Change search
Refine search result
1 - 40 of 40
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Berglund, Per
    et al.
    KTH, Superseded Departments, Biotechnology.
    Branneby, Cecilia
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Carlqvist, Peter
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Magnusson, Anders
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Aldol and Michael additions catalyzed by a rationally redesigned hydrolytic enzyme2003In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 226, no 2, p. U155-U156Article in journal (Refereed)
  • 2.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Svedendahl Humble, Maria
    Branneby, Cecilia
    7.18 C-X Bond Formation: Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications2012In: Comprehensive Chirality, Elsevier, 2012, Vol. 7, p. 390-401Chapter in book (Refereed)
    Abstract [en]

    Enantiomerically pure amines and amino acids are important building blocks in academic research as well as in industrial-scale chemical production. Transaminases are versatile enzymes providing access to such compounds of high enantiomeric excess. This chapter illustrates the available strategies with transaminases such as kinetic resolution or stereoselective synthesis and highlights many successful examples for amino acid and chiral amines synthesis. There are some known challenges linked to the use of transaminases, for example in terms of unfavorable equilibria and inhibition. Several successful examples to overcome these limitations are presented. Also, the classification of transaminases, mechanistic details, and various strategies for optimization are discussed.

  • 3.
    Berglund, Per
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Abedi, Vahak
    AstraZeneca.
    Wells, Andrew
    AstraZeneca.
    Federsel, Hans-Jürgen
    AstraZeneca.
    Omega-Transaminases Redesigned for Chiral Amine Synthesis2011In: BIT Life Sciences’ 2nd Symposium on Enzymes & Biocatalysis, Dalian, China: BIT Life Sciences , 2011Conference paper (Refereed)
  • 4.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2006Conference paper (Refereed)
  • 5.
    Branneby, Cecilia
    et al.
    Cambrex Karlskoga AB.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase-Catalyzed Aldol and Michael-Type Reactions2005In: Book of abstracts, 2005Conference paper (Refereed)
  • 6.
    Carlqvist, Peter
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, p. 331-336Article in journal (Refereed)
    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.

  • 7. Cassimjee, Karim Engelmark
    et al.
    Hendil-Forssell, Peter
    Volkov, Alexey
    Krog, Anne
    Malmo, Jostein
    Aune, Trond Erik V.
    Knecht, Wolfgang
    Miskelly, Iain R.
    Moody, Thomas S.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Streamlined Preparation of Immobilized Candida antarctica Lipase B2017In: ACS OMEGA, ISSN 2470-1343, Vol. 2, no 12, p. 8674-8677Article in journal (Refereed)
    Abstract [en]

    Candida antarctica lipase B (CalB) was efficiently expressed (6.2 g L-1) in Escherichia coli by utilizing an N-terminal tag cassette and the XylS/Pm expression system in a fed-batch bioreactor; subsequent direct binding to EziG from crude extracts resulted in an immobilized catalyst with superior activity to Novozym 435.

  • 8.
    Cassimjee, Karim Engelmark
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedi, Vahak
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation2012In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 28, p. 5466-5470Article in journal (Refereed)
    Abstract [en]

    For biocatalytic production of pharmaceutically important chiral amines the.-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum omega-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (similar to 5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

  • 9.
    Cassimjee, Karim Engelmark
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Miceli, Valentina
    KTH, School of Biotechnology (BIO), Biochemistry.
    Colomina, Carla Granados
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction2011In: ACS CATAL, ISSN 2155-5435, Vol. 1, no 9, p. 1051-1055Article in journal (Refereed)
    Abstract [en]

    Measurement of the active enzyme fraction in a given enzyme preparation is a requirement for accurate kinetic measurements and activity comparisons of, for example, engineered mutants. omega-Transaminases, enzymes capable of interconverting ketones and amines by use of pyridoxal-5'-phosphate (PIP), can be used for the production of pharmaceutically important chiral amines but are subject to engineering to meet the practical requirements in synthesis reactions. Therefore, an active site quantification method is needed. Such a method was developed by quantifying the amount of consumed substrate in a virtually irreversible half transamination reaction. (S)-1-phenylethylamine was converted to acetophenone, while the holo enzyme (E-PLP) was converted to apo enzyme with bound pyridoxamine-5'-phosphate (E:PMP). Further, the mass of active enzyme was correlated to the absorbance of the holo enzyme to achieve a direct measurement method. The active Chromobacterium violaceum omega-transaminase with bound PLP can be quantified at 395 nm with an apparent extinction coefficient of 8.1 mM(-1) cm(-1).

  • 10.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum2018In: Molecular Catalysis, ISSN 2468-8231, Vol. 446, p. 115-123Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5’-phosphate (PLP)dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer.The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

  • 11.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Characterization of the operational stability of a transaminase from Vibrio fluvialisManuscript (preprint) (Other academic)
  • 12.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Inactivation pathway underlying the operational instability of an amine transaminase from Chromobacterium violaceumManuscript (preprint) (Other academic)
  • 13.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Characterization of the stability of Vibrio fluvialis JS17 amine transaminase2018In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 282, p. 10-17Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.

