Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580.
Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.
University of Bayreuth, Bayreuth, Germany.
Frank
F
Hilbrig
Valérie
V
Jérôme
Mark
M
Salzig
Ruth
R
Freitag
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification; D015374:Biosensing Techniques / Q000379:methods*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D020582:Hyphomicrobium / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification
D017886:Durapatite; D010088:Oxidoreductases; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors