    Svartström, Olov
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Terrapon, Nicolas
    Lombard, Vincent
    de Bruijn, Ino
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmsten, Jonas
    Dalin, Ann-Marie
    El Muller, Emilie
    Shah, Pranjul
    Wilmes, Paul
    Henrissat, Bernard
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ninety-nine de novo assembled genomes from the moose (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation2017In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 11, no 11, p. 2538-2551Article in journal (Refereed)
    Abstract [en]

    The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

  • 145.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Branneby, C.
    Abedi, V.
    Wells, A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    CASCAT: Redesign of omega-Transaminases for Synthesis of Chiral Amines2010In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 150, p. S123-S124Article in journal (Other academic)
    Abstract [en]

    Transaminases (EC 2.6.1.18) are attractive biocatalysts for synthesis of chiral amines and alpha-amino acids. These enzymes catalyze transfer of an amine group from a donor substrate to an acceptor compound using the cofactor pyridoxal-5′-phoshate (PLP). omega-Transaminases are a versatile subgroup of the transaminases that does not require a carboxylic acid group in alpha-position (in contradiction toalpha-transaminases) and hence accept a wider spectrum of ketones or amines. The omega-transaminases are employed industrially for production of both R- and S-enantiopure amines.

    One bottleneck is the unfavourable equilibrium in such reactions run in the synthesis mode. We have developed a one-pot multi-enzyme system in a cascade fashion for equilibrium displacement by removing formed acetone.

    Another issue is the fact that most omega-transaminases show S-selectivity, however a few R-selective strains do exist. We have used an S-selective omega-transaminase variant from Arthrobacter citreus and created an R-selective variant by rational redesign using a homology enzyme model. This homology modelling/rational design approach was further explored on an omega-transaminase from Chromobacterium violaceum.

  • 146.
    Säll, Anna
    et al.
    Lund Univ, Dept Immunotechnol, Lund, Sweden.;Alligator Biosci, Lund, Sweden..
    Corbee, Daniel
    Lund Univ, Dept Immunotechnol, Lund, Sweden.;Camurus, Lund, Sweden..
    Vikstrom, Sara
    Lund Univ, Dept Immunotechnol, Lund, Sweden.;BioGaia AB, Lund, Sweden..
    Ottosson, Filip
    Lund Univ, Dept Immunotechnol, Lund, Sweden.;Lund Univ, Dept Clin Sci, Malmo, Sweden..
    Persson, Helena
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Lund Univ, Dept Immunotechnol, Lund, Sweden..
    Waldemarson, Sofia
    Lund Univ, Dept Immunotechnol, Lund, Sweden..
    Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 2, article id e0189116Article in journal (Refereed)
    Abstract [en]

    There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.

  • 147. Tan, Tien Chye
    et al.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gandini, Rosaria
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, p. e86736-Article in journal (Refereed)
    Abstract [en]

    Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/ V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the a-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the beta-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the beta-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose alpha-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.

  • 148.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Kracher, Daniel
    Gandini, Rosaria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sygmund, Christoph
    Kittl, Roman
    Haltrich, Dietmar
    Hallberg, B Martin
    Ludwig, Roland
    Divne, Christina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, p. 7542-7542Article in journal (Refereed)
    Abstract [en]

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  • 149.
    Torron, Susana
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinell, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Biocatalytic Synthesis of Epoxy Resins from Fatty Acids as a Versatile Route for the Formation of Polymer Thermosets with Tunable Properties2016In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 17, no 12, p. 4003-4010Article in journal (Refereed)
    Abstract [en]

    The work herein presented describes the synthesis and polymerization of series of bio-based epoxy resins prepared through lipase catalyzed transesterification. The epoxy-functional polyester resins with various architectures (linear, hi branched, and tetra-branched) were synthesized through condensation of fatty acids derived from epoxidized soybean oil and linseed oil with three different hydroxyl cores under bulk conditions. The selectivity of the lipases toward esterification/transesterification reactions allowed the formation of macromers with up to 12 epoxides in the backbone. The high degree of functionality of the resins resulted in polymer thermosets with T-g values ranging from 25 to over 100 degrees C prepared through cationic polymerization. The determining parameters of the synthesis and the mechanism for the formation of the species were determined through kinetic studies by H-1 NMR, SEC, and molecular modeling studies. The correlation between macromer structure and thermoset properties was studied through real-time FTIR measurements, DSC, and DMA.

  • 150.
    Torron, Susana
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Semlitsch, Stefan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Polymer Thermosets from Multifunctional Polyester Resins Based on Renewable Monomers2014In: Macromolecular Chemistry and Physics, ISSN 1022-1352, E-ISSN 1521-3935, Vol. 215, no 22, p. 2198-2206Article in journal (Refereed)
    Abstract [en]

    The use of monomers based on natural materials as a future supply of raw materials has gained more interest in the last decade. Sources ranging from wood to plant oils and algae are exploited as alternatives to traditional fossil-based resources for the synthesis of polymeric materials. The use of these raw materials is not only of interest because of its abundance, but also in terms of price, durability, and/or biodegradability. In the present study, a series of resins utilizing a monomer derived from birch bark is prepared. The thermoset resins are formed by reacting an epoxy-functional omega-hydroxy fatty acid with methacrylate monomers using enzyme catalysis to form multifunctional resins via a one-pot synthesis. The derived oligomers are crosslinked through different polymerization routes to produce thermosets with different properties and/or functionalities. This approach allows natural-based resins with tuned functionalities and mechanical and thermal properties to be obtained.

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