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Chen, S., Berglund, P. & Svedendahl Humble, M. (2018). The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum. Molecular Catalysis, 446, 115-123
Open this publication in new window or tab >>The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum
2018 (English)In: Molecular Catalysis, ISSN 2468-8231, Vol. 446, p. 115-123Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Biocatalysis, Dimeric enzymes, PLP-dependent enzymes, Pyridoxal-5’-phosphate (PLP), Schiff base
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-224452 (URN)10.1016/j.mcat.2017.12.033 (DOI)000426411900013 ()2-s2.0-85041891410 (Scopus ID)
Note

QC 20180320

Available from: 2018-03-18 Created: 2018-03-18 Last updated: 2018-03-20Bibliographically approved
Steffen-Munsberg, F., Matzel, P., Sowa, M. A., Berglund, P., Bornscheuer, U. T. & Höhne, M. (2016). Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases. Applied Microbiology and Biotechnology, 100, 4511-4521
Open this publication in new window or tab >>Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases
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2016 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, p. 4511-4521Article in journal (Refereed) Published
Abstract [en]

Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme’s function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved ‘flipping’ arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2016
Keywords
Enzyme catalysis, Transamination, Functional analysis, Structure activity relationship
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-180823 (URN)10.1007/s00253-015-7275-9 (DOI)000375057700022 ()26795966 (PubMedID)2-s2.0-84955291838 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme
Note

QC 20160201

Available from: 2016-01-24 Created: 2016-01-24 Last updated: 2017-11-30Bibliographically approved
Palo-Nieto, C., Afewerki, S., Anderson, M., Tai, C.-W., Berglund, P. & Córdova, A. (2016). Integrated Heterogeneous Metal/Enzymatic Multiple Relay Catalysis for Eco-Friendly and Asymmetric Synthesis. ACS Catalysis, 6, 3932-3940
Open this publication in new window or tab >>Integrated Heterogeneous Metal/Enzymatic Multiple Relay Catalysis for Eco-Friendly and Asymmetric Synthesis
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2016 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, p. 3932-3940Article in journal (Refereed) Published
Abstract [en]

Organic synthesis is in general performed using stepwise transformations where isolation and purification of key intermediates is often required prior to further reactions. Herein we disclose the concept of integrated heterogeneous metal/enzymatic multiple relay catalysis for eco-friendly and asymmetric synthesis of valuable molecules (e.g., amines and amides) in one-pot using a combination of heterogeneous metal and enzyme catalysts. Here reagents, catalysts, and different conditions can be introduced throughout the one-pot procedure involving multistep catalytic tandem operations. Several novel cocatalytic relay sequences (reductive amination/amidation, aerobic oxidation/reductive amination/amidation, reductive amination/kinetic resolution and reductive amination/dynamic kinetic resolution) were developed. They were next applied to the direct synthesis of various biologically and optically active amines or amides in one-pot from simple aldehydes, ketones, or alcohols, respectively.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
Keywords
heterogeneous metal, enzyme, reductive amination, relay catalysis, heterogeneous catalysis, tandem reactions, kinetic resolution, dynamic kinetic resolution
National Category
Organic Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-187377 (URN)10.1021/acscatal.6b01031 (DOI)000377326700066 ()2-s2.0-84973911460 (Scopus ID)
Note

QC 20160623

Available from: 2016-05-22 Created: 2016-05-22 Last updated: 2017-11-30Bibliographically 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
Córdova, A., Berglund, P., Anderson, M. & Afewerki, S. (2015). Efficient Synthesis Of Amines And Amides From Alcohols And Aldehydes By Using Cascade Catalysis. WO2015144902-A1.
Open this publication in new window or tab >>Efficient Synthesis Of Amines And Amides From Alcohols And Aldehydes By Using Cascade Catalysis
2015 (English)Patent (Other (popular science, discussion, etc.))
Abstract [en]

The present invention relates generally to an eco-friendly methodology for the conversion of alcohols and aldehydes to amines and amides using an integrated enzyme cascade system with metal-and organocatalysis. More specifically, the present invention relates to synthesis of capsaicinoids starting from vanillin alcohol and using a combination of an enzyme cascade system and catalysts. Furthermore, the method also relates to synthesis of capsaicinoids derivatives starting from vanillin alcohol derivatives and using a combination of an enzyme cascade system and catalysts.

