Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.
Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.
University of Stuttgart, Institute of Interfacial Process Engineering and Plasma Technology, Stuttgart, Germany.
Lisa
L
Blaschke
Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Wenke
W
Wagner
Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Christina
C
Werkmeister
Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Marion
M
Wild
Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Adrian
A
Gihring
University of Stuttgart, Institute of Interfacial Process Engineering and Plasma Technology, Stuttgart, Germany; Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.
Steffen
S
Rupp
University of Stuttgart, Institute of Interfacial Process Engineering and Plasma Technology, Stuttgart, Germany; Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany. Electronic address: susanne.zibek@igb.fraunhofer.de.
Susanne
S
Zibek
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C048530:formaldehyde dismutase
Biomethanol; Cofactor regeneration; Escherichia coli rhamnose promoter; Formaldehyde dismutase; Oxidoreductase; Pseudomonas putida