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  • 51.
    Eriksson, Magnus
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase-Catalyzed Syntheses of Telechelic Polyesters2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Telechelic polyesters have successfully been synthesized with lipase-catalyzed polymerization. The produced telechelics had a high degree of di­functionalization, high purity (requiring little or no workup) and controlled degree of polymerization. The syntheses were performed in one-pot one-step reaction systems. The use of protection/deprotection chemistry was not necessary, since the lipase selectivity was utilized in the syntheses. Two different types of lipase-catalyzed polymerizations were applied – ring-opening polymerization and polycondensation. In ring-opening polymerization telechelics were produced by a combination of initiation, α-functionalization, and linking through termination, w-func­tionalization. In polycondensation different types of end-cappers were used to synthesize telechelics. Several exampels of functional groups were used for end-functionalization - epoxide, methacrylate and tetraallyls. Enzyme kinetic schemes describing the different functionalization met­hods of polyesters are presented and discussed. Stoichiometry and different reaction conditions have been studied to understand the effects these functions have on the final structure of the synthesized telechelics. Polyesters are classified as biodegradable, and can also be synthesized from materials that can be extracted or fermented from renewable sources like plants. Lipase-catalysts have several beneficial attributes, like high selectivity, they are renewable and biodegradable, are non-toxic and metal-free and can operate under mild reaction conditions.

    The focus of this thesis has been on lipase-catalyzed syntheses and characterization of the produced telechelics, in addition some materials have been produced. Some uses of telechelics are surface modification, materials for block co-polymers, functional films and biomedical applications.

  • 52.
    Eriksson, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Boyer, Antoine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Sinigoi, Loris
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Trey, Stacy
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    One-Pot Enzymatic Route to Tetraallyl Ether Functional Oligoesters: Synthesis, UV Curing, and Characterization2010In: Journal of Polymer Science Part A: Polymer Chemistry, ISSN 0887-624X, E-ISSN 1099-0518, Vol. 48, no 23, p. 5289-5297Article in journal (Refereed)
    Abstract [en]

    An enzymatic one-pot route in bulk was used to synthesize tetraallyl ether (tAE) functional oligomers based on divinyl adipate, 1,4-butanediol and trimethylolpropane diallyl ether. By using lipase B from Candida antarctica as catalyst and varying the stoichiometric ratio of monomers, it was possible to reach targeted molecular weights (from 1300 to 3300 g mol(-1)) of allyl-ether functional polyesters. The enzyme catalyzed reaction reached completion (>98% conversion based on all monomers) within 24 h at 60 degrees C, under reduced pressure (72 mbar) resulting in similar to 90% yield after filtration. The tAE-functional oligoesters were photopolymerized, without any purification other than removal of the enzyme by filtration, with thiol functional monomers (dithiol, tetrathiol) in a 1: 1 ratio thiol-ene reaction. The photo-initiator, 2,2-dimethoxy-2-phenylacetophenone, was used to improve the rate of reaction under UV light. High conversions (96-99% within detection limits) were found for all thiol-ene films as determined by FT-Raman spectroscopy. The tAE-functional oligoesters were characterized by NMR, MALDI, and SEC. The UV-cured homopolymerized films and the thiol-ene films properties were characterized utilizing DSC and DMTA.

  • 53.
    Eriksson, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Fogelström, Linda
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    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.
    Trey, Stacy
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enzymatic One-Pot Route to Telechelic Polypentadecalactone Epoxide: Synthesis, UV Curing, and Characterization2009In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, no 11, p. 3108-3113Article in journal (Refereed)
    Abstract [en]

    In an enzymatic one-pot procedure immobilized lipase B from Candida antarctica was used to synthesize semicrystalline diepoxy functional macromonomers based on glycidol, pentadecalactone, and adipic acid. By changing the stoichiometry of the building blocks. macromonomers of controlled molecular weight front 1400 to 2700 g mol(-1) could be afforded. The enzyme-catalyzed reaction went to completion (conversion >= 95%) within 24 h at 60 degrees C. After removal of the enzyme, the produced macromonomers were used for photopolymerization without any purification. The macromonomers readily copolymerized cationically with a cycloaliphatic diepoxide (Cyracure UVR-6110; CA-dE) to high conversion. The cross-linked copolymers formed a durable film with a degree of crystallinity depending on the macromonomer size and amount of CA-dE used, without CA-dE the macromonomers homopolymerized only to a low degree. Combined with CA-dE conversions of 85-90% were determined by FT-Raman spectroscopy. The films became more durable once reinforced with CA-dE, increasing the cross-link density and reducing the crystallinity of the PDL segments in the films.

  • 54.
    Eriksson, Magnus G.
    et al.
    KTH, School of Industrial Engineering and Management (ITM), Machine Design (Dept.), Mechatronics.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Johansson, Mats
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Trey, Stacy M.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    One-pot enzymatic polycondensation to telechelic methacrylate-functional oligoesters used for film formation2011In: POLYM CHEM, ISSN 1759-9954, Vol. 2, no 3, p. 714-719Article in journal (Refereed)
    Abstract [en]

    Based on largely renewable monomers, an enzymatic one-pot polycondensation route towards functional oligomers with targeted molecular weights and end-groups was developed. This one-pot synthesis was performed by combining Candida antarctica lipase B (CALB), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol, and divinyl adipate under reduced pressure (72 mbar) at 60 degrees C. The polymerization went to completion (>95% conversion for all monomers) within 24 h and the fraction of methacrylate end-groups was >90%. Three targeted dimethacrylate functional oligomers with molecular weights of 920, 1700 and 2500 g mol(-1) (degrees of polymerization 4, 8, and 13 respectively) were synthesized. The oligomer products were characterized by NMR, MALDI-TOF MS and SEC. The dimethacrylate functional oligomers were further UV homopolymerized or combined with a tetrathiol crosslinker to demonstrate the potential to produce novel networks with tunable thermal properties dependent on chain length of the telechelic building blocks. This research is the first to demonstrate methacrylate functionalization and condensation polymerization in a one step process, which expands the growing toolbox for polymer/material chemists towards an increased throughput in available macromonomers used in material design.

  • 55.
    Eriksson, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    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.
    Trey, Stacy
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    One-step enzymatic polycondensation to telechelic methacrylate-functional polyesters used for film formationManuscript (preprint) (Other academic)
  • 56.
    Eriksson, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enzymatic one-pot polycondensation to telechelic epoxy oligomersManuscript (preprint) (Other academic)
  • 57.
    Eriksson, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nilsson, Camilla
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    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.
    Trey, Stacy
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    One-pot synthesis to functional free-standing polymer film for sensor applicationsManuscript (preprint) (Other academic)
  • 58.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enzyme substrate solvent interactions: a case study on serine hydrolases2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Reaction rates and selectivities were measured for transacylation of fatty acid esters in solvents catalysed by Candida antarctica lipase B and by cutinase from Humicola insolens. With these enzymes classical water-based enzymology can be expanded to many different solvents allowing large variations in interaction energies between the enzymes, the substrates and the surrounding. Further ,hydrolysis reactions catalysed by Bacillus subtilis esterase 2 were investigated.

    Thermodynamics analyses revealed that the enzyme contribution to reaction rate acceleration compared to acid catalysis was purely entropic. On the other hand, studies of differences in activation entropy and enthalpy between enantiomers and between homologous esters showed that high substrate specificity was favoured by enthalpic stabilisation.

    Solvent was found to have a profound effect on enzyme catalysis, affecting both reaction rate and selectivity. Differences in substrate solubility will impact enzyme specificity since substrate binding is an equilibrium between enzyme-bound substrate and substrate in free solution. In addition, solven tmolecules were found to act as enzyme inhibitors, showing both competitive and non-competitive behaviour.

