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  • 1. Bassanini, Ivan
    et al.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. CNR, Italy.
    Riva, Sergio
    Dicarboxylic esters: Useful tools for the biocatalyzed synthesis of hybrid compounds and polymers2015Inngår i: Beilstein Journal of Organic Chemistry, ISSN 2195-951X, E-ISSN 1860-5397, Vol. 11, s. 1583-1595Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Dicarboxylic acids and their derivatives (esters and anhydrides) have been used as acylating agents in lipase-catalyzed reactions in organic solvents. The synthetic outcomes have been dimeric or hybrid derivatives of bioactive natural compounds as well as functionalized polyesters.

  • 2.
    Berglund, Per
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Branneby, Cecilia
    Svedendahl Humble, Maria
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Carlqvist, Peter
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Magnusson, Anders
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Brinck, Tore
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Aldol and Michael additions catalyzed by a rationally redesigned hydrolytic enzyme2003Inngår i: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 226, nr 2, s. U155-U156Artikkel i tidsskrift (Fagfellevurdert)
  • 3.
    Berglund, Per
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Vallikivi, I.
    Fransson, Linda
    KTH, Tidigare Institutioner, Bioteknologi.
    Dannacher, H.
    Holmquist, Mats
    KTH, Tidigare Institutioner, Bioteknologi.
    Martinelle, Mats
    KTH, Tidigare Institutioner, Bioteknologi.
    Björkling, F.
    Parve, O.
    Hult, Karl
    KTH, Tidigare Institutioner, Bioteknologi.
    Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes1999Inngår i: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 10, nr 21, s. 4191-4202Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R)-enantiomer preference for the smallest substrate, 2-phenoxypropanoic acid ester, to an (S)-enantiomer preference for the homologous esters with longer acyl moieties. The E-values span the range from E=13 (R) to E=56 (S). A molecular modeling study identified two different substrate-binding modes for each enantiomer. We found that the enantiomers favored different modes. This discovery provided a model that offered a rational explanation for the observed switch in enantioselectivity. (C) 1999 Elsevier Science Ltd. All rights reserved.

  • 4.
    Bernhardt, Peter
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Kazlauskas, R. J.
    Molecular basis of perhydrolase activity in serine hydrolases2005Inngår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 44, nr 18, s. 2742-2746Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    (Chemical Equation Presented) Changing substrates: A mutation that forms a cis-proline-peptide bond in a loop structure close to the active site of an aryl esterase from Pseudomonas fluorescens converts the enzyme into a perhydrolase (see picture). The switch in activity is explained by a new hydrogen bond formed between a backbone carbonyl oxygen atom and the peroxy deacylation intermediate.

  • 5.
    Branneby, Cecilia
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Carlqvist, Peter
    KTH, Tidigare Institutioner                               , Kemi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Brinck, Tore
    KTH, Tidigare Institutioner                               , Kemi.
    Berglund, Per
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations2004Inngår i: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 31, nr 4-6, s. 123-128Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.

  • 6. Branneby, Cecilia
    et al.
    Carlqvist, Peter
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Brinck, Tore
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Berglund, Per
    KTH, Tidigare Institutioner, Bioteknologi.
    Rational redesign of a lipase to an aldolase2003Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, nr 28, s. 8633-8633Artikkel i tidsskrift (Fagfellevurdert)
  • 7.
    Branneby, Cecilia
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Carlqvist, Peter
    KTH, Tidigare Institutioner                               , Kemi.
    Magnusson, Anders
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Brinck, Tore
    KTH, Tidigare Institutioner                               , Kemi.
    Berglund, Per
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Carbon-Carbon Bonds by Hydrolytic Enzymes2003Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 125, nr 4, s. 874-875Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases

  • 8.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2006Konferansepaper (Fagfellevurdert)
  • 9.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2005Inngår i: Book of abstracts, 2005Konferansepaper (Fagfellevurdert)
  • 10. Cammenberg, Maria
    et al.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Park, Seongsoon
    Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate2006Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, nr 11, s. 1745-1749Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 11. Carlqvist, P.
    et al.
    Eklund, R.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Brinck, Tore
    KTH, Tidigare Institutioner                               , Kemi.
    Rational design of a lipase to accommodate catalysis of Baeyer-Villiger oxidation with hydrogen peroxide2003Inngår i: Journal of Molecular Modeling, ISSN 1610-2940, E-ISSN 0948-5023, Vol. 9, nr 3, s. 164-171Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal mol(-1) and the second step by 19.5 kcal mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required.

