Protein engineering of chymosin and expression in Trichoderma reesei.

Chymosin (EC 3.4.23.4) is an aspartic proteinase containing three disulphide bridges, which occurs naturally in the fourth stomach of the unweaned calf The mature enzyme initiates milk clotting by the specific cleavage of Ic-casein between the Phe105-Met106 bond [l-41. This high specificity forms the basis for its extensive use in the process of cheese making. The primary sequence of the protein contains three functional regions. The hydrophobic leader pre-sequence directs the newly synthesized polypeptide through the membrane and upon cleavage gives rise to the zymogen, prochymosin (Mr = 40 777; 365 amino acids), the inactive state being maintained by the N-terminal propeptide [S]. Under the acidic conditions found in the stomach the precursor undergoes autocatalytic activation to chymosin (Mr = 35 600; 323 amino acids) releasing the 42 amino acid propeptide in a two step process [6-81. The two naturally occurring chymosins A and B differ by a single amino acid substitution of an aspartate to a glycine at position 244, the A form being less abundant, approximately 5% [ 51. Structural analysis has been reported for calf chymosin B [9, lo]. The enzyme is bi-lobal with a deep, extended cleft, each lobe having a similar fold. The principal catalytic residues are Asp 215 and Asp 32 (pepsin numbering, [ 111) which the crystal structure shows to be maintained close together in a local arrangement exhibiting a two-fold related network of hydrogen bonds involving Thr 2 18, Ser 35, NHGly 217, NHGly 34. This network is considered to be responsible in part for determining the state of ionization of the catalytic carboxylates whose titration is inferred from the bell-shaped pH/ activity curves. The pH optima for calf stomach chymosins A and B have been reported to be 4.2 and 3.7 respectively using synthetic substrates [ 121. The cleavage of a number of synthetic substrates by chymosin exhibit a range of pH optima [13-161. The complementary DNA for both chymosins have been cloned and expressed in Escherichia coli [ 17-22]. Direct expression at high levels results in the production of highly refractive inclusion bodies [23, 241. Although these can be easily isolated, subsequent recovery of the entrapped pro-