Crystallization and chemical modification of disulfide bond of calf chymosin

In order to carry out protein engineering of chymosin, purification, crystallization and chemical modification of calf chymosin were undertaken. The resolution of iso-enzymes A,B and the degraded product, fraction C was improved and the period of purification was shortened by using fast protein liquid chromatograph (FPLC). Crystal of approximately cubic shape was obtained from 10 mg/ml purified chymosin B at 4 degrees C after successive dialysis against 1.4, 1.8 and 2.0 M NaCl in 0.1 M sodium phosphate buffer. One disulfide bond located on the surface of chymosin molecule was reduced by 0.72 mM dithiothreitol (DTT) at pH 6.3. The reduced disulfide bond was identified as Cys250-Cys283 by using the method of cyanogen bromide cleavage and peptide separation. The reduction of one disulfide bond resulted in the loss of about 25% activity. Carboxymethylated and mercurated derivatives exhibit the similar activity to that of the reduced enzyme suggesting that Cys250-Cys283 plays some role in keeping the conformation with full activity.