    In several homologous data series enthalpy-entropy compensation relationships were encountered. A possible extrathermodynamic relationship between enthalpy and entropy can easily be lost under co-varying errors propagated from the experiments. From the data in this thesis, one instance was found of a real enthalpy-entropy compensation that could be distinguished from statistical errors, while other examples could not be verified.

  • 59.
    Fransson, Linda
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bernhardt, Peter
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    On the benefit of an active siteManuscript (preprint) (Other academic)
  • 60.
    Fransson, Linda
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry. KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Laurell, Anna
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Widyan, Khalid
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Wingstrand, Erica
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Moberg, Christina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Minor Enantiomer Recycling-Effect of Two Reinforcing Catalysts on Product Yield and Enantiomeric Excess2010In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 2, no 6, p. 683-693Article in journal (Refereed)
    Abstract [en]

    Kinetic modeling of a recycling procedure in which the minor product enantiomer from an enantioselective catalytic reaction is selectively retransformed to starting material by a second chiral catalyst demonstrates that the enantiomeric excess of the product is not affected by the relative amounts of the two catalysts, but that the yield increases when the amount of the catalyst for the product-forming reaction is increased. The yield, but not the enantiomeric excess, is also affected by the initial substrate concentration. The recycling process is compared to sequential processes in which either the second catalyst is added after completion of the first reaction or in which the two catalysts are added simultaneously. In the sequential processes, high enantioselectivity can be obtained at the expense of product yield, whereas under recycling conditions both high enantiomeric excess and high yield can be achieved. Experimental data from a recycling procedure providing qualitative support for results from kinetic modeling are presented.

  • 61. Gardossi, Lucia
    et al.
    Poulsen, Pout B.
    Ballesteros, Antonio
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedas, Vytas K.
    Vasic-Racki, Durda
    Carrea, Giacomo
    Magnusson, Anders
    Schmid, Andreas
    Wohlgemuth, Roland
    Halling, Peter J.
    Guidelines for reporting of biocatalytic reactions2010In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 28, no 4, p. 171-180Article, review/survey (Refereed)
    Abstract [en]

    Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.

  • 62. Gharizadeh, B.
    et al.
    Oggionni, M.
    Zheng, B. Y.
    Akom, E.
    Pourmand, N.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wallin, K. L.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Type-specific multiple sequencing primers - A novel strategy for reliable and rapid genotyping of human papilloma viruses by pyrosequencing technology2005In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 7, no 2, p. 198-205Article in journal (Refereed)
    Abstract [en]

    DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

  • 63.
    Gharizadeh, Baback
    et al.
    Stanford Univ, Stanford Genome Technol Ctr.
    Akhras, Michael
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nourizad, Nader
    KTH, School of Biotechnology (BIO), Biochemistry.
    Ghaderi, Mehran
    Univ Örebro, Dept Caring Sci, Div Biomed.
    Yasuda, Kenji
    Univ Tokyo, Grad Sch Arts & Sci, Dept Life Sci.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pourmand, Nader
    Stanford Univ, Stanford Genome Technol Ctr.
    Methodological improvements of pyrosequencing technology2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 124, no 3, p. 504-511Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.

  • 64.
    Graber, Marianne
    et al.
    Univ La Rochelle, Lab Biotechnol & Chim Bioorgan.
    Irague, Romain
    Univ La Rochelle, Lab Biotechnol & Chim Bioorgan.
    Rosenfeld, Eric
    Univ La Rochelle, Lab Biotechnol & Chim Bioorgan.
    Lamare, Sylvain
    Univ La Rochelle, Lab Biotechnol & Chim Bioorgan.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Solvent as a competitive inhibitor for Candida antarctica lipase B2007In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1774, no 8, p. 1052-1057Article in journal (Refereed)
    Abstract [en]

    In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanot, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and I -propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K-il were changed to "K-i", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.

