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Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
KTH, Skolan för bioteknologi (BIO), Biokemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-3073-5641
Vise andre og tillknytning
2012 (engelsk)Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, nr 28, s. 5466-5470Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2012. Vol. 10, nr 28, s. 5466-5470
Emneord [en]
OPTICALLY-ACTIVE AMINES; ASYMMETRIC-SYNTHESIS; CHIRAL AMINES; SUBSTRATE-SPECIFICITY; RESONANCE COMPONENTS; CHEMICAL-REACTIVITY; AMINOTRANSFERASE; SUBSTITUENT; IDENTIFICATION; BIOCATALYSIS
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-99250DOI: 10.1039/c2ob25893eISI: 000305764600020Scopus ID: 2-s2.0-84863611002OAI: oai:DiVA.org:kth-99250DiVA, id: diva2:541827
Merknad
QC 20120724Tilgjengelig fra: 2012-07-24 Laget: 2012-07-23 Sist oppdatert: 2020-01-29bibliografisk kontrollert
Inngår i avhandling
1. Amine Transaminases in Biocatalytic Amine Synthesis
Åpne denne publikasjonen i ny fane eller vindu >>Amine Transaminases in Biocatalytic Amine Synthesis
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Henrik Land, 2016. s. 101
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Emneord
Amine Transaminase, Biocatalysis, Transamination, Reductive Amination, Enzyme, Enzyme Engineering, Equilibrium Displacement, Screening, Enzyme Stability
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-194112 (URN)978-91-7729-164-0 (ISBN)
Disputas
2016-11-25, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20161017

Tilgjengelig fra: 2016-10-17 Laget: 2016-10-17 Sist oppdatert: 2019-08-21bibliografisk kontrollert

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