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  • 1. Aaldering, L. J.
    et al.
    Poongavanam, V.
    Langkjær, N.
    Natarajan Arul, Murugan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Jørgensen, P. T.
    Wengel, J.
    Veedu, R. N.
    Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 8, p. 755-763Article in journal (Refereed)
    Abstract [en]

    The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  • 2.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformation: By Uwe T. Bornscheuer and Romas J. Kazlauskas2006In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, no 8, p. 1280-Article, book review (Other academic)
  • 3. Bunyapaiboonsri, T.
    et al.
    Ramström, Olof
    KTH, Superseded Departments, Chemistry.
    Lohmann, S.
    Lehn, J. M.
    Peng, L.
    Goeldner, M.
    Dynamic deconvolution of a pre-equilibrated dynamic combinatorial library of acetylcholinesterase inhibitors2001In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 2, no 6, p. 438-444Article in journal (Refereed)
    Abstract [en]

    A dynamic combinatorial library composed of interconverting acylhydrazones has been generated and screened towards inhibition of acetylcholinesterase from the electric ray Torpedo marmorata. Starting from a small set (13) of initial hydrazide and aldehyde building blocks, a library containing possibly 66 different species was obtained in a single operation. Of all possible acylhydrazones formed, active compounds containing two terminal cationic recognition groups separated by an appropriate distance, permitting two-site binding, could be rapidly identified by using a dynamic deconvolution-screening procedure, based on the sequential removal of starting building blocks. A very potent bis-pyridinium inhibitor (K (i)= 1.09 nM, alphaK(i) = 2.80 nM) was selected from the process and the contribution of various structural features to inhibitory potency was evaluated.

  • 4. Cammenberg, Maria
    et al.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Park, Seongsoon
    Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate2006In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, no 11, p. 1745-1749Article in journal (Refereed)
    Abstract [en]

    One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze, aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used, ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A moecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar to that in the acylation with methyoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In with 1-phenylethanol afforded the same rate with all acyl donors.

  • 5.
    Caraballo, Rémi
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Sakulsombat, Morakot
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Towards Dynamic Drug Design: Identification and Optimization of β-Galactosidase Inhibitors from a Dynamic Hemithioacetal System2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 11, p. 1600-1606Article in journal (Refereed)
    Abstract [en]

    A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of -galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C-glycosides. Inhibition studies provided important structural information for the two groups, and 1-thiobenzyl--D-galactopyranoside demonstrated the best inhibitory effects.

  • 6.
    Carlqvist, Peter
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, p. 331-336Article in journal (Refereed)
    Abstract [en]

    Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

  • 7.
    Dorau, Robin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gorbe, Tamas
    Humble, Maria Svedendahl
    Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 4, p. 338-346Article in journal (Refereed)
    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.

  • 8.
    Engfeldt, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Renberg, Björn
    KTH, School of Biotechnology (BIO).
    Brumer, Harry
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

  • 9.
    Eriksson, Adam
    et al.
    KTH, School of Chemical Science and Engineering (CHE).
    Kürten, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 23, p. 2301-2305Article in journal (Refereed)
    Abstract [en]

    Terpenes represent one of the most diversified classes of natural products with potent biological activities. The key to the myriad of polycyclic terpene skeletons with crucial functions in organisms from all kingdoms of life are terpene cyclase enzymes. These biocatalysts enable stereospecific cyclization of relatively simple, linear, prefolded polyisoprenes by highly complex, partially concerted, electrophilic cyclization cascades that remain incompletely understood. Herein, additional mechanistic light is shed on terpene biosynthesis by kinetic studies in mixed H2O/D2O buffers of a classII bacterial ent-copalyl diphosphate synthase. Mass spectrometry determination of the extent of deuterium incorporation in the bicyclic product, reminiscent of initial carbocation formation by protonation, resulted in a large kinetic isotope effect of up to seven. Kinetic analysis at different temperatures confirmed that the isotope effect was independent of temperature, which is consistent with hydrogen tunneling.

