Enhanced fusidic acid transdermal delivery achieved by newly isolated and optimized Bacillus cereus Keratinase

The expanding interest in bioremediation of poorly degradable wastes has led to the discovery of many microbial enzymes capable of degrading recalcitrant substances such as keratinaceous wastes that are produced in vast quantities on daily basis. Such enzymes don't only work as a bioremediation tool but also have multiple beneficial applications. Hence, environmental samples were collected from sewage water, soils, animal bodies and feces in order to isolate keratinase producing organisms. Keratinolytic isolates were isolated from sewage water; soils; animal bodies; animal feces, and identified both traditionally and molecularly through 16S-rRNA sequencing to be Bacillus cereus strain. Produced keratinase was purified by centrifugation, ammonium sulfate precipitation, and HPLC, then assayed using Azokeratine based analysis. keratinase quantification yielded a 420 ± 1.63 U/mL. Optimum production was obtained at 40 °C, pH 7, 3 days incubation, 0.5 % substrate, 0.4 g/l magnesium ion, 2% v/v inoculum, 0.5 g/l NaCl, 0.4 g/l K₂HPO₄, and 0.3 g/l KH₂PO₄. Production was increased by 1.9 fold after acclimatization to reach 809 ± 2.49 U/mL in only 2 days. Thermal and pH stability testing revealed the effectiveness of the isolated keratinase over a wide range of temperatures at neutral pH. Finally, isolated keratinase enhanced fusidic acid topical penetration to treat induced deep skin bacterial infection in mice. A 1.4 fold decrease in treatment period and a 2 log cycle reduction in the viable count of Staphylococcus aureus were noticed in keratinase/fusidic acid treated mice compared to mice treated with fusidic acid alone. This study shed some light on a simple keratinase production optimization technique and suggested a promising medical application of this enzyme as a drug delivery agent.