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Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines
Uppsala university.ORCID iD: 0000-0003-3073-5641
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. (Biocatalysis)ORCID iD: 0000-0003-4359-1659
Technishe Universität Darmstadt.
Technishe Universität Darmstadt.
(English)Manuscript (preprint) (Other academic)
Keywords [en]
Transaminase, Chromobacterium violaceum, kinetic resolution, enzyme engineering
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-261073OAI: oai:DiVA.org:kth-261073DiVA, id: diva2:1356289
Note

QC 20191011

Available from: 2019-10-01 Created: 2019-10-01 Last updated: 2019-10-11Bibliographically approved
In thesis
1. Transaminase Biocatalysis: Applications and Fundamental Studies
Open this publication in new window or tab >>Transaminase Biocatalysis: Applications and Fundamental Studies
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications.

From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine.

From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes.

Abstract [sv]

Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar.

Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin.

Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras.

Abstract [it]

La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni.

Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine.

Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 88
Series
TRITA-CBH-FOU ; 2019:47
Keywords
biocatalysis, enzymatic cascade, transaminase, amine, racemization, PLP, enzyme engineering, Chromobacterium violaceum, protein stability, enzyme stability, molecular dynamics, structural biology, enzyme technology, chiral amines, kinetic resolution
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-260146 (URN)978-91-7873-312-5 (ISBN)
Public defence
2019-10-24, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
EU, Horizon 2020, 634200
Note

QC 2019-10-01

Available from: 2019-10-01 Created: 2019-09-26 Last updated: 2019-10-01Bibliographically approved

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