Cloning and characterisation of a new 2-deoxy-D-ribose-5-phosphate aldolase from Rhodococcus erythropolis.
A new 2-deoxy-D-ribose-5-phoshate aldolase (DERA) gene was cloned from Rhodococcus erythropolis strain DSM 311, recombinantly expressed in Escherichia coli, and purified via affinity chromatography which yielded a homo-dimeric enzyme of 44.3 kDa as apparent by size exclusion chromatography. To characterise the enzyme, investigations about pH and temperature tolerance, stability, as well as analyses on resistance to organic solvents and acetaldehyde were performed. In addition, kinetic constants of the new DERA(RE) were compared to respective values of the DERA from E. coli (DERA(EC)). Stability of DERA(RE) turned out to be a crucial factor: The pH for optimal DERA(RE) activity was determined to be 7.0, whereas the highest stability was achieved at pH 9.0 with a half-life of approximately 20 days. The optimal temperature for DERA(RE) activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being 2.5× higher compared to DERA(EC) under the exposure of 300 mM acetaldehyde. Hence it has the potential as a new promising biocatalyst with applications in organic synthesis.
Institut für Bioorganische Chemie der Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Stetternicher Forst, Geb. 15.8, D-52426 Jülich, Germany.
Irene
I
Kullartz
Jörg
J
Pietruszka
D000079:Acetaldehyde; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008661:Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; D000079:Acetaldehyde