Chromosomal Integration of KerA Gene in Bacillus megaterium For Stable Keratinase Production

Abstract: In order to develop a stable strain of Bacillus megaterium for Keratinase production, the Keratinase gene (KerA) of Bacillus lichiniformis ATCC 53757 and SPlipA gene from plasmid pHIS1525.SPlipA (Bacillus megaterium origin) were PCR amplified and constructed to give a gene cassette called SPK . Then the gene cassette SPK was cloned into the Integration vector, pMUTIN-GFP+ 6192bps and transformed in Bacillus megaterium ATCC 14945. The chromosomal integration was created using homologous single crossing over mechanism. The strong natural promoter from the chromosomal locus of the SPK not only produced the increased extracellular enzyme, but also functions as a non inducible promoter which does not require any inducer for the production of the enzyme in the new integrant strain. The integrant strain was subjected to feather degradation test and found that it could totally digest the feather meal in complete seven days, resulting in a rich fermentation broth.