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Changing or improving the enantioselectivity of ω-transaminase towards (R)-amines, utilizing a semi-rational design approach
KTH, School of Biotechnology (BIO).
2013 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

This thesis gives a brief insight on how protein engineering is made with ω-Transaminases - enzymes that are used to create chiral amines which are included in many pharmaceuticals, fine chemicals and agrochemicals - in order to find ω-Transaminase variants that have potential for scale up in industrial processes.

Several ways to produce (S)-amines with ω-Transaminases exist today as most characterized ω-Transaminases are (S)-selective. The (R)-selective ω-Transaminases are in the other hand rare and in 2003, only 1 (R)-selective ω-Transaminases was known. In 2012, the group of Svedendahl Humble et al. to change the enantioselectivity for the substrate 2-aminotetralin from E = 3.9 (S) to E = 63 (R) by introducing two poiny mutations (F88A/A231F) in the active site of Chromobacterium violaceum ω-Transaminase.

By using the same variant (F88A/A231F) as a starting template, two new residues in the active site were targeted for site directed mutagenesis that hopefully would give variatns with increased E-caalue for (R)-2-aminotetralin or with changed enantiopreference, frpm (S) to (R), for other stubstrates.

This report covers most of the steps, starting from the rational design of the active site and ends up with screening and kinetics of the possible hits using one template substrate, 1-aminotetralin.

Place, publisher, year, edition, pages
Keyword [en]
Biocatalysis, protein engineering applied enzymology, aminotransferase, enzyme kinetics
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-150477OAI: diva2:744712
Available from: 2015-04-17 Created: 2014-09-04 Last updated: 2015-09-22Bibliographically approved

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