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Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
KTH, Skolan för bioteknologi (BIO), Biokemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
KTH, Skolan för bioteknologi (BIO), Biokemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.
Visa övriga samt affilieringar
2011 (Engelska)Ingår i: ACS CATAL, ISSN 2155-5435, Vol. 1, nr 9, s. 1051-1055Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Measurement of the active enzyme fraction in a given enzyme preparation is a requirement for accurate kinetic measurements and activity comparisons of, for example, engineered mutants. omega-Transaminases, enzymes capable of interconverting ketones and amines by use of pyridoxal-5'-phosphate (PIP), can be used for the production of pharmaceutically important chiral amines but are subject to engineering to meet the practical requirements in synthesis reactions. Therefore, an active site quantification method is needed. Such a method was developed by quantifying the amount of consumed substrate in a virtually irreversible half transamination reaction. (S)-1-phenylethylamine was converted to acetophenone, while the holo enzyme (E-PLP) was converted to apo enzyme with bound pyridoxamine-5'-phosphate (E:PMP). Further, the mass of active enzyme was correlated to the absorbance of the holo enzyme to achieve a direct measurement method. The active Chromobacterium violaceum omega-transaminase with bound PLP can be quantified at 395 nm with an apparent extinction coefficient of 8.1 mM(-1) cm(-1).

Ort, förlag, år, upplaga, sidor
2011. Vol. 1, nr 9, s. 1051-1055
Nyckelord [en]
aminotransferase, chiral amines, pyridoxal-5 '-phosphate, PLP, biocatalysis, enzyme kinetics
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-41299DOI: 10.1021/cs200315hISI: 000294704500010Scopus ID: 2-s2.0-80052431209OAI: oai:DiVA.org:kth-41299DiVA, id: diva2:444071
Anmärkning
QC 20110927Tillgänglig från: 2011-09-27 Skapad: 2011-09-26 Senast uppdaterad: 2020-05-11Bibliografiskt granskad
Ingår i avhandling
1. ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
Öppna denna publikation i ny flik eller fönster >>ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Biocatalysis offers an alternative to classic chemistry by using enzymes, the protein catalysts of Nature, for production of fine chemicals. Evolution has created enzymes capable of catalysis at moderate temperature of a specific reaction in the presence of a plethora of compounds in the aqueous cell environment. The focal point of biocatalysis is to utilise these traits in vitro, for creation of valuable molecules.

The ω-transaminase is an enzyme capable of producing chiral amines, compounds used to great extent in pharmaceuticals. Much effort has in recent years been invested in the research and engineering of this enzyme type since the catalysed reaction offers an advantageous alternative to classical techniques. Nevertheless, there is a need for method development, adaptation of the enzyme and increased understanding of the catalytic mechanism for feasibility as an effective biocatalyst for unnatural substrates. This thesis addresses a chosen set of obstacles as a contribution to meeting the demands at hand. ω-Transaminase from Chromobacterium violaceum and Arthrobacter citreus was used.

Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced. To accurately compare their kinetic constants an active site quantification method is required but has not been available. Here such a method is presented (Paper 1) which encompasses a virtually irreversible half transamination reaction.

In stereoselective synthesis the ω-transaminase catalysed equilibrium reaction inherently results in incomplete conversion. An equilibrium displacement system is presented (Paper II) where isopropylamine is the amino donor for transamination of acetophenone and derivatives thereof, coupled to an enzymatic cascade reaction.

For many unnatural substrates the specificity and enantiospecificity is insufficient. Rationally redesigned variants were produced with improved properties for chosen substrates (Paper III and IV). The catalytic contributions of field and resonance of a variant compared to the wild type were investigated (Paper IV) for increased knowledge of the mechanism.

For rational redesign of an enzyme the three-dimensional structure is required, of which only a few are available for the ω-transaminases. X-ray crystallographic structures of the holo and apo form of Chromobacterium violaceum ω-transaminase were made (Paper V) which revealed significant structural rearrangements upon coenzyme binding which may be of consequence for future engineering.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2012. s. x, 57
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2012:13
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-92516 (URN)978-91-7501-242-1 (ISBN)
Disputation
2012-04-20, FR4 (Oscar Kleins Auditorium) AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20120402Tillgänglig från: 2012-04-02 Skapad: 2012-04-02 Senast uppdaterad: 2012-04-02Bibliografiskt granskad

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Humble, Maria SvedendahlBerglund, Per

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