  • 65. Gustavsson, M. T.
    et al.
    Persson, P. V.
    Iversen, T.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase2005In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 6, no 1, p. 196-203Article in journal (Refereed)
    Abstract [en]

    A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M-r xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.

  • 66.
    Hamberg, Anders
    KTH, School of Biotechnology (BIO), Biochemistry.
    Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved.

    In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase.

    In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches.

    In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance.

    In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.

  • 67.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kempka, Martin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, p. 167-172Article in journal (Refereed)
    Abstract [en]

     A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

  • 68.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lundgren, Stina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Penhoat, Maël
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Moberg, Christina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    High-Throughput Enzymatic Method for Enantiomeric Excess Determination of O-Acetylated Cyanohydrins2006In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 7, p. 2234-2235Article in journal (Refereed)
  • 69.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lundgren, Stina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Wingstrand, Erica
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Moberg, Christina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    High Throughput Synthesis and Analysis of Acylated Cyanohydrins2007In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 13, no 15, p. 4334-4341Article in journal (Refereed)
    Abstract [en]

    The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.

  • 70.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Magnusson, Anders
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hu, Francis J.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    Selective monoacylation of diols by substrate assisted catalysis in T40A CALBManuscript (preprint) (Other academic)
  • 71.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Magnusson, Anders
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hu, Francis J.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Selective Monoacylation of Diols by Substrate Assisted Catalysis in T40A Candida antarctica Lipase B2013In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 3, p. 743-747Article in journal (Refereed)
    Abstract [en]

    The selectivity towards diols over monoesters in the esterification of diols catalysed by lipase B from Candida antarctica (CALB) was improved by the single point mutation T40A in the enzyme's oxyanion hole. Substrate-assisted catalysis was suggested from molecular modelling of the tetrahedral intermediate in esterification of 1,2-ethanediol catalysed by T40A CALB. The non-reacting hydroxyl group of the diol forms a hydrogen bond to the oxyanion in the transition state, replacing that deleted in mutation. Monoester yields in transacylation reactions were monitored over time to compare the selectivities for wild-type and T40A CALB. The results showed increased selectivities towards the diols tested over their corresponding monoesters as a result of the T40A mutation with substrate-assisted catalysis as a plausible explanation.

  • 72.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Maurer, S.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational engineering of Candida antarctica lipase B for selective monoacylation of diols2012In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 48, no 80, p. 10013-10015Article in journal (Refereed)
    Abstract [en]

    The enzyme Candida antarctica lipase B was subjected to site directed mutagenesis suggested by molecular modelling. The selectivity for the enzyme increased towards a range of diols over their corresponding monoesters as an effect of the mutations.

  • 73.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Maurer, Steffen
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational engineering of CALB for selective monoacylation of diolsManuscript (preprint) (Other academic)
  • 74. Hauer, Bernhard
    et al.
    Engelmark Cassimjee, David Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Process for producing polyamines2009Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    The present invention relates to a process for the production of a polyamine involving the use of enzymes; in particular to a process performed in aqueous environment; to the polyamines produced by said method; as well as the use of said polyamines for manufacturing paper, for immobilizing enzymes, or for preparing pharmaceutical or cosmetical compositions. The invention also relates to a novel method for in situ regeneration of cofactors NAD(P)+.

  • 75.
    Hedfors, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase chemoselectivity - kinetics and applications2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

     

    A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.

     

  • 76.
    Hedfors, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase selectivity in functional polyester synthesis2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzyme selectivity means that the enzyme´s preferences towards competing substrates will be different. In this thesis, the enzyme selectivity has been studied for utilization in synthesis of functionalized macromonomers. The aim was to study how the inherent –or introduced – selectivity of lipases can be used to introduce thiol‐ or enefunctionalities into short polyesters. Thiol‐ and ene‐functionalized renewable organic precursor molecules in combination with thiol‐ene click chemistry opens up for a sustainable material production. Lipases do not normally affect ene‐moieties and the preference towards thiols is low, enabling introduction of these functional groups for further modifications. In addition, lipases have been shown to be good catalysts in the formation of polyesters, both via ring‐opening and polycondensation polymerization.

