Characteristic Of Cystaine Protease Inhibitor And Its Change During Autolysis Of Sea Cucumber(Stichopus Japonicus)

Sea cucumber is easily subjected to autolysis after fishing and during processing. Endogenous proteases play critical role on autolysis. Previous studies demonstrated endogenous cysteine protease(ECP) was one the main proteases involved in autolysis. However, there was no report on the activity change of the cysteine protease inhibitors(ECPI). So, in this dissertation, physicochemical properties of ECPI and its activity profile during autolysis were investigated. One of ECP gene, cystatin, was obtained before in our lab. The recombinant cystatin protein was used to prepare its antibody, which was applied to locate the change of cystatin expression during autolysis by immunohistochemical staining.The reverse zymograghy method was optimized for the detection of ECPI in sea cucumber. The factors needed to be optimized included papain addition, incubation p H and temperature. The optimized condition was 400μg/L of papain addition at pH7.0 and 40℃. Under the above optimized conditions, the change of ECPI activity in sea cucumber was detected during autolysis at 4℃, 10℃, 20℃ and 30℃. At the same time, zymography was used to examine the change of ECP activity in the presence and absence of E-64; which was the specific inhibitor of cyteine protease. SDS-PAGE is applied to investigate the autolysis degree of sea cucumber. Results showed that significant degradation of protein occurred in the dermis during autolysis of sea cucumber. The activity of ECP demonstrated an increase trend during autolysis, but the activity of ECPI was relatively stable.An ECPI gene, cystatin, had been cloned before. Recombinant cystatin protein was expressed and separated from the E coli cells which had been transfected with cystatin-p ET21 b plasmid. The recombinant cystatin was evaluated for the optimal pH and temperature by reverse zymography. The result was consistent with that of the endogenous ECPI. The specific antibody against recombinant cystatin was prepared, which allowed us to locate the cystatin in the dermis of sea cucumber by immunohistochemical staining method. Result of the sea cucumber autolyzed for 48 h at 20℃ showed that the expression of cystatin did not changed significantly with that of fresh sea cucumber. This was consistent with the result that zymography. This mode, in which ECP increased and ECPI kept stable, suggested a significant molecular-interaction change between ECP and ECPI, or the regulation effect of ECPI on ECP changed significantly. Moreover, the section of the sea cucumber autolyzed for 48 h at 20℃ also showed an increment of the cystatin-positive cells, suggested these cell migrated from other part to the endodermis. This migration might connect with the hypoxia and catching stress. Above all, the molecular-interaction change between ECP and ECPI deserve further investigation to explain the increase activity of ECP and the relative stable activity of ECPI.