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  • 1.
    Cassimjee, Karim Engelmark
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kourist, Robert
    Lindberg, Diana
    Larsen, Marianne Wittrup
    KTH, School of Biotechnology (BIO), Biochemistry.
    Thanh, Nguyen Hong
    KTH, School of Biotechnology (BIO), Biochemistry.
    Widersten, Mikael
    Bornscheuer, Uwe T.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    One-step enzyme extraction and immobilization for biocatalysis applications2011In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 4, p. 463-469Article in journal (Refereed)
    Abstract [en]

    An extraction/immobilization method for His(6)-tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His 6 tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

  • 2.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kourist, Robert
    University of Greifswald, Germany.
    Lindberg, Diana
    Uppsala university, SE.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry.
    Widersten, Mikael
    Uppsala university, SE.
    Bornscheuer, Uwe T
    University of Greifswald, DE.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    A One Step Enzyme Extraction and Immobilization Method for Organic and Aqueous Solvents2008In: Biocat2008 / [ed] Ralf Grote, Garabed Antranikian, Hamburg, Germany: TuTech Innovation GmbH , 2008Conference paper (Refereed)
  • 3.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Kourist, Robert
    University of Greifswald, Germany.
    Lindberg, Diana
    Uppsala university, SE.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Widersten, Mikael
    Uppsala university, SE.
    Bornscheuer, Uwe T
    University of Greifswald, DE.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    A One Step General Enzyme Immobilization Method for Organic and Aqueous Solvents2008In: Book of Abstracts, 2008Conference paper (Refereed)
  • 4. Irani, Mehdi
    et al.
    Törnvall, Ulrika
    Genheden, Samuel
    Larsen, Marianne Wittrup
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hatti-Kaul, Rajni
    Ryde, Ulf
    Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 7, p. 1280-1289Article in journal (Refereed)
    Abstract [en]

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

  • 5.
    Larsen, Marianne Wittrup
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Zielinska, Dorota F.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hidalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 796-801Article in journal (Refereed)
    Abstract [en]

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

  • 6.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression systemManuscript (preprint) (Other academic)
  • 7.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Zielinska, Dorota F.
    KTH, School of Biotechnology (BIO).
    Martinelle, Mats
    Hildalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    KTH, School of Biotechnology (BIO).
    Suppression of water asa nucleophile in Pseudozyma (Candida) antarctica lipase B catalysis.Manuscript (preprint) (Other academic)
  • 8.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kroutil, W
    Gruber, C.C.
    A tunnel provides the active site of a lipase withsubstrate water.Manuscript (preprint) (Other academic)
  • 9. Neubauer, A.
    et al.
    Golson, R.
    Ukkonen, K.
    Krause, M.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Neubauer, P.
    Vasala, A.
    Controlling nutrient release in cell cultivation2009In: Genetic Engineering and Biotechnology News, ISSN 1935-472X, Vol. 29, no 11, p. 50-51Article in journal (Refereed)
  • 10.
    Takwa, Mohamad
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide2011In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 47, no 26, p. 7392-7394Article in journal (Refereed)
    Abstract [en]

    Based on molecular modelling, the enzyme Candida antarctica lipase B was redesigned as a catalyst for the ring opening polymerization of D, D-lactide. Two mutants with 90-fold increased activity as compared to the wild-type enzyme were created. In a preparative synthesis of poly(D,D-lactide) the mutants greatly improved the rate and the degree of polymerization.

  • 11.
    Wittrup Larsen, Marianne
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Expression of a lipase in prokaryote and eukaryote host systems allowing engineering2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pseudozyma (Candida) antarctica lipase B (PalB) was expressed in Escherichia coli facilitating protein engineering. The lack of glycosylation was evaluated for a deeper understanding of the difficulties in expressing PalB in E. coli. Different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3), Origami 2(DE3), and coexpression of groES and groEL. Periplasmic expression resulted 5.2 mg/L active PalB at 16 °C in shake flasks. This expression level was improved by using the EnBase technology, enabling fed-batch cultivation in 24-deep well scale. The feed rate was titrated with the addition of α-amylase, which slowly releases glucose as energy source. Different media were evaluated where the EnBase mineral salt medium resulted in 7.0 mg/L of active PalB.

    Protein secreted directly into the media was obtained using the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for screening and production of PalB in P. pastoris. A protease sensitive fusion protein CBM-PalB (cellulose-binding module) was used as a model system. When optimised, the expression system resulted in 46 mg/L lipase in 72 hours in shake flask, 37 mg/L lipase in 28 hours in 96-deep-well plate format, and 2.9 g PalB per 10 L bioreactor cultivation.

    The E. coli expression system was used to express a small focused library of PalB variants, designed to prevent water from entering the active site through a hypothesised tunnel. Screening of the library was performed with a developed assay, allowing for simultaneous detection of both transacylation and hydrolytic activity. From the library a mutant S47L, in which the inner part of the tunnel was blocked, was found to catalyse transacylation of vinyl butyrate in 20 mM butanol 14 times faster than hydrolysis. Water tunnels, assisting water in reaching the active sites, were furthermore found by molecular modelling in many hydrolases. Molecular modelling showed a specific water tunnel in PalB. This was supported by experimental data, where the double mutant Q46A S47L catalysed transacylation faster than hydrolysis compared to the wild type PalB.

  • 12.
    Wittrup Larsen, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems2008In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, p. 90-97Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in A pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7lac promoter.

    Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2 mg/L culture at 16 degrees C, which is an improvement to previous reports.

    The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC-ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.

1 - 12 of 12
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