  • 10.
    Kourist, Robert
    et al.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Bartsch, Sebastian
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Understanding promiscuous amidase activity of an esterase from Bacillus subtilis2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 1, p. 67-69Article in journal (Refereed)
    Abstract [en]

    Water works. Bacillus subtilis esterase BS2 is a promiscuous esterase that shows amidase activity. This amidase activity was shown to depend on a hydrogen-bond network with the substrate amide hydrogen (indicated by arrow). When this stabilising hydrogen bond network was removed by a point mutation, the amide activity was significantly lowered in comparison with the esterase activity. (Figure Presented)

  • 11.
    Land, Henrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed)
    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.

  • 12.
    Larsen, Marianne Wittrup
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Zielinska, Dorota F.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hidalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 796-801Article in journal (Refereed)
    Abstract [en]

    A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mm butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked, tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.

  • 13.
    Lindgren, Joel
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ekblad, Caroline
    Abrahmsén, Lars
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    A Native Chemical Ligation Approach for Combinatorial Assembly of Affibody Molecules2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 7, p. 1024-1031Article in journal (Refereed)
    Abstract [en]

    Affinity molecules labeled with different reporter groups, such as fluorophores or radionuclides, are valuable research tools used in a variety of applications. One class of engineered affinity proteins is Affibody molecules, which are small (6.5 kDa) proteins that can be produced by solid phase peptide synthesis (SPPS), thereby allowing site-specific incorporation of reporter groups during synthesis. The Affibody molecules are triple-helix proteins composed of a variable part, which gives the protein its binding specificity, and a constant part, which is identical for all Affibody molecules. In the present study, native chemical ligation (NCL) has been applied for combinatorial assembly of Affibody molecules from peptide fragments produced by Fmoc SPPS. The concept is demonstrated for the synthesis of three different Affibody molecules. The cysteine residue introduced at the site of ligation can be used for directed immobilization and does not interfere with the function of the investigated proteins. This strategy combines a high-yield production method with facilitated preparation of proteins with different C-terminal modifications.

  • 14.
    Lindgren, Joel
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability2014In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 14, p. 2132-2138Article in journal (Other academic)
    Abstract [en]

    Protein-based pharmaceuticals typically display high selectivity and low toxicity, but are also characterized by low oral availability, mainly because of enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal wall. One way to increase the proteolytic stability of peptides and proteins is by intramolecular crosslinking, such as the introduction of disulfide bridges. However, disulfide bridges are at risk of thiol-disulfide exchange or reduction during production, purification, and/or therapeutic use, whereas thioether bridges are expected to be stable under the same conditions. In this study, thioether crosslinking was investigated for a 46 aa albumin-binding domain (ABD) derived from streptococcal protein G. ABD binds with high affinity to human serum albumin (HSA) and has been proposed as a fusion partner to increase the in vivo half-lives of therapeutic proteins. In the study, five ABD variants with single or double intramolecular thioether bridges were designed and synthesized. The binding affinity, secondary structure, and thermal stability of each protein was investigated by SPR-based biosensor analysis and CD spectroscopy. The proteolytic stability in the presence of the major intestinal proteases pepsin (found in the stomach) and trypsin in combination with chymotrypsin (found in pancreatin secreted to the duodenum by the pancreas) was also investigated. The most promising crosslinked variant, ABD_CL1, showed high thermal stability, retained high affinity in binding to HSA, and showed dramatically increased stability in the presence of pepsin and trypsin/chymotrypsin, compared to the ABD reference protein. This suggests that the intramolecular thioether crosslinking strategy can be used to increase the stability towards gastrointestinal enzymes.

  • 15.
    Léonard, Valérie
    et al.
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lamare, Sylvain
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Graber, Marianne
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol2007In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 6, p. 662-667Article in journal (Refereed)
    Abstract [en]

    The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rote and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol(-1), and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 Jmol(-1)K(-1).

  • 16. Löfgren, J.
    et al.
    Görbe, T.
    Oschmann, M.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Bäckvall, J. -E
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Article in journal (Refereed)
    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.

  • 17.
    Magnusson, Anders O.
    et al.
    KTH, School of Biotechnology (BIO).
    Rotticci Mulder, Johanna C.
    KTH, School of Biotechnology (BIO).
    Santagostino, Alberto
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO).
    Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1051-1056Article in journal (Refereed)
    Abstract [en]

    The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1)m(-1), respectively. The substrate selectivity changed over 400000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.

