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Publications (10 of 11) Show all publications
Land, H., Campillo-Brocal, J. C., Svedendahl Humble, M. & Berglund, P. (2019). B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization. ChemBioChem (Print), 20(10), 1297-1304
Open this publication in new window or tab >>B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization
2019 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed) Published
Abstract [en]

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2019
Keywords
Biocatalysis, Enzymes, Enzyme Stabilization, Protein engineering, Molecular modeling
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-243023 (URN)10.1002/cbic.201800749 (DOI)2-s2.0-85063236398 (Scopus ID)
Funder
Carl Tryggers foundation
Note

QC 20190225

Available from: 2019-02-01 Created: 2019-02-01 Last updated: 2019-05-27Bibliographically approved
Land, H., Hendil-Forssell, P., Martinelle, M. & Berglund, P. (2016). One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones. catalysis science & technology, 6, 2897-2900
Open this publication in new window or tab >>One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
2016 (English)In: catalysis science & technology, ISSN 2044-4753, Vol. 6, p. 2897-2900Article in journal (Refereed) Published
Abstract [en]

An efficient one-pot one-step biocatalytic amine transaminase/acyl transferase cascade for the formation of amides from the corresponding aldehydes and ketones in aqueous solution has been developed. N-benzyl-2-methoxyacetamide has been synthesized utlilizing the developed cascade in conversions up to 97%. The cascade was also evaluated for the synthesis of chiral amides.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2016
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-185329 (URN)10.1039/C6CY00435K (DOI)000375545600004 ()2-s2.0-84967261237 (Scopus ID)
Note

QC 20160422

Available from: 2016-04-16 Created: 2016-04-16 Last updated: 2016-11-24Bibliographically 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
Steffen-Munsberg, F., Vickers, C., Kohls, H., Land, H., Mallin, H., Nobili, A., . . . Bornscheuer, U. T. (2015). Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications. Biotechnology Advances, 33(5), 566-604
Open this publication in new window or tab >>Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications
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2015 (English)In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 5, p. 566-604Article in journal (Refereed) Published
Abstract [en]

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

Keywords
Annotation, Biocatalysis, Bioinformatics, Enzyme discovery, PLP-dependent enzymes, Protein function, Transaminase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-162120 (URN)10.1016/j.biotechadv.2014.12.012 (DOI)000358467500011 ()25575689 (PubMedID)2-s2.0-84931009357 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 289350
Note

QC 20150827

Available from: 2015-03-22 Created: 2015-03-22 Last updated: 2017-12-04Bibliographically approved
Scheidt, T., Land, H., Anderson, M., Chen, Y., Berglund, P., Yi, D. & Fessner, W.-D. (2015). Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases. Advanced Synthesis and Catalysis, 357(8), 1721-1731
Open this publication in new window or tab >>Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
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2015 (English)In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, p. 1721-1731Article in journal (Refereed) Published
Abstract [en]

omega-Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)- or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify omega-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify omega-transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase-catalyzed transacylation to furnish enantiomerically pure (R)and (S)-configured amines. The latter are instrumental for the rapid enantioselectivity typing of omega-transaminases. This method was used to characterize two novel (S)-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

Place, publisher, year, edition, pages
John Wiley & Sons, 2015
Keywords
biocatalysis, chiral amines, high-throughput screening, protein engineering, reductive amination
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-172236 (URN)10.1002/adsc.201500215 (DOI)000355235700013 ()2-s2.0-84930226708 (Scopus ID)
Note

QC 20150825

Available from: 2015-08-25 Created: 2015-08-14 Last updated: 2017-12-04Bibliographically approved
Wang, B., Land, H. & Berglund, P. (2013). An efficient single-enzymatic cascade for asymmetric synthesis of chiral amines catalyzed by omega-transaminase. Chemical Communications, 49(2), 161-163
Open this publication in new window or tab >>An efficient single-enzymatic cascade for asymmetric synthesis of chiral amines catalyzed by omega-transaminase
2013 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 49, no 2, p. 161-163Article in journal (Refereed) Published
Abstract [en]

An efficient single-enzymatic cascade approach for the asymmetric synthesis of chiral amines has been developed, which applies the amino donor 3-aminocyclohexa-1,5-dienecarboxylic acid spontaneously tautomerizing to reach reaction completion with excellent ee values.

Keywords
Optically-Active Amines, Amination, Coli
National Category
Biological Sciences Other Chemistry Topics
Identifiers
urn:nbn:se:kth:diva-109597 (URN)10.1039/c2cc37232k (DOI)000311938800014 ()
Note

QC 20130109

Available from: 2013-01-09 Created: 2013-01-08 Last updated: 2017-12-06Bibliographically approved
Steffen-Munsberg, F., Vickers, C., Thontowi, A., Schätzle, S., Tumlirsch, T., Svedendahl Humble, M., . . . Höhne, M. (2013). Connecting Unexplored Protein Crystal Structures to Enzymatic Function. ChemCatChem, 5(1), 150-153
Open this publication in new window or tab >>Connecting Unexplored Protein Crystal Structures to Enzymatic Function
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2013 (English)In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 1, p. 150-153Article in journal (Refereed) Published
Keywords
Amines, Asymmetric synthesis, Enantioselectivity, Enzyme catalysis, Protein structures
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-117838 (URN)10.1002/cctc.201200544 (DOI)000314238400016 ()2-s2.0-84871649774 (Scopus ID)
Funder
EU, European Research Council, KBBE-2011-5 289350
Note

QC 20130207

Available from: 2013-02-07 Created: 2013-02-05 Last updated: 2017-12-06Bibliographically approved
Steffen-Munsberg, F., Vickers, C., Thontowi, A., Schätzle, S., Meinhardt, T., Svedendahl Humble, M., . . . Höhne, M. (2013). Revealing the Structural Basis of Promiscuous Amine Transaminase Activity. ChemCatChem, 5(1), 154-157
Open this publication in new window or tab >>Revealing the Structural Basis of Promiscuous Amine Transaminase Activity
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2013 (English)In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 1, p. 154-157Article in journal (Refereed) Published
Keywords
Amines, Catalytic promiscuity, Chirality, Dual substrate recognition, Enzyme catalysis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-117823 (URN)10.1002/cctc.201200545 (DOI)000314238400017 ()2-s2.0-84871644215 (Scopus ID)
Funder
EU, European Research Council, KBBE-2011-5 289350
Note

QC 20130207

Available from: 2013-02-07 Created: 2013-02-05 Last updated: 2017-12-06Bibliographically 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
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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
Engelmark Cassimjee, K., Svedendahl Humble, M., Land, H., Abedi, V. & Berglund, P.Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton Parameterisation.
Open this publication in new window or tab >>Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton Parameterisation
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-92322 (URN)
Note
QS 2012Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2012-04-02Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3073-5641

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