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Stabilization of an amine transaminase for biocatalysis
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0003-3073-5641
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-9577-832X
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0003-2371-8755
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. Vol. 124, p. 20-28
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-180821DOI: 10.1016/j.molcatb.2015.11.022ISI: 000370458100003Scopus ID: 2-s2.0-84949440870OAI: oai:DiVA.org:kth-180821DiVA, id: diva2:897101
Note

QC 20160126. QC 20160319

Available from: 2016-01-24 Created: 2016-01-24 Last updated: 2018-03-19Bibliographically approved
In thesis
1. Amine Transaminases in Biocatalytic Amine Synthesis
Open this publication in new window or tab >>Amine Transaminases in Biocatalytic Amine Synthesis
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. p. 101
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Keywords
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:nbn:se:kth:diva-194112 (URN)978-91-7729-164-0 (ISBN)
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
2. Stability and inactivation mechanisms of two transaminases
Open this publication in new window or tab >>Stability and inactivation mechanisms of two transaminases
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the past decades, more and more enzymes are employed as biocatalysts in industrial processes because of their advantages, such as high efficiency, substrate selectivity and stereoselectivity. Among them, amine transaminases (ATAs) are pyridoxal 5’-phosphate (PLP) dependent enzymes. ATAs have gained attention for their excellent performance in chiral amine synthesis, and their broad substrate acceptance. However, the low operational stability of amine transaminases still limits their application in industry.

The amine transaminase from Chromobacterium violaceum (Cv-ATA) has been selected for further investigation for its relatively low operational stability. Co-solvents and various additives have been added to the enzyme storage solution to improve its storage stability at various temperatures. Co-lyophilization of Cv-ATA with surfactants has been applied to improve its enzymatic activity in neat organic solvents.

As a PLP-dependent dimeric enzyme, the Cv-ATA is not primarily inactivated due to tertiary structural changes. Instead, both dimer dissociation and PLP release may affect the enzyme stability. Therefore, the inactivation pathway of the Cv-ATA during operational conditions was explored. The unfolding of the enzyme was detected by several methods, and the detection of fluorescence intensity spectrum of tryptophan is extensively applied for its high sensitivity. The phosphate group of PLP can be coordinated into the phosphate group binding cup, which may influence the enzyme structural stability. Therefore, the effect of both PLP and inorganic phosphate ions (present in phosphate buffer) on the enzyme stability was explored.

The amine transaminase from Vibrio fluvialis (Vf-ATA) is another amine transaminase, which catalyses the same biocatalytic reaction and has a similar substrate scope as Cv-ATA. However, there is still a lack of data on the stability of Vf-ATA. Consequently, the operational stability of Vf-ATA in various environments was studied.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. p. 56
Series
TRITA-CBH-FOU ; 2018:10
Keywords
Amine Transaminase, Operational Stability, Inactivation Pathway, Enzyme Unfolding, Phosphate Group Binding Cup
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-224538 (URN)978-91-7729-716-1 (ISBN)
Public defence
2018-04-11, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180320

Available from: 2018-03-20 Created: 2018-03-19 Last updated: 2018-03-27Bibliographically approved

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Land, HenrikBerglund, PerSvedendahl Humble, Maria

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