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Tools in biocatalysis: enzyme immobilisation on silica and synthesis of enantiopure amines
KTH, School of Biotechnology (BIO), Biochemistry.
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.

Place, publisher, year, edition, pages
2010. , 27 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:11
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-12936OAI: oai:DiVA.org:kth-12936DiVA: diva2:319717
Presentation
2010-05-28, 15:00 (English)
Opponent
Supervisors
Note
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2011-11-29Bibliographically approved
List of papers
1. Silica-immobilized His(6)-tagged enzyme: Alanine racemase in hydrophobic solvent
Open this publication in new window or tab >>Silica-immobilized His(6)-tagged enzyme: Alanine racemase in hydrophobic solvent
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.

Keyword
Geobacillus stearothermophilus, enzyme surface, enzyme immobilization, His(6)-tag, bacillus-stearothermophilus, inhibition, resolution, esters
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-17276 (URN)10.1002/bit.21587 (DOI)000252511800023 ()17680683 (PubMedID)2-s2.0-38449117584 (Scopus ID)
Note
QC 20100525 QC 20111128Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. One-step enzyme extraction and immobilization for biocatalysis applications
Open this publication in new window or tab >>One-step enzyme extraction and immobilization for biocatalysis applications
Show others...
2011 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 4, 463-469 p.Article in journal (Refereed) Published
Abstract [en]

An extraction/immobilization method for His(6)-tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His 6 tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

Keyword
Condition promiscuity, Enzyme in organic solvent, Enzymatic synthesis, HisSi Immobilization, His(6)-tag
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-39187 (URN)10.1002/biot.201000357 (DOI)000289214500011 ()2-s2.0-79953685174 (Scopus ID)
Available from: 2011-09-08 Created: 2011-09-08 Last updated: 2017-12-08Bibliographically approved
3. Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration
Open this publication in new window or tab >>Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration
Show others...
2010 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 46, no 30, 5569-5571 p.Article in journal (Refereed) Published
Abstract [en]

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Keyword
OPTICALLY-ACTIVE AMINES, ASYMMETRIC-SYNTHESIS, OMEGA-TRANSAMINASES, CHIRAL AMINES
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12935 (URN)10.1039/c0cc00050g (DOI)000280145000048 ()
Note
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2017-12-12Bibliographically approved

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