Journal of Applied Sciences Research, 3(8): 695-699, 2007 © 2007, INSInet Publications Corresponding Author: Magda A. El-Bendary, Microbial Chemistry Department, National Research Centre, Dokki, Giza, Egypt. 695 Purification and Characterization of Milk Clotting Enzyme Produced by Bacillus sphaericus Magda A. El-Bendary, Maysa E. Moharam and Thanaa H. Ali Microbial Chemistry Department, National Research Centre, Dokki, Giza, Egypt. Abstract: Milk clotting enzyme (MCE) produced by Egyptian Bacillus sphaericus NRC 24 was partially purified and characterized. MCE was obtained by fractional precipitation with acetone, followed by the chromatography of the most active fraction on DEAE-Sephadex A-25 and finally on Sephadex G-100 with 48 purification fold and specific activity about 648148 U/mg protein. The maximum enzyme activity was at a wide range of pHs (5.7-7.5) and 55ºC. The clotting activity of the purified enzyme was stimulated 2 with increasing CaCl concentration up to 0.25%. However, a gradual reduction of the activity was observed by increasing NaCl concentration between 5-20%. Zinc ions had a stimulating effect on the purified enzyme; while Ni and Hg ions had an inhibitory effect on the purified enzyme. Key words: Bacillus sphaericus, Milk clotting enzyme , purification INTRODUCTION Milk coagulation is the basic step in cheese manufacturing. Milk clotting enzymes are the primary active agents in cheese making, which involves the enzyme-mediated cleavage of kaba-casein at the peptide bond Phe 105-Met 106 that renders the casein micelles unstable and eventually causes aggregation that yields a clot and a gel afterwards . [1] Chymosin (EC 3.4.23.4) is a milk clotting enzyme (MCE) obtained from the fourth stomach of the unweaned calf. Problems associated with animal slaughtering have necessitated finding other alternatives to calf chymosin. In this regard, various alternatives are used for chymosin production; these sources are animals, plants and microorganisms. These enzymes are purified and characterized by a number of authors . [1-8] During a study on rennin-like enzymes produced by Bacillus sphaericus, an Egyptian isolate, B. sphaericus NRC 24, showed a high milk clotting activity and was considered a novel and promising producer . Therefore, in the present study an attempt [9] was made to purify and characterize the milk clotting enzyme produced by B. sphaericus NRC 24. MATERIALS AND METHODS Microorganism And Enzyme Production: B. sphaericus NRC 24 was used in this study. Fodder yeast (4%) supplemented with NYSM salts was [10] inoculated with the test organism and incubated for three days at 30°C on orbital shaker at 150 rpm. The cells were harvested by centrifugation and the supernatant (crude enzyme source) was used for purification experiments. Purification of MCE: The crude enzyme solution was fractioned by acetone at 30, 50 and 70%. The active fraction with high milk clotting activity (MCA) was further purified by passing through a column (1.5 x 40cm) of DEAE-Sephadex A-25 pre-equilibrated with 0.02 M phosphate buffer at pH 6. Elution of protein was then carried out by batch wise addition of 40 ml portions of increasing molarities (0.0 –0.4 M) of NaCl in 0.02 M phosphate buffer at pH 6. Fractions of 5 ml each were collected at room temperature (25°C) at a flow rate of about 20 ml/h and analyzed for MCA and protein content. The active fractions were dialyzed against distilled water and concentrated via lypholization. The concentrated enzyme was loaded on to a Sephadex G-100 column (2cm x 46cm) pre- washed with 0.02M phosphate buffer at pH 6. Fractions of 5 ml each were collected at room temperature at a flow rate of about 30 ml/h. The active enzyme fractions were pooled and stored at 4°C for further studies. Enzyme Assay: The enzyme was assayed as described by Greenberg with some modification. The enzyme [11] source (0.2 ml) was added to 2 ml of substrate solution 2 (12% skim milk powder in 0.01M CaCl ). The time necessary for the formation of curd fragment was measured. Milk clotting activity is expressed in term of Soxhlet unit.