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Silica-immobilized His(6)-tagged enzyme: Alanine racemase in hydrophobic solvent
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.ORCID iD: 0000-0002-9577-832X
2008 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 3, 712-716 p.Article in journal (Refereed) Published
Abstract [en]

A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His(6)-tagging.

Place, publisher, year, edition, pages
2008. Vol. 99, no 3, 712-716 p.
Keyword [en]
Geobacillus stearothermophilus, enzyme surface, enzyme immobilization, His(6)-tag, bacillus-stearothermophilus, inhibition, resolution, esters
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-17276DOI: 10.1002/bit.21587ISI: 000252511800023PubMedID: 17680683ScopusID: 2-s2.0-38449117584OAI: diva2:335319
QC 20100525 QC 20111128Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-11-28Bibliographically approved
In thesis
1. Tools in biocatalysis: enzyme immobilisation on silica and synthesis of enantiopure amines
Open this publication in new window or tab >>Tools in biocatalysis: enzyme immobilisation on silica and synthesis of enantiopure amines
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents two techniques in the field of biocatalysis:

An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol.

An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines.

Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development.


Abstract [sv]

I denna avhandling presenteras två tekniker inom ämnet biokatalys:

En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod.

Ett jämviksförskjutningssystem som möjliggör fullständig omsätt-ning i reaktioner med ω-transaminaser där isopropylamin är amino-donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer.

Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling.

27 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2010:11
National Category
Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-12936 (URN)
2010-05-28, 15:00 (English)
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2011-11-29Bibliographically approved

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