Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.