High level expression of a recombinant amylosucrase gene and selected properties of the enzyme.
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Your vote was cast
Thank you for your feedback
Thank you for your feedback
MetadataShow full item record
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.
CitationHigh level expression of a recombinant amylosucrase gene and selected properties of the enzyme. 2011, 89 (6):1821-9 Appl. Microbiol. Biotechnol.
AffiliationHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
The following license files are associated with this item:
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by-nc-sa/4.0/
- Characterisation of the activator effect of glycogen on amylosucrase from Neisseria polysaccharea.
- Authors: Potocki de Montalk G, Remaud-Simeon M, Willemot RM, Monsan P
- Issue date: 2000 May 1
- Cloning and characterization of the gene for amylosucrase from Neisseria polysaccharea: production of a linear alpha-1,4-glucan.
- Authors: Büttcher V, Welsh T, Willmitzer L, Kossmann J
- Issue date: 1997 May
- Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme.
- Authors: De Montalk GP, Remaud-Simeon M, Willemot RM, Planchot V, Monsan P
- Issue date: 1999 Jan
- Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea.
- Authors: Jung JH, Seo DH, Ha SJ, Song MC, Cha J, Yoo SH, Kim TJ, Baek NI, Baik MY, Park CS
- Issue date: 2009 Sep 8
- Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase.
- Authors: Albenne C, Skov LK, Mirza O, Gajhede M, Feller G, D'Amico S, André G, Potocki-Véronèse G, van der Veen BA, Monsan P, Remaud-Simeon M
- Issue date: 2004 Jan 2