pH Sensitive Phase Transition and Enzyme Activity of Trypsin-Chitosan Conjugant
Trypsin-chitosan conjugant was prepared by coupling trypsin with chitosan(CS) through the method of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide/N-hudroxysuccinamide(EDC/NHS) coupling reactions.The resulting conjugant had borne both characteristics of pH-sensitive phase transitional behavior and enzyme catalytic activity.The critical phase transitional pH point(CPSP) of the conjugant was determined by turbid titration method.The results show that CPSP value increase with the decrease of chitosan molecular weight.The CPSP value of CS and its trypsin conjugant could reach 6.36 when the chitosan with molecular weight of 8 × 104 was used.The near neutral CPSP is in favor of stabilizing the enzyme activity during the cyclic procedure of phase transition.Trypsin-CS conjugant could preserve 69% of initial enzyme activity after 30 days storage,and 79% of enzyme activity was observed after 6 cyclic phase transitional procedure.Trypsin-CS conjugant could catalyze the degradation of BSA in homogeneous phase when the pH of solution is slightly less than CPSP value.The conjugant could be recovered by precipitating it from solution when the pH is higher than CPSP value.HPLC results demonstrated that the self degradation of trypsin was significantly inhibited and the interference from the self-degraded peptide could be eliminated in the peptide fragment mapping.