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Amine Transaminases in Biocatalytic Amine Synthesis
KTH, School of Biotechnology (BIO), Industrial Biotechnology. KTH Royal Institute of Technology.ORCID iD: 0000-0003-3073-5641
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity.

For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines.

Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed.

Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed.

One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents.

Place, publisher, year, edition, pages
Stockholm: Henrik Land , 2016. , 101 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Keyword [en]
Amine Transaminase, Biocatalysis, Transamination, Reductive Amination, Enzyme, Enzyme Engineering, Equilibrium Displacement, Screening, Enzyme Stability
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-194112ISBN: 978-91-7729-164-0 (print)OAI: oai:DiVA.org:kth-194112DiVA: diva2:1037614
Public defence
2016-11-25, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20161017

Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2016-10-17Bibliographically approved
List of papers
1. Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
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, 5466-5470 p.Article 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.

Keyword
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
2. An efficient single-enzymatic cascade for asymmetric synthesis of chiral amines catalyzed by omega-transaminase
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, 161-163 p.Article 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.

Keyword
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
3. One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
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, 2897-2900 p.Article 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
4. Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
Open this publication in new window or tab >>Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
Show others...
2015 (English)In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, 1721-1731 p.Article 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
Keyword
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
5. Stabilization of an amine transaminase for biocatalysis
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, 20-28 p.Article 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: 2017-11-30Bibliographically approved

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