  • 14.
    Chen, Shan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Stabilization of an amine transaminase for biocatalysis2016In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 124, p. 20-28Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.

  • 15.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedi, Vahak
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton ParameterisationManuscript (preprint) (Other academic)
  • 16.
    Engelmark Cassimjee, Karim
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational Redesign of omega-Transaminase2010In: Biocat2010, Hamburg, Germany: TuTech Verlag , 2010Conference paper (Refereed)
  • 17.
    Land, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Uppsala University, Sweden.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Pharem Biotech AB, Sweden.
    YASARA: A tool to obtain structural guidance in biocatalytic investigations2018In: Protein Engineering: Methods and Protocols, Humana Press, 2018, Vol. 1685, p. 43-67Chapter in book (Refereed)
    Abstract [en]

    In biocatalysis, structural knowledge regarding an enzyme and its substrate interactions complements and guides experimental investigations. Structural knowledge regarding an enzyme or a biocatalytic reaction system can be generated through computational techniques, such as homology- or molecular modeling. For this type of computational work, a computer program developed for molecular modeling of proteins is required. Here, we describe the use of the program YASARA Structure. Protocols for two specific biocatalytic applications, including both homology modeling and molecular modeling such as energy minimization, molecular docking simulations and molecular dynamics simulations, are shown. The applications are chosen to give realistic examples showing how structural knowledge through homology and molecular modeling is used to guide biocatalytic investigations and protein engineering studies.

  • 18. Löfgren, J.
    et al.
    Görbe, T.
    Oschmann, M.
    Svedendahl, Maria
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Bäckvall, J. -E
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Article in journal (Refereed)
    Abstract [en]

    Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.

  • 19.
    Löfgren, Johanna
    et al.
    Stockholm Univ, Dept Organ Chem, Stockholm, Sweden..
    Gorbe, Tamas
    Stockholm Univ, Dept Organ Chem, Stockholm, Sweden..
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Bäckvall, Jan-Erling
    Stockholm Univ, Dept Organ Chem, Stockholm, Sweden..
    Towards improved activity of Candida antarctica Lipase A (CalA) against tertiary alcohols2017In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal (Other academic)
  • 20.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Biocatalytic Promiscuity2011In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 19, p. 3391-3401Article, review/survey (Refereed)
    Abstract [en]

    Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio-, chemo- and stereo-selective transformations. Enzyme promiscuity allows optimisation of industrial processes that require reaction conditions different from those in nature. Many enzymes can be used in reactions completely different from the reaction the enzyme originally evolved to perform. Such catalytically promiscuous reactions can be secondary activities hidden behind a native activity and might be discovered either in screening for that particular activity or, alternatively, by chance. Recently, researchers have designed enzymes to show catalytic promiscuity. It is also possible to design new enzymes from scratch by computer modelling (de novo design), but most work published to date starts from a known enzyme backbone. Promiscuous activity might also be induced or enhanced by rational design or directed evolution (or combinations thereof). Enzyme catalytic promiscuity provides fundamental knowledge about enzyme/substrate interactions and the evolution of new enzymes. New enzymes are required by industry, which needs to optimise chemical processes in an environmentally sustainable way. In this review various aspects of enzyme catalytic promiscuity are considered from a biocatalytic perspective.

  • 21.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedu, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase2012In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 4, no 8, p. 1167-1172Article in journal (Refereed)
    Abstract [en]

    Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2-propylamine. Most known ?-transaminases are (S)-selective and there is, therefore, a need of (R)-selective enzymes. We report the successful rational design of an (S)-selective ?-transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ?-transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ?-transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site-directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15-fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2-aminotetralin.

  • 22.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Håkansson, Maria
    Kimbung, Yengo R
    Walse, Björn
    Abedi, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Logan, Derek T
    Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, p. 779-792Article in journal (Refereed)
    Abstract [en]

    The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: •  -transaminases and -transaminases bind by dynamic light scattering (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction).

  • 23.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO).
    Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B2009Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

  • 24.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lipase and ω-Transaminase: Biocatalytic Investigations2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition.

  • 25.
    Svedendahl, Maria
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Carlqvist, Peter
    KTH, Superseded Departments, Chemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Michael-type additions catalyzed by a rationally redesigned lipase2004Conference paper (Refereed)
  • 26.
    Svedendahl, Maria
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Branneby, Cecilia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Carlqvist, Peter
    KTH, Superseded Departments, Chemistry.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Brinck, Tore
    KTH, Superseded Departments, Chemistry.
    Berglund, Per
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Expanding the Synthetic Scope of Hydrolytic Enzymes: Catalysis of Aldol- and Michael-Type Additions2004Conference paper (Refereed)
  • 27.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Lindberg, Lina
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Reversed Enantiopreference of an omega-Transaminase by a Single-Point Mutation2010In: CHEMCATCHEM, ISSN 1867-3880, Vol. 2, no 8, p. 976-980Article in journal (Refereed)
    Abstract [en]

