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  Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells

Janke, R., Genzel, Y., Freund, S., Wolff, M. W., Grammel, H., Rühmkorf, C., et al. (2010). Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells. Journal of Biotechnology, 150(3), 396-403. doi:10.1016/j.jbiotec.2010.09.963.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0013-90FB-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-A2BC-2
Genre: Journal Article

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 Creators:
Janke, R.1, Author              
Genzel, Y.1, Author              
Freund, S.1, 2, Author              
Wolff, M. W.1, 3, Author              
Grammel, H.4, Author              
Rühmkorf, C.1, 5, Author              
Seidemann, J.1, 6, Author              
Wahl, A.1, Author              
Reichl, U.1, 3, Author              
Affiliations:
1Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society, ou_1738140              
2International Max Planck Research School (IMPRS), Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society, ou_1738143              
3Otto-von-Guericke-Universität Magdeburg, External Organizations, ou_1738156              
4Systems Biology, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society, ou_1738155              
5TU München, Freising, persistent:22              
6University of Applied Sciences Jena, Germany, persistent:22              

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 Abstract: The GUT1 gene of the halotolerant yeast Pichia farinosa, encoding glycerokinase (EC 2.7.1.30), was expressed in Pichia pastoris. A purification factor of approximately 61-fold was achieved by a combination of nickel affinity and anion exchange chromatography. The specific activity of the final preparation was 201.6 units per mg protein with a yield of about 21%. A nearly homogeneous enzyme preparation was confirmed by SDS-polyacrylamide gels and mass spectrometry analysis. Glycerol stabilized the purified enzyme for long-term storage at −80 ◦C. The pH and temperature optima were in the range of 6.5-7.0 and 45-50 ◦C, respectively. ATP was the most effective phosphoryl group donor tested. Additionally, the enzyme phosphorylated glycerol also with ITP, UTP, GTP and CTP. The Km values of the enzyme for ATP and ITP were 0.428 and 0.845mM, respectively. The kinetic properties of the enzyme with respect to UTP, GTP, and CTP suggested that glycerokinase exhibited negative cooperativity as double reciprocal plots showed a biphasic response to increasing nucleoside triphosphate concentrations. The application as a coupling enzyme in the determination of pyruvate kinase activity in cell extracts of Madin–Darby canine kidney cells showed good reproducibility when compared with a commercially available preparation of bacterial glycerokinase. © 2010 Elsevier B.V. All rights reserved. [accessed November 30th, 2010]

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Language(s): eng - English
 Dates: 2010
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: eDoc: 518411
Other: 41/10
DOI: 10.1016/j.jbiotec.2010.09.963
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Title: Journal of Biotechnology
Source Genre: Journal
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Publ. Info: Elsevier
Pages: - Volume / Issue: 150 (3) Sequence Number: - Start / End Page: 396 - 403 Identifier: ISSN: 0168-1656
CoNE: /journals/resource/954925484698