PMID: 9546178Apr 18, 1998Paper

Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli

Applied and Environmental Microbiology
S WittH M Kalisz

Abstract

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; beta-D-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10 degrees C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar c...Continue Reading

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Citations

Oct 27, 2011·Journal of Diabetes Science and Technology·Stefano FerriKoji Sode
Dec 4, 2012·International Journal of Molecular Sciences·Yohei HoraguchiKoji Sode
Sep 29, 2009·Applied Biochemistry and Biotechnology·Yao GuoZhaoxin Lu
Nov 18, 2004·Journal of Applied Microbiology·E LeiterI Pócsi
Nov 21, 2007·Applied Biochemistry and Biotechnology·Dimitris G HatzinikolaouDimitris Kekos
May 4, 2010·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Olivier CourjeanNicolas Mano
Apr 27, 2007·Glycoconjugate Journal·Amirreza Faridmoayer, Christine H Scaman
Sep 28, 2018·Microbiology and Molecular Biology Reviews : MMBR·Bastien BissaroVincent G H Eijsink
Aug 4, 2018·Journal of Diabetes Science and Technology·Ryan BoehmIrada Isayeva
Apr 12, 2021·Biotechnology and Applied Biochemistry·Seyyed Hossein KhatamiAhmad Movahedpour
Apr 29, 2019·Bioelectrochemistry·Nicolas Mano
Jun 3, 2021·Acta Crystallographica. Section D, Structural Biology·Leona ŠvecováJan Dohnálek
Jan 13, 2018·Chemical Reviews·Elvira RomeroAndrea Mattevi
Sep 7, 2007·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Maxim Zakhartsev, Carmen Momeu
Feb 12, 2019·Langmuir : the ACS Journal of Surfaces and Colloids·Gustav Ferrand-Drake Del CastilloAndreas Dahlin
May 7, 2005·Langmuir : the ACS Journal of Surfaces and Colloids·Alessandro NapoliJeffrey A Hubbell
Apr 9, 2009·Bioelectrochemistry·Soren Demin, Elizabeth A H Hall
Apr 21, 2009·Biotechnology Advances·Sandip B BankarLaxmi Ananthanarayan

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