    Altering the characteristics of an active-site loop in an (S)-selective omega-transaminase from Arthrobacter citreus (variant CNB05-01) influences the enantioselectivity. This active-site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal-5'-phosphate (PLP) in the so called "phosphate group binding cup". Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site-directed mutagenesis aiming to increase the enzyme enantioselectivity for 4-fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4-fluorophenylacetone or 4-nitroacetophenone in asymmetric synthesis using a whole-cell system approach. Three of the five variants show increased enantioselectivity for 4-fluorophenylacetone compared to CNB05-01. Variant CNB05-01/Y331C increases the enantioselectivity from 98% ee to over 99.5% ee. A single-point mutation, V328A, turn the (S)-selective omega-transaminase into an (R)-selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting (R) selectivity for 4-fluorophenylacetone and retaining (S) selectivity for 4-nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.

  • 28.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Lindberg, Lina
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Reversed enantiopreference of an ω-transaminase by a single-point mutationManuscript (preprint) (Other academic)
  • 29.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Carlqvist, Peter
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Allnér, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Frise, Anton
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 15, p. 2443-2451Article in journal (Refereed)
    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.

  • 30.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Abedi, Vahak
    AstraZeneca.
    Federsel, Hans-Jürgen
    AstraZeneca.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    From S to R: Key Residues Controlling Enantiomer Preference and Activity in omega-Transaminase2011Conference paper (Refereed)
  • 31.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Branneby, C.
    Abedi, V.
    Wells, A.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    CASCAT: Redesign of omega-Transaminases for Synthesis of Chiral Amines2010In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 150, p. S123-S124Article in journal (Other academic)
    Abstract [en]

    Transaminases (EC 2.6.1.18) are attractive biocatalysts for synthesis of chiral amines and alpha-amino acids. These enzymes catalyze transfer of an amine group from a donor substrate to an acceptor compound using the cofactor pyridoxal-5′-phoshate (PLP). omega-Transaminases are a versatile subgroup of the transaminases that does not require a carboxylic acid group in alpha-position (in contradiction toalpha-transaminases) and hence accept a wider spectrum of ketones or amines. The omega-transaminases are employed industrially for production of both R- and S-enantiopure amines.

    One bottleneck is the unfavourable equilibrium in such reactions run in the synthesis mode. We have developed a one-pot multi-enzyme system in a cascade fashion for equilibrium displacement by removing formed acetone.

    Another issue is the fact that most omega-transaminases show S-selectivity, however a few R-selective strains do exist. We have used an S-selective omega-transaminase variant from Arthrobacter citreus and created an R-selective variant by rational redesign using a homology enzyme model. This homology modelling/rational design approach was further explored on an omega-transaminase from Chromobacterium violaceum.

  • 32.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    Cambrex Karlskoga AB.
    Sjöstrand, Ulf
    Cambrex Karlskoga AB.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Rational Redesign of ω-Transaminases2010Conference paper (Refereed)
  • 33.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploring Promiscuous Activities in a Lipase2009Conference paper (Refereed)
  • 34.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Fast carbon-carbon bond formation by a promiscuous lipase2005In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 51, p. 17988-17989Article in journal (Refereed)
  • 35.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Catalytic Promiscuity of a Lipase for Direct Epoxidation Reactions2007In: / [ed] Vicente Gotor, 2007Conference paper (Refereed)
  • 36.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Promiscuous Reactions in Candida antarctica lipase B2008Conference paper (Refereed)
  • 37.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Jovanovic, Biljana
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    A Non-Hydrolytic Lipase Mutant with Michael Addition Activity for Esters in Water2008In: Biocat2008 / [ed] Ralf Grote, Garabed Antranikian, 2008Conference paper (Refereed)
  • 38.
    Svedendahl, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Jovanovic, Biljana
    KTH, School of Biotechnology (BIO), Biochemistry.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water2009In: CHEMCATCHEM, ISSN 1867-3880, Vol. 1, no 2, p. 252-258Article in journal (Refereed)
    Abstract [en]

    Suppression of,the,native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

  • 39.
    Vongvilai, Pornrapee
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Linder, Mats
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Sakulsombat, Morakot
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Racemase Activity of B. cepacia Lipase Leads to Dual-Function Asymmetric Dynamic Kinetic Resolution of alpha-Aminonitriles2011In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 50, no 29, p. 6592-6595Article in journal (Refereed)
    Abstract [en]

    Applaudable promiscuity: Racemase-type activity discovered for B. cepacia lipase with N-substituted α-aminonitriles is proposed to involve a C-C bond-breaking/forming mechanism in the hydrolase site of the enzyme, as supported by experimental data and calculations. This promiscuous activity in combination with the transacylation activity of the enzyme enabled the asymmetric synthesis of N-methyl α-aminonitrile amides in high yield (see scheme).

  • 40. Wikmark, Ylva
    et al.
    Humble, Maria Svedendahl
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Stockholm University, Sweden .
    Backvall, Jan-E.
    Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent2015In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 14, p. 4284-4288Article in journal (Refereed)
    Abstract [en]

    A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His(6)-tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.

1 - 40 of 40
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf