Extraction, purification, kinetic characterization and immobilization of urease from Bacillus sphaericus MTCC 5100

Urease is a well-known enzyme, that catalyzes the urea hydrolysis to ammonia and carbon dioxide. Industrial application of this enzyme in the beverages, fertilizer and pharmaceutical. In this research, microbial urease was purified, characterized and immobilized on metal electrode for biosensor application. Enzyme was purified step by step process, salt precipitation followed by ion exchange chromatography followed by gel filtration chromatography. The purification fold was achieved 6.67 with specific activity 32.5 IU. The estimated molecular weight and Km of the enzyme were 185 kDa and 2.0±0.5 mM respectively. SDS-PAGE was also performed to check the subunit of the purified enzyme and it was found to be three subunits 70 kDa, 60 kDa and 55 kDa. Immobilization of purified urease was performed by cross linking using glutaraldehyde on metal electrode for biosensor development. The storage stability was achieved up to 60 days when store at 4 °C in PBS buffer.