Research On Recombinant Human Chymotrypsin

Chymotrypsin (EC3.4.21.1) belongs to the serine proteases family, whose zymogen is cut into two parts by trypsin between Arg15and Ile16(Bovine chymotrypsin A number) and connected by disulfide bonds. After that the zymogen was cut out of two dipeptides in Leu13, Tyr146and Asn148by chymotrypsin digested, ie Serl4-Arg15and Tyr147-Asn148.As a result, the enzyme formed its active form with three disulfide-linked polypeptide chains. Chymotrypsin hydrolyzed protein at the positions of Tyr, Trp and Phe carboxyl terminal peptide bond specifically, and it also hydrolyzed carboxyl terminal of Leu peptide bond. The recombinant human chymotrypsin (hCT), the autolysis site mutant mhCT (F1141) and A chain deleted mutant mhCT (A) were expressed in E.coli, and activated to gain the active ones. At last, the relationship between the three dimensional structure and the catalytic ability was discussed.For the recombinant hCT,the optimum pH7.5to8.5, the optimum temperature37℃. The Michaels constant was0.076mmol/L with BTEE as the substrate, the maximum UV absorption wavelength was280nm.The purity was96.9%by HPLC analysis.The recombinant hCT was stable under acidic pH3-5, while poor stablity in the vicinity of the optimum pH. The effect of metal ions on the enzyme activity was different under pH4.0with that under pH8.0, minor affection under pH4.0while greater under pH8.0. Ca2+is added to stabilize the enzyme activity.After refolding, the chymotrypsinoen was activated by its weak activity instead of trypsin. It is referred to self-activated hCT in this paper. Self-activated hCT had the optimum pH value of5.5to6.5,and the optimum temperature of50℃.The Michaels constant was0.12mmol/L with BTEE as the substrate.the enzyme activity was greatly inpact by metal ions, such as enzyme could be inactivated with the present of1mmol/L Fe3+or Cu2+.The A chain deleted chymotrypsin was constructed by substituting the A chain sequence of chymotrypsin by the propeptide of trypsin. For the mutant chymotrypsin, A chain was deleted due to no disulfide bond connected with B chain after activated by trypsin. The mhCT(A) mutant owned the optimum pH value of7.0to8.5, indicating that the mutations resulted in a wide range of variation.The mhCT (A) was sensitive to temperature, and had poor stability. The Michaelis constant was0.047mmol/L with BTEE as the substrate.The autolysis site Phe114was mutated to He to obtain the mhCT (F1141) mutant. The Michaelis constant was0.078mmo1/L with BTEE as the substrate. After the mutation, the stability under high temperature was increased and inactivation after several frozen-thaws was decreased. The mhCT (F114I) mutant was well of stability under acidic pH and had residual activity of40%, while the recombinant hCT had only10%when kept in25℃with the same time at pH9.0.