National Category
Organic Chemistry
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-180012 (URN)
External cooperation:
Patent
WO2015144902-A1
Note

QS 2016

Available from: 2016-01-05 Created: 2016-01-05 Last updated: 2016-09-01Bibliographically 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
Kohls, H., Anderson, M., Dickerhoff, J., Weisz, K., Cordova, A., Berglund, P., . . . Hoehne, M. (2015). Selective Access to All Four Diastereomers of a 1,3-Amino Alcohol by Combination of a Keto Reductase- and an Amine Transaminase-Catalysed Reaction. Advanced Synthesis and Catalysis, 357(8), 1808-1814
Open this publication in new window or tab >>Selective Access to All Four Diastereomers of a 1,3-Amino Alcohol by Combination of a Keto Reductase- and an Amine Transaminase-Catalysed Reaction
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2015 (English)In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, p. 1808-1814Article in journal (Refereed) Published
Abstract [en]

The biocatalytic synthesis of chiral amines has become a valuable addition to the chemists' tool-box. However, the efficient asymmetric synthesis of functionalised amines bearing more than one stereocentre, such as 1,3-amino alcohols, remains challenging. By employing a keto reductase (KRED) and two enantiocomplementary amine transaminases (ATA), we developed a biocatalytic route towards all four diastereomers of 4-amino-1-phenylpentane-2-ol as a representative molecule bearing the 1,3-amino alcohol functionality. Starting from a racemic hydroxy ketone, a kinetic resolution using an (S)-selective KRED provided optically active hydroxy ketone (86% ee) and the corresponding diketone. Further transamination of the hydroxy ketone was performed by either an (R)- or an (S)-selective ATA, yielding the (2R,4R)- and (2R,4S)-1,3-amino alcohol diastereomers. The remaining two diastereomers were accessible in two subsequent asymmetric steps: the diketone was reduced regio- and enantioselectively by the same KRED, which yielded the (S)-configured hydroxy ketone. Eventually, the subsequent transamination of the crude product with (R)- and (S)-selective ATAs yielded the remaining (2S,4R)and (2S,4S)-diastereomers, respectively.

Keywords
amine transaminase, amino alcohols, enzyme catalysis, keto reductase
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-172237 (URN)10.1002/adsc.201500214 (DOI)000355235700020 ()2-s2.0-84930202995 (Scopus ID)
Note

QC 20150825

Available from: 2015-08-25 Created: 2015-08-14 Last updated: 2017-12-04Bibliographically approved
Gaffney, D., Abdallah, N. H., Cooney, J. C., Laffir, F. R., Engelmark Cassimjee, K., Berglund, P., . . . Magner, E. (2014). Preparation and characterisation of a Ni2+/Co2+-cyclam modified mesoporous cellular foam for the specific immobilisation of His(6)-alanine racemase. Journal of Molecular Catalysis B: Enzymatic, 109, 154-160
Open this publication in new window or tab >>Preparation and characterisation of a Ni2+/Co2+-cyclam modified mesoporous cellular foam for the specific immobilisation of His(6)-alanine racemase
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2014 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 109, p. 154-160Article in journal (Refereed) Published
Abstract [en]

Nickel and cobalt cyclam modified mesocellular foam (MCF) materials were prepared and characterised. The metal cyclam modified materials displayed reduced surface areas and pore diameters in comparison to MCF. The modified materials were used to specifically anchor a histidine tagged form of the enzyme, alanine racemase (HT-AlaR). Non-specific adsorption was predominantly hydrophobic//hydrophilic in nature and could be significantly reduced in the presence of 2% polyethylene glycol. The activity of HT-AlaR immobilised on Ni and Co-MCF was essentially the same as that of the free enzyme, demonstrating that enzymes can be specifically immobilised within the pores of mesoporous materials in a stable and catalytically active manner.

Keywords
Mesoporous silicates, Metal cyclam, His-tagged alanine racemase
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-156108 (URN)10.1016/j.molcatb.2014.08.011 (DOI)000343854100021 ()2-s2.0-84907207324 (Scopus ID)
Note

QC 20150112

Available from: 2015-01-12 Created: 2014-11-21 Last updated: 2017-12-05Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9577-832X

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