  • 12.
    Carlqvist, Peter
    et al.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
    Svedendahl, Maria
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Brinck, Tore
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions2005Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, s. 331-336Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 13. Engstrom, Karin
    et al.
    Vallin, Michaela
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Backvall, Jan-E.
    Kinetic resolution of diarylmethanols using a mutated variant of lipase CALB2012Inngår i: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 68, nr 37, s. 7613-7618Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An enzymatic kinetic resolution of diarylmethanols via acylation has been developed. This was achieved by the use of a mutated variant of CALB that accepts larger substrates compared to the wild type. By the use of diarylmethanols with two differently sized aryl groups, enantioselective transformations were achieved. A larger size-difference led to a higher enantioselectivity. In addition, substrates with electronically different aryl groups, such as phenyl and pyridyl, also gave an enantioselective reaction. The highest E value was observed with a substrate where steric and electronic effects were combined.

  • 14.
    Engström, Karin
    et al.
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem.
    Vallin, Michaela
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Syrén, Per-Olof
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Bäckvall, Jan-E.
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem.
    Mutated variant of Candida antarctica lipase B in (S)-selective dynamic kinetic resolution of secondary alcohols2011Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, nr 1, s. 81-82Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An (S)-selective dynamic kinetic resolution of secondary alcohols, employing a mutated variant of Candida antarctica lipase B (CalB) gave products in 84-88% yield and in 90-97% ee.

  • 15.
    Eriksson, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Boyer, Antoine
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Ytbehandlingsteknik.
    Sinigoi, Loris
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Johansson, Mats
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Ytbehandlingsteknik.
    Malmström, Eva
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Ytbehandlingsteknik.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Trey, Stacy
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Ytbehandlingsteknik.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    One-Pot Enzymatic Route to Tetraallyl Ether Functional Oligoesters: Synthesis, UV Curing, and Characterization2010Inngår i: Journal of Polymer Science Part A: Polymer Chemistry, ISSN 0887-624X, E-ISSN 1099-0518, Vol. 48, nr 23, s. 5289-5297Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 16.
    Eriksson, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Fogelström, Linda
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Malmström, Eva
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Johansson, Mats
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Trey, Stacy
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Enzymatic One-Pot Route to Telechelic Polypentadecalactone Epoxide: Synthesis, UV Curing, and Characterization2009Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 11, s. 3108-3113Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 17.
    Eriksson, Magnus G.
    et al.
    KTH, Skolan för industriell teknik och management (ITM), Maskinkonstruktion (Inst.), Mekatronik.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Malmström, Eva
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Johansson, Mats
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Trey, Stacy M.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    One-pot enzymatic polycondensation to telechelic methacrylate-functional oligoesters used for film formation2011Inngår i: POLYM CHEM, ISSN 1759-9954, Vol. 2, nr 3, s. 714-719Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 18.
    Eriksson, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Enzymatic one-pot polycondensation to telechelic epoxy oligomersManuskript (preprint) (Annet vitenskapelig)
  • 19.
    Eriksson, Magnus
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Nilsson, Camilla
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Malmström, Eva
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Johansson, Mats
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Trey, Stacy
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Ytbehandlingsteknik.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    One-pot synthesis to functional free-standing polymer film for sensor applicationsManuskript (preprint) (Annet vitenskapelig)
  • 20.
    Fransson, Linda
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Bernhardt, Peter
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    On the benefit of an active siteManuskript (preprint) (Annet vitenskapelig)
  • 21.
    Fransson, Linda
    et al.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi. KTH, Skolan för bioteknologi (BIO), Biokemi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Laurell, Anna
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Widyan, Khalid
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Wingstrand, Erica
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Moberg, Christina
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Minor Enantiomer Recycling-Effect of Two Reinforcing Catalysts on Product Yield and Enantiomeric Excess2010Inngår i: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 2, nr 6, s. 683-693Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 22. Gardossi, Lucia
    et al.
    Poulsen, Pout B.
    Ballesteros, Antonio
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Svedas, Vytas K.
    Vasic-Racki, Durda
    Carrea, Giacomo
    Magnusson, Anders
    Schmid, Andreas
    Wohlgemuth, Roland
    Halling, Peter J.
    Guidelines for reporting of biocatalytic reactions2010Inngår i: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 28, nr 4, s. 171-180Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

