Change search
Link to record
Permanent link

Direct link
BETA
Alternative names
Publications (10 of 34) Show all publications
Löfgren, J., Görbe, T., Oschmann, M., Svedendahl, M. & Bäckvall, J.-E. -. (2019). Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A. ChemBioChem (Print)
Open this publication in new window or tab >>Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A
Show others...
2019 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Article in journal (Refereed) In press
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.

Place, publisher, year, edition, pages
Wiley-VCH Verlag, 2019
Keywords
biocatalysis, enzymes, kinetic resolution, protein engineering, tertiary alcohols
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-252237 (URN)10.1002/cbic.201800792 (DOI)000474071700014 ()2-s2.0-85064051728 (Scopus ID)
Note

QC 20190610

Available from: 2019-06-10 Created: 2019-06-10 Last updated: 2019-10-29Bibliographically approved
Chen, S., Land, H., Berglund, P. & Svedendahl Humble, M. (2016). Stabilization of an amine transaminase for biocatalysis. Journal of Molecular Catalysis B: Enzymatic, 124, 20-28
Open this publication in new window or tab >>Stabilization of an amine transaminase for biocatalysis
2016 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 124, p. 20-28Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2016
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-180821 (URN)10.1016/j.molcatb.2015.11.022 (DOI)000370458100003 ()2-s2.0-84949440870 (Scopus ID)
Note

QC 20160126. QC 20160319

Available from: 2016-01-24 Created: 2016-01-24 Last updated: 2018-03-19Bibliographically approved
Wikmark, Y., Humble, M. S. & Backvall, J.-E. (2015). Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent. Angewandte Chemie International Edition, 54(14), 4284-4288
Open this publication in new window or tab >>Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent
2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 14, p. 4284-4288Article in journal (Refereed) Published
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.

Keywords
biocatalysis, kinetic resolution, lipase A, protein engineering, secondary alcohols
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-165210 (URN)10.1002/anie.201410675 (DOI)000351679600024 ()25676632 (PubMedID)2-s2.0-84925582964 (Scopus ID)
Note

QC 20150507

Available from: 2015-05-07 Created: 2015-04-24 Last updated: 2017-12-04Bibliographically approved
Berglund, P., Svedendahl Humble, M. & Branneby, C. (2012). 7.18 C-X Bond Formation: Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications. In: Comprehensive Chirality: (pp. 390-401). Elsevier, 7
Open this publication in new window or tab >>7.18 C-X Bond Formation: Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications
2012 (English)In: 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.

Place, publisher, year, edition, pages
Elsevier, 2012
Keywords
Amino acids, Aminotransferase, Chiral amines, Enzyme engineering, Equilibrium displacement, Product inhibition, Pyridoxal 5'-phosphate
National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:kth:diva-162995 (URN)10.1016/B978-0-08-095167-6.00723-0 (DOI)2-s2.0-84899893722 (Scopus ID)978-008095168-3 (ISBN)
Note

QC 20150327. QC 20160207

Available from: 2015-03-27 Created: 2015-03-26 Last updated: 2016-02-07Bibliographically approved
Cassimjee, K. E., Humble, M. S., Land, H., Abedi, V. & Berglund, P. (2012). Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation. Organic and biomolecular chemistry, 10(28), 5466-5470
Open this publication in new window or tab >>Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
Show others...
2012 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 28, p. 5466-5470Article in journal (Refereed) Published
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.

Keywords
OPTICALLY-ACTIVE AMINES; ASYMMETRIC-SYNTHESIS; CHIRAL AMINES; SUBSTRATE-SPECIFICITY; RESONANCE COMPONENTS; CHEMICAL-REACTIVITY; AMINOTRANSFERASE; SUBSTITUENT; IDENTIFICATION; BIOCATALYSIS
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-99250 (URN)10.1039/c2ob25893e (DOI)000305764600020 ()2-s2.0-84863611002 (Scopus ID)
Note
QC 20120724Available from: 2012-07-24 Created: 2012-07-23 Last updated: 2017-12-07Bibliographically approved
Svedendahl Humble, M., Engelmark Cassimjee, K., Håkansson, M., Kimbung, Y. R., Walse, B., Abedi, V., . . . Logan, D. T. (2012). Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.. The FEBS Journal, 279(5), 779-792
Open this publication in new window or tab >>Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
Show others...
2012 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, p. 779-792Article in journal (Refereed) Published
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).

National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-74820 (URN)10.1111/j.1742-4658.2012.08468.x (DOI)000300665900009 ()22268978 (PubMedID)2-s2.0-84857650697 (Scopus ID)
Note
QC 20120309Available from: 2012-02-03 Created: 2012-02-03 Last updated: 2017-12-08Bibliographically approved
Svedendahl Humble, M., Engelmark Cassimjee, K., Abedu, V., Federsel, H.-J. & Berglund, P. (2012). Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase. ChemCatChem, 4(8), 1167-1172
Open this publication in new window or tab >>Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase
Show others...
2012 (English)In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 4, no 8, p. 1167-1172Article in journal (Refereed) Published
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.

Keywords
Amination, Biocatalysis, Enantiopreference, Enantiospecificity, Protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-92286 (URN)10.1002/cctc.201100487 (DOI)000306907700020 ()2-s2.0-84864374814 (Scopus ID)
Note

QC 20120905

Available from: 2012-03-30 Created: 2012-03-30 Last updated: 2017-12-07Bibliographically approved
Cassimjee, K. E., Humble, M. S., Miceli, V., Colomina, C. G. & Berglund, P. (2011). Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction. ACS CATAL, 1(9), 1051-1055
Open this publication in new window or tab >>Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
Show others...
2011 (English)In: ACS CATAL, ISSN 2155-5435, Vol. 1, no 9, p. 1051-1055Article in journal (Refereed) Published
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).

Keywords
aminotransferase, chiral amines, pyridoxal-5 '-phosphate, PLP, biocatalysis, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-41299 (URN)10.1021/cs200315h (DOI)000294704500010 ()
Note
QC 20110927Available from: 2011-09-27 Created: 2011-09-26 Last updated: 2012-04-02Bibliographically approved
Svedendahl Humble, M. & Berglund, P. (2011). Biocatalytic Promiscuity. European Journal of Organic Chemistry (19), 3391-3401
Open this publication in new window or tab >>Biocatalytic Promiscuity
2011 (English)In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 19, p. 3391-3401Article, review/survey (Refereed) Published
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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2011
Keywords
Enzyme catalysis, Biocatalysis, Biotransformations, Catalytic promiscuity, Protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-37553 (URN)10.1002/ejoc.201001664 (DOI)000293133100001 ()2-s2.0-79959757238 (Scopus ID)
Note

QC 20110815

Available from: 2011-08-15 Created: 2011-08-15 Last updated: 2017-12-08Bibliographically approved
Svedendahl, M., Engelmark Cassimjee, K., Abedi, V., Federsel, H.-J. & Berglund, P. (2011). From S to R: Key Residues Controlling Enantiomer Preference and Activity in omega-Transaminase. Paper presented at Biotrans2011, Giardini Naxos, Italy, October 2 – 6 2011.
Open this publication in new window or tab >>From S to R: Key Residues Controlling Enantiomer Preference and Activity in omega-Transaminase
Show others...
2011 (English)Conference paper, Published paper (Refereed)
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-74488 (URN)
Conference
Biotrans2011, Giardini Naxos, Italy, October 2 – 6 2011
Note
QC 20120206Available from: 2012-02-03 Created: 2012-02-03 Last updated: 2012-02-06Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2371-8755

Search in DiVA

Show all publications