Abstract
Genes for the hydrocarbon-degrading enzymes – alkane 1-monooxygenase (AlkM) and catechol 1,2-dioxygenase (CatA) – were amplified from three environmental strains of Acinetobacter baumannii from Pasig River and oil sludge sample. Each gene has been previously cloned into a pBAD/Thio-TOPO plasmid expression vector and the recombinant pBAD/Thio-TOPO plasmids were introduced into E coli TOP10 cells via transformation. This study was conducted to determine if AlkM and CatA proteins will be expressed by the recombinant E coli TOP10 cells, to determine the optimized conditions for protein expression, and to perform purification of expressed AlkM and CatA fusion proteins. The pilot expression resulted in the production of the putative 62 kDa AlkM-thioredoxin and the 50 kDa CatA-thioredoxin fusion proteins, and the identity of the expressed proteins was verified to be AlkM and CatA using western blotting. The study showed that best AlkM and CatA expression was observed when recombinant cells were grown in Super broth using 6-h post-induction time with at least 0.2% arabinose. Purified AlkM and CatA fusion proteins were obtained using affinity chromatography and subsequent size exclusion chromatography with Sephadex G-75 column. The preliminary activity of the purified CatA fusion enzyme observed at 1.128 units/mg was much lower than activities reported for other species, while an AlkM activity assay could not be performed because AlkM fusion proteins from recombinant E coli did not solubilize after purification. These suggest the need to perform an extensive study to address AlkM solubility problem and optimize parameters for enzyme activity of expressed CatA and AlkM. Successful expression of AlkM and Cat A genes from a human bacterial pathogen in non-pathogenic E coli host cells is an initial step to the possible use of recombinant cells in producing AlkM and CatA enzymes from a pathogenic bacterial species in addressing problems of oil contamination
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