    In paper I Candida antarctica lipase B was used to end‐functionalize poly(ε‐caprolactone) with free thiols in a one‐pot reaction. The advantage of using achemoselective lipase as catalyst was that no protection of the thiol was needed. The chemoselectivity displayed by Candida antarctica lipase B turned out to be 88 000 in favour of the alcohol (paper II). Rhizomucor miehei lipase showed less pronounced chemoselectivity. The largest contribution to the selectivities was derived from the more than two orders of magnitude higer KM towards the thiol compared to the alcohol. Thiols can be cross‐linked with enes in radical reactions to form networks, enabling formation of materials.

    One promising renewable molecule containing an acrylate moiety is itaconic acid. In paper III the selectivity towards the two esters in dimethyl itaconate was investigated and the active site of Candida antarctica lipase B was redesigned to generate variants with increased and decreased selectivity. One variant showed 14‐fold higher selectivity and could regioselectively add dimethyl itaconate onto a diol. This variant could be used in end‐functionalizations of polymers, introducing acrylate‐ester end‐groups.

    The enzyme selectivity towards lactones and their corresponding polyesters is of importance when designing a ring‐opening polymerization reaction. In paper IV Candida antarctica lipase B was found to prefer ω‐pentadecalactone and polyesters over ε‐caprolactone ten‐fold, while Humicola insolens cutinase preferred ε‐caprolactone and its corresponding polyester four‐fold over ω‐pentadecalactone and its polyester. From a selectivity point of view, Candida antarctica lipase B and Humicola insolens cutinase would be equally good in ring‐opening polymerization of ω‐pentadecalactone, while in the case of ε‐caprolactone Humicola insolens cutinase would be the preferred choice.

  • 77.
    Hedfors, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Takwa, Mohamad
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Selectivity towards itaconic acid esters by Candida antarctica lipase B and variantsManuscript (preprint) (Other academic)
  • 78.
    Hedfors, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase chemoselectivity towards alcohol and thiol acyl acceptors in a transacylation reaction2010In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 66, no 1-2, p. 120-123Article in journal (Refereed)
    Abstract [en]

    The lipase chemoselectivity towards an alcohol and a thiol was investigated for the two lipases Candida antarctica lipase B (CalB) and Rhizomucor miehei lipase (Rml). Hexanol and hexanethiol were used as acyl acceptors in a transacylation reaction with ethyl octanoate in cyclohexane. CalB showed the highest chemoselectivity ratio (k(cat)/K-M)(OH)/(k(cat)/K-M)(SH), of 88,000 while the ratio for Rml was 1200. That could be compared with the ratio, k(OH)/k(SH), of 120 for the non-catalyzed reaction. Thus, the enzyme contribution to the chemoselectivity between hexanol and hexanethiol was 730 for CalB and 10 for Rml. High K-M values displayed towards hexanethiol (above 1.8 M) were the largest contribution to the selectivity. No saturation was achieved. The K-M values were more than two orders of magnitude higher than those of hexanol.

  • 79.
    Hedfors, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Takwa, Mohamad
    KTH, School of Biotechnology (BIO), Biochemistry.
    Spinelli, Pietro
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Competition between lactones and polyesters in enzyme catalyzed ring-opening polymerizationManuscript (preprint) (Other academic)
  • 80.
    Hedfors, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Östmark, Emma
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Malmström, Eva E.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Thiol end-functionalization of poly(epsilon-caprolactone), catalyzed by Candida antarctica lipase B2005In: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 38, no 3, p. 647-649Article in journal (Refereed)
    Abstract [en]

    The use of Candida antarctica Lipase B (CALB) chemoselective catalyst in the Thiol End-Functionalization of Poly(ε-caprolacetone) was discussed. Thiol-functionalization of poly(ε-caprolacetone)(PCL) was made by an initiation reaction catalyzed by CALB in bulk. 2-Mercaptoethanol (1) was used to initiate the enzyme-assisted ring opening polymerization of ε-caprolacetone(2) to give the desired thiol-functionalized polymer. The structure of the terminated PCL was confirmed by 13C nuclear magnetic resonance .

