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One-step enzyme extraction and immobilization for biocatalysis applications
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
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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.

Place, publisher, year, edition, pages
2011. Vol. 6, no 4, 463-469 p.
Keyword [en]
Condition promiscuity, Enzyme in organic solvent, Enzymatic synthesis, HisSi Immobilization, His(6)-tag
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-39187DOI: 10.1002/biot.201000357ISI: 000289214500011Scopus ID: 2-s2.0-79953685174OAI: oai:DiVA.org:kth-39187DiVA: diva2:439510
Available from: 2011-09-08 Created: 2011-09-08 Last updated: 2017-12-08Bibliographically 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.

Publisher
27 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:11
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12936 (URN)
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

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Berglund, Per

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