  • 18.
    Nilsson, Anders
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindgren, Joel
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 20, p. 2056-2062Article in journal (Refereed)
    Abstract [en]

    Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three-helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid-phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N-terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C-terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface-plasmon-resonance-based (SPR-based) biosensor studies of the protein domains showed that the three-helix structure and high-affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.

  • 19.
    Pei, Zhichao
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Yu, Hui
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Theurer, Matthias
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Waldén, Annelie
    KTH, School of Biotechnology (BIO).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO).
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Photogenerated Carbohydrate Microarrays2007In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 2, p. 166-168Article in journal (Refereed)
    Abstract [en]

    Chemical Equation Presented) Sugars in a row. A new strategy for carbohydrate microarrays based on photochemical ligation of perfluorophenylazide-derivatized carbohydrates to PEO surfaces is presented. It constitutes a controllable and robust method of array fabrication, on the carbohydrate chemistry and on the surface-chemistry levels, and the resulting carbohydrate arrays can be efficiently used to reveal the recognition patterns of carbohydrate-binding proteins.

  • 20.
    Perols, Anna
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Arcos Famme, Melina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions2015In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 17, p. 2522-2529Article in journal (Other academic)
    Abstract [en]

    Antibodies are extensively used both as research tools and in the clinics for diagnostics and therapy. For many applications the antibodies are labeled, e.g. with detection probes or cytotoxic agents that improve the therapeutic effect. Labeling is typically performed using amine-reactive probes targeting lysine residues accessible on the surface of the protein, resulting in a heterogeneously labeled antibody. One alternative strategy for site-specific labeling is to use the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc-region of IgG. By introducing the photoactivable amino acid benzoylphenylalanine (BPA) in the Z domain, a covalent bond can be formed between the Z domain and the antibody by UV irradiation, to produce a site-specifically labeled product. The Z domain with BPA in position 32,  Z32BPA, was synthesized by solid phase peptide synthesis and was further functionalized for alkyne-azide cycloaddition reactions to give alkyne-Z32BPA and azide-Z32BPA for Cu(I)-catalyzed click reaction, and DBCO-Z32BPA for Cu-free strain-promoted click reaction. The reactivity of the functionalized Z domains in Cu(I)-catalyzed and strain-promoted alkyne-azide cycloaddition reactions was analyzed using MALDI-TOF MS and RP-HPLC, showing fast reaction. To further evaluate the concept, the Z32BPA variants were conjugated to the human IgG1 antibody trastuzumab and biotinylated by Cu(I)-catalyzed or strain-promoted alkyne-azide cycloaddition reactions. Western blot analysis of the biotinylated antibodies, using streptavidin-HRP for detection, demonstrated that all Z-antibody conjugates could be site-specifically labeled by the alkyne-azide cycloaddition reactions.

  • 21.
    Ramström, Olof
    et al.
    KTH, Superseded Departments, Chemistry.
    Lehn, J. M.
    In situ generation and screening of a dynamic combinatorial carbohydrate library against concanavalin A2000In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 1, no 1, p. 41-48Article in journal (Refereed)
    Abstract [en]

    Dynamic combinatorial chemistry (DCC) is a recently introduced approach that is based on the generation of combinatorial libraries by reversible interconversion of the library constitutents. In this study, the implementation of such libraries on carbohydrate-lectin interactions was examined. The dynamic carbohydrate libraries were generated from a small set (four or six compounds) of initial carbohydrate dimers through mild disulfide interchange, and selection was performed under two conditions defining either adaptive or pre-equilibrated libraries. Upon initiation, libraries were formed that contained comparable amounts of 10 or 21 individual dimeric species, dynamically interchanging during the scrambling process. They were probed with respect to binding to the plant lectin concanavalin A, either present during library generation or added after equilibration. The libraries could be generated easily both in the presence and absence of the receptor, and a bis-mannose structure was preferentially bound and selected from the mixture. Scrambling of the library in the presence of the receptor resulted in slightly higher yields than when the receptor was added after scrambling, indicating that the receptor to some extent acts as a thermodynamic trap during library generation. The present results illustrate the extention of the DCC approach to carbohydrate recognition groups, the generation of isoenergetic dynamic libraries, and the implementation of either adaptive or pre-equilibrated procedures.