  • 23.
    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, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Solvent as a competitive inhibitor for Candida antarctica lipase B2007Inngår i: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1774, nr 8, s. 1052-1057Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 24. Gustavsson, M.
    et al.
    Lehtio, J.
    Denman, S.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Martinelle, Mats
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris2001Inngår i: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 14, nr 9, s. 711-715Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wildtype lipase.

  • 25. Gustavsson, M. T.
    et al.
    Persson, P. V.
    Iversen, T.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase2005Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 6, nr 1, s. 196-203Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 26.
    Gustavsson, Malin T.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Persson, P. V.
    Iversen, T.
    Hult, Karl
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Martinelle, Mats
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein2004Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 5, nr 1, s. 106-112Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulose-binding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surfaces. The epsilon-caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(epsilon-caprolactone) to the surface hydroxyl groups but rather from surface-deposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant.

  • 27.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Kempka, Martin
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Sjödahl, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Roeraade, Johan
    KTH, Skolan för kemivetenskap (CHE), Kemi, Analytisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests2006Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, nr 2, s. 167-172Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 28.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lundgren, Stina
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Penhoat, Maël
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Moberg, Christina
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    High-Throughput Enzymatic Method for Enantiomeric Excess Determination of O-Acetylated Cyanohydrins2006Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, nr 7, s. 2234-2235Artikkel i tidsskrift (Fagfellevurdert)
  • 29.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lundgren, Stina
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Wingstrand, Erica
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Moberg, Christina
    KTH, Skolan för kemivetenskap (CHE), Kemi, Organisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    High Throughput Synthesis and Analysis of Acylated Cyanohydrins2007Inngår i: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 13, nr 15, s. 4334-4341Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 30.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Magnusson, Anders
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hu, Francis J.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    Selective monoacylation of diols by substrate assisted catalysis in T40A CALBManuskript (preprint) (Annet vitenskapelig)
  • 31.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Magnusson, Anders
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hu, Francis J.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Selective Monoacylation of Diols by Substrate Assisted Catalysis in T40A Candida antarctica Lipase B2013Inngår i: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, nr 3, s. 743-747Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 32.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Maurer, S.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Rational engineering of Candida antarctica lipase B for selective monoacylation of diols2012Inngår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 48, nr 80, s. 10013-10015Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 33.
    Hamberg, Anders
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Maurer, Steffen
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Rational engineering of CALB for selective monoacylation of diolsManuskript (preprint) (Annet vitenskapelig)
  • 34.
    Hedfors, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Hendil-Forssell, Peter
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Syrén, Per-Olof
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Takwa, Mohamad
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Selectivity towards itaconic acid esters by Candida antarctica lipase B and variantsManuskript (preprint) (Annet vitenskapelig)
  • 35.
    Hedfors, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Lipase chemoselectivity towards alcohol and thiol acyl acceptors in a transacylation reaction2010Inngår i: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 66, nr 1-2, s. 120-123Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 36.
    Hedfors, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Takwa, Mohamad
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Spinelli, Pietro
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Competition between lactones and polyesters in enzyme catalyzed ring-opening polymerizationManuskript (preprint) (Annet vitenskapelig)
  • 37.
    Hedfors, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Östmark, Emma
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Malmström, Eva E.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Thiol end-functionalization of poly(epsilon-caprolactone), catalyzed by Candida antarctica lipase B2005Inngår i: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 38, nr 3, s. 647-649Artikkel i tidsskrift (Fagfellevurdert)
    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 .

