Production and characterization of milk-clotting enzyme from Bacillus amyloliquefaciens JNU002 by submerged fermentation

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS-PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 degrees C, and the ratio would be even higher (22,992) at 70 degrees C. The milk-clotting reaction and proteolytic reaction were prevented at 75 degrees C. This enzyme was stable at pH 4-6 and below 40 degrees C, and this was convenient for storage and transportation.