High level expression of a recombinant amylosucrase gene and selected properties of the enzyme.

2.50
Hdl Handle:
http://hdl.handle.net/10033/620822
Title:
High level expression of a recombinant amylosucrase gene and selected properties of the enzyme.
Authors:
Schneider, Jens; Fricke, Christin; Overwin, Heike; Hofer, Bernd ( 0000-0002-5174-4021 )
Abstract:
Two high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD(600). To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
High level expression of a recombinant amylosucrase gene and selected properties of the enzyme. 2011, 89 (6):1821-9 Appl. Microbiol. Biotechnol.
Journal:
Applied microbiology and biotechnology
Issue Date:
Mar-2011
URI:
http://hdl.handle.net/10033/620822
DOI:
10.1007/s00253-010-3000-x
PubMed ID:
21113589
Type:
Article
Language:
en
ISSN:
1432-0614
Appears in Collections:
Publications of the research group Chemical Biology (CBIO)

Full metadata record

DC FieldValue Language
dc.contributor.authorSchneider, Jensen
dc.contributor.authorFricke, Christinen
dc.contributor.authorOverwin, Heikeen
dc.contributor.authorHofer, Bernden
dc.date.accessioned2017-02-16T13:30:15Z-
dc.date.available2017-02-16T13:30:15Z-
dc.date.issued2011-03-
dc.identifier.citationHigh level expression of a recombinant amylosucrase gene and selected properties of the enzyme. 2011, 89 (6):1821-9 Appl. Microbiol. Biotechnol.en
dc.identifier.issn1432-0614-
dc.identifier.pmid21113589-
dc.identifier.doi10.1007/s00253-010-3000-x-
dc.identifier.urihttp://hdl.handle.net/10033/620822-
dc.description.abstractTwo high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD(600). To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subject.meshChromatography, Affinityen
dc.subject.meshEscherichia colien
dc.subject.meshGene Expressionen
dc.subject.meshGenetic Vectorsen
dc.subject.meshGlucoseen
dc.subject.meshGlucosyltransferasesen
dc.subject.meshModels, Molecularen
dc.subject.meshNeisseriaen
dc.subject.meshOligosaccharidesen
dc.subject.meshProtein Structure, Tertiaryen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshSubstrate Specificityen
dc.titleHigh level expression of a recombinant amylosucrase gene and selected properties of the enzyme.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalApplied microbiology and biotechnologyen

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