Cloning and gene expression of alkane hydroxylase gene from Pseudomonas putida

Pseudomonas putida is a bacterium that has ability to growth on limited substrates that mainly is alkanes. The ability to use wide range of hydrocarbons is advantage of this bacterium to other bacteria community. P. putida have alkane hydroxylase system (alk-B) for alkane biodegradation. In this study alk-B gene from P. putida was amplified. Blunt cloning first done in pBluescript plasmid then sticky end cloning was carried out in pET-26 vector. For expression of this recombinant gene E. coli BL-21 bacterium were used. The activity of recombinant enzyme was done by reduction of absorbance by oxidation of NADPH. The results of this study show that alk-B gene was inserted in pBluescript plasmid. Colony PCR confirmed this insertion. This gene successfully cloned in pET-26a vector. By increasing the time after induction by IPTG expression of alk-B protein were increased. Recombinant protein has activity. Transfer of this gene to fast growth bacterium and wide substrate confirm production of robust biocatalysts in the future.