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  • 1.
    Engström, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Enantioselective biotransformations using engineered lipases from Candida antarctica2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described. 

    In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type.

    An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties.

    In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended.

    First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved.

  • 2.
    Engström, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Protein engineering of enzymes for improved enantioselectivity and application of engineered enzymes in organic synthesis2010Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
  • 3.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Johnston, Eric V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Verho, Oscar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Gustafson, Karl P. J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Shakeri, Mozaffar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Tai, Cheuk-Wai
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Bäckvall, Jan-E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Co-immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme2013Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 52, nr 52, s. 14006-14010Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Surpassing nature: A hybrid catalyst in which Candida antarctica lipase B and a nanopalladium species are co-immobilized into the compartments of mesoporous silica is presented. The metal nanoparticles and the enzyme are in close proximity to one another in the cavities of the support. The catalyst mimics a metalloenzyme and was used for dynamic kinetic resolution of a primary amine in high yield and excellent enantioselectivity.

  • 4.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Nyhlén, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Sandström, Anders G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-Erling
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters2010Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, nr 20, s. 7038-7042Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of α-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45−276, which is a large improvement compared to 2−20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95−99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

  • 5.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Nyhlén, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Sandström, Anders G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-Erling
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Enantioselective Kinetic Resolution of p-Nitrophenyl 2-Phenylpropanoate by a Variant of Candida antarctica Lipase A Developed by Directed Evolution2010Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, nr 20, s. 7038-7042Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of α-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45−276, which is a large improvement compared to 2−20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95−99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

  • 6.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Shakeri, Mozaffar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Dynamic Kinetic Resolution of β-Amino Esters by a Heterogeneous System of a Palladium Nanocatalyst and Candida antarctica Lipase A2011Ingår i: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, nr 10, s. 1827-1830Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A dynamic kinetic resolution (DKR) of β-amino esters have been developed by the use of a heterogeneous racemization catalyst and an immobilized enzyme that accepts aromatic, heteroaromatic and aliphatic substrates. The reaction conditions were optimized to yield an efficient catalytic system without by-product formation. The products are obtained in 96–99 % ee and high yields

  • 7.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Vallin, Michaela
    Hult, Karl
    Bäckvall, Jan-E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Kinetic resolution of diarylmethanols using a mutated variant of lipase CALB2012Ingår i: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 68, nr 37, s. 7613-7618Artikel i tidskrift (Refereegranskat)
    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.

  • 8.
    Engström, Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Vallin, Michaela
    Syrén, Per-Olof
    Hult, Karl
    Bäckvall, Jan-E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Mutated variant of Candida antarctica lipase B in (S)-selective dynamickinetic resolution of secondary alcohols2011Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, nr 1, s. 81-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 9.
    Sandström, Anders G.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Engström, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Nyhlén, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Kasrayan, Alex
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-Erling
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid2009Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 22, nr 7, s. 413-420Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).

  • 10.
    Sandström, Anders G.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Wikmark, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Engström, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Nyhlén, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library2012Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, nr 1, s. 78-83Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.

  • 11.
    Shakeri, Mozaffar
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Engström, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Sandström, Anders G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Bäckvall, Jan-Erling
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Highly enantioselective resolution of β-amino esters by Candida antarctica lipase A immobilized in mesocellular foam: application to dynamic kinetic resolution2010Ingår i: ChemCatChem, ISSN 1867-3899, Vol. 2, nr 5, s. 534-538Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Candida antarctica lipase A (CALA) immobilized in functionalized mesocellular foam in the presence of sucrose, followed by lyophilization, led to a dramatic increase in the enantioselectivity as well as an improved thermostability of the enzyme. The immobilized lipase was used for kinetic resolution (KR) and dynamic kinetic resolution (DKR) of the β-amino ester, ethyl 3-amino-3-phenylpropanoate. The temperature of optimum activity of CALA shifted from 20–30 °C to 80–90 °C on immobilization in the MCF. An “enantiomeric ratio” E (E=νA/νB; νA and νB are the rate constants for entantiomers A and B) of 69 and a conversion of 43 % in 1 h were obtained at 80 °C, whereas non-immobilized CALA lost its activity at T≥50 °C. The obtained immobilized CALA showed an E value of greater than 500 at 22 °C. Combination of the immobilized CALA with a ruthenium complex, acting as a racemization catalyst, allowed for a successful DKR of ethyl 3-amino-3-phenylpropanoate resulting in 85 % conversion and 89 % ee.

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