  • 81.
    Hedin, Eva M. K.
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 50, p. 16658-16671Article in journal (Refereed)
    Abstract [en]

    The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyi-sn-glycero-3-phosphati-dylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 1418514196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TILL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.

  • 82. Heinze, Birgit
    et al.
    Kourist, Robert
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis2007In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 3, p. 125-131Article in journal (Refereed)
    Abstract [en]

    Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.

  • 83.
    Henriksson, Gunnar
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Zhang, Liming
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Nilsson, Thomas
    Inst för virkeslära Sveriges Lantbruksuniversitet, Uppsala.
    Ohlsson, Anna
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Torkil
    KTH, School of Biotechnology (BIO), Glycoscience.
    Inhomogenity inlignin structure between different cell wall layers i  conifers and hardwood2006In: Fifth Plant Biomechanics Conference, 2006, p. 145-150Conference paper (Refereed)
  • 84. Holmquist, M.
    et al.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Creation of a synthetically useful lipase with higher than wild-type enantioselectivity and maintained catalytic activity1999In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 1, no 5, p. 763-765Article in journal (Refereed)
    Abstract [en]

    Formula presented Wild type I: 89.9% ee (E=32) Wild type II: 79.8% ee (E=10) Lipase hybrid: 95.4% ee (E=54) We have found that two Geotrichum candidum lipase isozymes have remarkably different abilities to differentiate between enantiomers of ethyl 2-methyldecanoate. By rational recombination of selected portions of the two isozymes, we have created a novel lipase with an enantioselectivity superior to that of the best wild-type parent isozyme. Site-directed mutagenesis identified two key amino acid residues responsible for the improved enantioselectivity without compromised total activity of the reengineered enzyme.

  • 85. HOLMQUIST, M
    et al.
    MARTINELLE, M
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    CLAUSEN, IG
    PATKAR, S
    SVENDSEN, A
    HULT, K
    TRP89 IN THE LID OF HUMICOLA-LANUGINOSA LIPASE IS IMPORTANT FOR EFFICIENT HYDROLYSIS OF TRIBUTYRIN1994In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 29, no 9, p. 599-603Article in journal (Refereed)
    Abstract [en]

    To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. Ah Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V-max = 6000 +/- 300 mmol.min(-1).g(-1) and an apparent K-m = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent K-m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid of H. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.

  • 86.
    Hult, Karl
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Enzyme promiscuity: mechanism and applications2007In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 25, no 5, p. 231-238Article, review/survey (Refereed)
    Abstract [en]

    Introductory courses in biochemistry teach that enzymes are specific for their substrates and the reactions they catalyze. Enzymes diverging from this statement are sometimes called promiscuous. It has been suggested that relaxed substrate and reaction specificities can have an important role in enzyme evolution; however, enzyme promiscuity also has an applied aspect. Enzyme condition promiscuity has, for a long time, been used to run reactions under conditions of low water activity that favor ester synthesis instead of hydrolysis. Together with enzyme substrate promiscuity, it is exploited in numerous synthetic applications, from the laboratory to industrial scale. Furthermore, enzyme catalytic promiscuity, where enzymes catalyze accidental or induced new reactions, has begun to be recognized as a valuable research and synthesis tool. Exploiting enzyme catalytic promiscuity might lead to improvements in existing catalysts and provide novel synthesis pathways that are currently not available.

