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  • 1.
    Branneby, Cecilia
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Epoxidation catalyzed by a CALB mutantManuskript (preprint) (Annet vitenskapelig)
  • 2.
    Branneby, Cecilia
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Exploiting enzyme promiscuity for rational design2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area.

    Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity.

    In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant.

    In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

  • 3.
    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.

  • 4.
    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

  • 5.
    Branneby, Cecilia
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Park, Seongsoon
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Investigation of Substrate Specificity of Geobacillus stearothermophilus Alanine RacemaseManuskript (Annet vitenskapelig)
  • 6.
    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)
  • 7.
    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)
  • 8.
    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.

  • 9.
    Cassimjee, Karim Engelmark
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Trummer, Martin
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Silica-immobilized His(6)-tagged enzyme: Alanine racemase in hydrophobic solvent2008Inngår i: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, nr 3, s. 712-716Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His(6)-tagging.

  • 10.
    Engelmark Cassimjee, Karim
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    High Yield Transamination with Isopropyl Amine as Donor, by Employment of YADH and in situ Cofactor Regeneration2009Konferansepaper (Fagfellevurdert)
  • 11.
    Engelmark Cassimjee, Karim
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    High Yield Transamination with Isopropyl Amine as Donor, by Employment of YADH and in situ Cofactor Regeneration2009Inngår i: Book of abstracts, 2009Konferansepaper (Fagfellevurdert)
  • 12.
    Svedendahl, Maria
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Carlqvist, Peter
    KTH, Tidigare Institutioner, Kemi.
    Brinck, Tore
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Hult, Karl
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Berglund, Per
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Michael-type additions catalyzed by a rationally redesigned lipase2004Konferansepaper (Fagfellevurdert)
  • 13.
    Svedendahl, Maria
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Branneby, Cecilia
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Carlqvist, Peter
    KTH, Tidigare Institutioner, Kemi.
    Hult, Karl
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Brinck, Tore
    KTH, Tidigare Institutioner, Kemi.
    Berglund, Per
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Expanding the Synthetic Scope of Hydrolytic Enzymes: Catalysis of Aldol- and Michael-Type Additions2004Konferansepaper (Fagfellevurdert)
  • 14.
    Svedendahl, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Carlqvist, Peter
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
    Branneby, Cecilia
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Allnér, Olof
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
    Frise, Anton
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Brinck, Tore
    Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory2008Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, nr 15, s. 2443-2451Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

  • 15.
    Svedendahl, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Engelmark Cassimjee, Karim
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Rational Redesign of ω-Transaminases2010Konferansepaper (Fagfellevurdert)
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