  • 38.
    Hedin, Eva . M. K.
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Fransson, L.inda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy2002Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 48, s. 14185-14196Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry, 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using, a water-soluble spin relaxation agent. chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (< 0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.

  • 39.
    Hedin, Eva M. K.
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    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 spectroscopy2005Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 50, s. 16658-16671Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 40.
    Hedin, Eva M. K.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Mouritsen, Ole G.
    Hoyrup, P.
    Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins2004Inngår i: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 60, nr 2, s. 117-138Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.

  • 41.
    Hedin, Eva. M. K.
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Patkar, S. A.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Vind, J.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Svendsen, A.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Berglund, Per
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Selective reduction and chemical modification of oxidized lipase cysteine mutants2002Inngår i: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 80, nr 6, s. 529-539Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.

  • 42. Heinze, Birgit
    et al.
    Kourist, Robert
    Fransson, Linda
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Bornscheuer, Uwe T.
    Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis2007Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, nr 3, s. 125-131Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 43.
    Hult, Karl
    et al.
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Berglund, Per
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Engineered enzymes for improved organic synthesis2003Inngår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 14, nr 4, s. 395-400Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Recent developments to modify enzymes for use in organic synthesis have targeted several areas. These include altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate specificity, and improving substrate specificity, such as enantioselectivity in kinetic resolutions. Such modifications can be achieved either by rational redesign, which requires knowledge of the enzyme structure, or by random mutagenesis methods followed by screening. Both strategies of enzyme engineering can be successful and are very useful for improving the utility of enzymes for applied catalysis. Several examples illustrating these concepts in a variety of enzyme classes have appeared recently.

  • 44.
    Hult, Karl
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Enzyme promiscuity: mechanism and applications2007Inngår i: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 25, nr 5, s. 231-238Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

  • 45.
    Jahic, Mehmedalija
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Rotticci-Mulder, Johanna C.
    Martinelle, Mats
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hult, Karl
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner, Bioteknologi.
    Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein2002Inngår i: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 24, nr 6, s. 385-393Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O-2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.

  • 46.
    Kourist, Robert
    et al.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Bartsch, Sebastian
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Fransson, Linda
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Bornscheuer, Uwe T.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Understanding promiscuous amidase activity of an esterase from Bacillus subtilis2008Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, nr 1, s. 67-69Artikkel i tidsskrift (Fagfellevurdert)
    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)

  • 47.
    Larsen, Marianne Wittrup
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Zielinska, Dorota F.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hidalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis2010Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, nr 6, s. 796-801Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 48.
    Larsen Wittrup, Marianne
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO).
    Jahic, Mehmedalija
    KTH, Skolan för bioteknologi (BIO).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression systemManuskript (preprint) (Annet vitenskapelig)
  • 49.
    Larsen Wittrup, Marianne
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Zielinska, Dorota F.
    KTH, Skolan för bioteknologi (BIO).
    Martinelle, Mats
    Hildalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    KTH, Skolan för bioteknologi (BIO).
    Suppression of water asa nucleophile in Pseudozyma (Candida) antarctica lipase B catalysis.Manuskript (preprint) (Annet vitenskapelig)
  • 50.
    Larsen Wittrup, Marianne
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Martinelle, Mats
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Kroutil, W
    Gruber, C.C.
    A tunnel provides the active site of a lipase withsubstrate water.Manuskript (preprint) (Annet vitenskapelig)
123 1 - 50 of 105
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