  • 87. Högberg, Hans-Erik
    et al.
    Edlund, Helen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hedenström, Erik
    WATER ACTIVITY INFLUENCES ENANTIOSELECTIVITY IN A LIPASE-CATALYZED RESOLUTION BY ESTERIFICATION IN AN ORGANIC-SOLVENT1993In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 4, no 10, p. 2123-2126Article in journal (Refereed)
    Abstract [en]

    The enantioselectivity of Candida rugosa lipase-mediated esterification of 2-methylalkanoic acids with n-alcohols in cyclohexane is dependent on water activity.

  • 88. Irani, Mehdi
    et al.
    Törnvall, Ulrika
    Genheden, Samuel
    Larsen, Marianne Wittrup
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hatti-Kaul, Rajni
    Ryde, Ulf
    Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 7, p. 1280-1289Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB,(M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.

  • 89. Javanmard, Mehdi
    et al.
    Babrzadeh, Farbod
    KTH, School of Biotechnology (BIO), Biochemistry.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Davis, Ronald W.
    Improvement in cell capture throughput using parallel bioactivated microfluidic channels2012In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 14, no 4, p. 625-629Article in journal (Refereed)
    Abstract [en]

    Optimization of targeted cell capture with microfluidic devices continues to be a challenge. On the one hand, microfluidics allow working with microliter volumes of liquids, whereas various applications in the real world require detection of target analyte in large volumes, such as capture of rare cell types in several ml of blood. This contrast of volumes (microliter vs. ml) has prevented the emergence of microfluidic cell capture sensors in the clinical setting. Here, we study the improvement in cell capture and throughput achieved using parallel bioactivated microfluidic channels. The device consists of channels in parallel with each other tied to a single channel. We discuss fabrication and testing of our devices, and show the ability for an improvement in throughput detection of target cells.

  • 90.
    Karlsson, Sara
    et al.
    KTH.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Malmström, Eva
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Ledarskap i kravfylld tid: utveckling genom utvärdering vid KTH2011Conference paper (Other academic)
  • 91.
    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)

  • 92.
    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.

  • 93.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression systemManuscript (preprint) (Other academic)
  • 94.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Zielinska, Dorota F.
    KTH, School of Biotechnology (BIO).
    Martinelle, Mats
    Hildalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    KTH, School of Biotechnology (BIO).
    Suppression of water asa nucleophile in Pseudozyma (Candida) antarctica lipase B catalysis.Manuscript (preprint) (Other academic)
  • 95.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kroutil, W
    Gruber, C.C.
    A tunnel provides the active site of a lipase withsubstrate water.Manuscript (preprint) (Other academic)
  • 96. Laytragoon-Lewin, Nongnit
    et al.
    Nilsson, Per J.
    Castro, Juan
    Gharizadeh, Baback
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Glimelius, Bengt
    Elmberger, Goran
    Turesson, Ingela
    Svensson, Christer
    Human papillomavirus (HPV), DNA aberrations and cell cycle progression in anal squamous cell carcinoma patients2007In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 27, no 6C, p. 4473-4479Article in journal (Refereed)
    Abstract [en]

    Human papillomavirus (HP) infections of the genital tract are sexually transmitted and prevalent worldwide. In this study, the role of HPV in 72 patients with anal squamous cell carcinoma was investigated. Patients and Methods: Polymerase chain reaction (PCR) in combination with in situ hybridization was used to identify HPV-DNA in the patients biopsies. The HPV typing was conducted by pyrosequencing. Cell cycle and DNA content were analysed by cytometry. Results: Ninety percent of the carcinoma biopsies carried high-risk oncogenic HPV in their malignant cells. Eighty-one percent of these demonstrated a single infection with HPV16, 18 or 33 and 19% were double infected with HPV16 and HPV18 Accumulations of viral genes were seen at the necrotic area of the tumours. The HPV genome in the tumour cell influenced significant the host cell cycle progression, but not DNA aberrations. Within these patients, HPV status in the malignant cells was not found to be associated with patient survival time. Conclusion: High-risk oncogenic HPV may play an important role in the initiation of host cell proliferation in anal squamous cell carcinoma. However, infection with HPV may not have any direct influence itself on the clinical outcome of these patients considering the treatments currently available.

