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B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden. (fotosyntesgruppen)ORCID iD: 0000-0003-3073-5641
KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden.
Pharem Biotech AB, Biovat Pk,Forskargatan 20 J, S-15136 Sodertalje, Sweden.
KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden.ORCID iD: 0000-0002-9577-832X
2019 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed) Published
Abstract [en]

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 degrees C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 degrees C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2019. Vol. 20, no 10, p. 1297-1304
Keywords [en]
biocatalysis, enzyme stabilization, enzymes, molecular modeling, protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-389825DOI: 10.1002/cbic.201800749ISI: 000471316400013PubMedID: 30637901OAI: oai:DiVA.org:uu-389825DiVA, id: diva2:1339454
Funder
Carl Tryggers foundation , CTS 16:456Carl Tryggers foundation , CTS 18:803Available from: 2019-07-29 Created: 2019-07-29 Last updated: 2019-07-29Bibliographically approved

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