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Inactivation pathway underlying the operational instability of an amine transaminase from Chromobacterium violaceum
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-0135-8114
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-9577-832X
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0003-2371-8755
(English)Manuscript (preprint) (Other academic)
Keywords [en]
Enzyme stability, Oligomerisation, Pyridoxal phosphate, fluorescence, pH regulation, protein folding, Aminotransferase, Biocatalysis
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-224537OAI: oai:DiVA.org:kth-224537DiVA, id: diva2:1191652
Note

QC 20180320

Available from: 2018-03-19 Created: 2018-03-19 Last updated: 2018-03-20Bibliographically approved
In thesis
1. 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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Berglund, PerSvedendahl, Maria

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