  • 97. Leonard-Nevers, Valerie
    et al.
    Marton, Zsuzsanna
    Lamare, Sylvain
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Graber, Marianne
    Understanding water effect on Candida antarctica lipase B activity and enantioselectivity towards secondary alcohols2009In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 59, no 1-3, p. 90-95Article in journal (Refereed)
    Abstract [en]

    The effect of water activity (a(w)) on Candida antarctica lipase B (CALB) activity and enantioselectivity towards secondary alcohols was assessed. Experimental results for the resolution of racemic pentan-2-ol, hexan-3-ol, butan-2-ol and octan-4-ol by immobilized CALB-catalyzed acylation with methyl propanoate were obtained by using a solid/gas reactor. Water and substrate adsorption mechanism on immobilized CALB were then studied using moisture sorption analyzer and inverse gas chromatography, and the effective hydration state of the biocatalyst when varying aw was defined. The data showed a pronounced aw effect on both activity and enantioselectivity. If secondary alcohol follows the steric rules for being efficiently resolved, water at very low aw increased enantioselectivity by acting predominantly as an enantioselective inhibitor, making the stereospecificity pocket smaller. When increasing aw, water decreased enantioselectivity, due to an unfavourable increase of the entropic term T Delta(R-S)Delta S-double dagger of the differential free energy of activation. The "turning point" at which water changed from one predominant role to another would correspond to aw allowing full coverage of polar groups of the immobilized biocatalyst by water molecules.

  • 98. Lindback, E.
    et al.
    Gharizadeh, B.
    Ataker, F.
    Airell, A.
    Jalal, S.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Wretlind, B.
    DNA gyrase gene in Neisseria gonorrhoeae as indicator for resistance to ciprofloxacin and species verification2005In: International Journal of STD and AIDS (London), ISSN 0956-4624, E-ISSN 1758-1052, Vol. 16, no 2, p. 142-147Article in journal (Refereed)
    Abstract [en]

    We have identified a unique region of eight amino acids in the quinolone resistance-determining region in the gyrA gene in Neisseria gonorrhoeae as an indicator of resistance to fluoroquinolones. We sequenced that region by the Pyrosequencing(TM) technology in 46 N. gonorrhoeae strains and 11 urine samples positive in AMPLICOR(TM) N. gonorrhoeae polymerase chain reaction (Roche Diagnostics), with corresponding isolates of N. gonorrhoeae. The results showed that 28 samples with minimum inhibitory concentration (MIC) of ciprofloxacin > 1 mg/L had mutations in positions 91 and 95. Fifteen samples with MIC 0.125-1.0 mg/L had either one or both of the mutations. The 14 susceptible samples had no mutations. The target region also discriminates N. gonorrhoeae from other species of Neisseria. Our conclusion is that gyrA is an indicator of resistance to ciprofloxacin in N. gonorrhoeae and sequencing by Pyrosequencing technology is a suitable tool for analysis of DNA in urine samples.

  • 99. Lindback, E.
    et al.
    Gharizadeh, B.
    Ataker, F.
    Airell, A.
    Jalal, S.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Wretlind, B.
    Quinolone-resistant Neisseria gonorrhoeae - response2005In: International Journal of STD and AIDS (London), ISSN 0956-4624, E-ISSN 1758-1052, Vol. 16, no 12, p. 835-836Article in journal (Refereed)
  • 100.
    Liu, Rong
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Högberg, Hans-Erik
    Mittuniversitetet.
    Chemoenzymatic preparation of isomerically pure 3-bromo-2-butyl ethyl malonate esterManuscript (Other academic)
1234 51 - 100 of 167
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