  • 22. Rotticci, D.
    et al.
    Rotticci-Mulder, J. C.
    Denman, S.
    Norin, T.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improved enantioselectivity of a lipase by rational protein engineering2001In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 2, no 10, p. 766-770Article in journal (Refereed)
    Abstract [en]

    A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity, 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutants gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity, The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

  • 23. Seitz, Miriam
    et al.
    Syren, Per-Olof
    Steiner, Lisa
    Klebensberger, Janosch
    Nestl, Bettina M.
    Hauer, Bernhard
    Synthesis of Heterocyclic Terpenoids by Promiscuous Squalene-Hopene Cyclases2013In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, no Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved., p. 436-439Article in journal (Refereed)
  • 24. Seitz, Miriam
    et al.
    Syrén, Per-Olof
    Steiner, Lisa
    Klebensberger, Janosch
    Nestl, Bettina
    Hauer, Bernhard
    Synthesis of heterocyclic terpenoids by promiscuous squalene-hopene cyclases2013In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, p. 436-439Article in journal (Refereed)
  • 25. Skjot, Michael
    et al.
    De Maria, Leonardo
    Chatterjee, Robin
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svendsen, Allan
    Patkar, Sharnkant A.
    Ostergaard, Peter R.
    Brask, Jesper
    Understanding the Plasticity of the alpha/beta Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 3, p. 520-527Article in journal (Refereed)
    Abstract [en]

    The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha 5 and alpha 10. The simulation also confirmed the function of helix alpha 5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C. branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E > 50), which is a common structure of the profen drug family.

  • 26.
    Stamm, Arne
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Biundo, Antonino
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schmidt, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brücher, Jörg
    Holmen AB, Dev, S-89180 Östersund, Sweden.
    Lundmark, Stefan
    Perstorp AB, Innovat, Perstorp Ind Pk, S-28480 Perstorp, Sweden.
    Olsén, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bornscheuer, Uwe T
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Dept Biotechnol & Enzyme Catalysis, Felix Hausdorff Str 4, D-17487 Greifswald, Germany.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    A retrobiosynthesis-based route to generate pinene-derived polyesters2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, p. 1664-1671Article in journal (Refereed)
    Abstract [en]

    Significantly increased production of biobased polymers is aprerequisite to replace petroleum-based materials towardsreaching a circular bioeconomy. However, many renewablebuilding blocks from wood and other plant material are notdirectly amenable for polymerization, due to their inert backbonesand/or lack of functional group compatibility with thedesired polymerization type. Based on a retro-biosyntheticanalysis of polyesters, a chemoenzymatic route from (@)-apinenetowards a verbanone-based lactone, which is furtherused in ring-opening polymerization, is presented. Generatedpinene-derived polyesters showed elevated degradation andglass transition temperatures, compared with poly(e-decalactone),which lacks a ring structure in its backbone. Semirationalenzyme engineering of the cyclohexanone monooxygenasefrom Acinetobacter calcoaceticus enabled the biosynthesis ofthe key lactone intermediate for the targeted polyester. As aproof of principle, one enzyme variant identified from screeningin a microtiter plate was used in biocatalytic upscaling,which afforded the bicyclic lactone in 39% conversion in shakeflask scale reactions.

  • 27.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Carlqvist, Peter
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Allnér, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Frise, Anton
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 15, p. 2443-2451Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

  • 28.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Aumailley, Lucie
    Besenmatter, Werner
    Gounine, Farida
    Svendsen, Allan
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 5, p. 645-648Article in journal (Refereed)
  • 29.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 802-810Article in journal (Refereed)
    Abstract [en]

    Acrylates represent a class of 4-unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme-catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least-motion mechanism upon the sp(2) to sp(3) substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low-energy s-sis/s-trans conformations. Apparent k(cat) values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutinase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.

  • 30.
    Vallin, Michaela
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Mutant Lipase-Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec-Alcohols: A Thermodynamic Analysis of Enantioselectivity2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 3, p. 411-416Article in journal (Refereed)
    Abstract [en]

    The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

  • 31. Veld, Martijn A. J.
    et al.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Palmans, Ania R. A.
    Meijer, E. W.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lactone Size Dependent Reactivity in Candida Antarctica Lipase B: A Molecular Dynamics and Docking Study2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 8, p. 1330-1334Article in journal (Refereed)
1 - 31 of 31
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