Optimization of the production and characterization of milk-clotting enzyme from Bacillus subtilis isolated from marine sponge

Abstract

Background and objectives Milk-clotting enzyme (MCE) has important applications in the dairy industry and in the cheese-manufacturing process. Because of the increase in cheese consumption and the low supply of calf rennets, there is a need for a suitable rennet substitute from an appropriate source. The present investigation aims at microbial production of MCE and medium optimization for maximal enzyme production by the most potent strain. Partial purification and the properties of the partially purified enzyme are also studied. Materials and methods In the present study several microorganisms were tested for production of the MCE. MCE/caseinase ratio was investigated and was used as the key parameter for selection of the most potent strain and for medium optimization. Medium optimization experiments were carried out in an attempt to increase the enzyme productivity by the most potent strain. The produced enzyme was partially purified using ammonium sulfate at 50% concentration and the properties of the partially purified enzyme were investigated. Results and conclusion Among all the tested bacteria the marine sponge Pseudoceratina arabica isolated bacterium (isolate I) showed the highest milk-clotting activity. This isolate was used for MCE production throughout this study and it was identified as Bacillus subtilis KU710517. Maximum enzyme productivity (581.8 U/ml) and maximum MCE/caseinase ratio were obtained using a medium containing 50 g/l wheat bran with xylose and yeast extract as carbon and nitrogen sources. For the partially purified enzyme, the optimal temperature and pH were 85°C and 5.0, respectively. The enzyme was thermally stable at 45°C and retained 100% activity after 90 min. At 50°C for the same period of time it retained about 82% activity. However, at 60°C, the enzyme lost about 70% of its original activity after 30 min. The activation energy (Ea) of the enzyme was calculated as 6.95 kcal/mol. Km and Vmax values were 4.6 mg/ml and 2933 U/mg protein, respectively.