The AlkB monooxygenase of Pseudomonas oleovorans. Synthesis, stability, and level in recombinant Escherichia coli and the native host

We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk+ recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli. The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amts. of AlkB as P. oleovorans (1.5-2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about five-fold) more AlkB. The AlkB synthesis and degrdn. rates in batch cultures of the 3 strains were detd. by isotopic-labeling and immunol. techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were ~ 7, 12.5, and 45 micro g mg protein-1 h-1, resp. The half-lives of AlkB were estd. to be 80, 3, and 15 for P. oleovorans, E. coli HB101 (pGEc47), and E. coli W3110 (pGEc47), resp. Thus, the intracellular AlkB level in each of the 3 strains was the result of their AlkB synthesis and degrdn. rates. The AlkB level during batch growth was modeled by exptl. derived parameters for AlkB synthesis and degrdn., and showed good agreement with AlkB levels detd. by immunoblotting in all strains investigated. [on SciFinder (R)]