Recovery And Purification Of CPIs Of Grass Carp Surimi Wash Water By Affinity Packing Of CNBr-Papain-Sepharose 4B

Cysteine protease inhibitors?CPIs?,a class of natural protease inhibitors,can inhibit cysteine endopeptidase.It exists widely in animals,plants and microorganisms.It has potential utilization value in the fields of food and medicine industry.In this paper,affinity filler?CNBr-Papain-Speharose 4B?was firstly prepared wild papain as ligand and its stability was detected.Then the affinity filler was used to recover CPIs from the grass carp surimi washing water?SWW?,subsequently the physicochemical properties and biological activities of CPIs were systematically studied.The results not only establish necessary experiment foundation for the efficient and rapid recovery and purification of CPIs in wash water,but also provide a scientific reference for systematic identification of CPIs.The experimental results were as follows:First,the crude extract of CPIs from the SWW was subjected to CNBr-Papain-Speharose 4B affinity chromatography.The peak II of highest purification efficiency and specific activity,was determined to be the target peak combined with the characterization of reversed-phase enzymogram.Peak III gave two bands of 22kDa and 20kDa on SDS-PAGE and was further separated into two sharp peaks at C18RP-HPLC,Cystatin-I and Cystatin-II,purified with the 21.57 fold,0.93%yield and 12.16 fold,0.51%yield respectivelyBoth Cystatin-I and Cystatin-II of SWW were identified to be single component by TSK gel G2000 SW_XL HPLC,and their molecular weights?MW?were respectively22.0kDa and 19.5kDa.The two CPIs were competitive inhibitors.They were able to significantly inhibit Papain,Cathepsin B/L,and partially inhibit Chymotrpsin,but not Trypsin.Their inhibitory constants?Ki?for Papain were respectively 6.0nmol and16.25 nmol.The MW and isometric pI of Cystatin-I and Cystatin-II were analyzed by bidirectional electrophoresis,and were demonstrated to be 22kDa,8.6 and 19.7kDa,8.6 respectively.The data obtained from RPLC-MS/MS mass detection revealed,that the two CPIs both contained the characteristic conservative sequence QVAAG of the Cystatins superfamily.Based on these characteristics,Cystatin-I and Cystatin-II may belong to the Cystatins?family II?.An affinity filler was prepared:After the activation of Sepharose 4B by CNBr and being coupled with the solution Papain,affinity filler was further characterized by ultraviolet?UV?and infrared?IR?spectroscopy.There were obvious UV absorption peaks in 280nm and typical amides in 1672cm^-1,1547cm^-1and 1249cm^-1.Thus,it was testified that Papain was coupled to CNBr-Sepharose 4B respectively.The adsorption rate of recombinant silver carp Cystatin?family II,20.9kDa?was used as an index to screen the optimal coupling density of Speharose 4B and Papain,and the optimal ratio of CNBr-Speharose 4B to papain was 1:7?w/v?.The thermal stability and pH stability of the filler were measured by the adsorption rate of Cystatin.The result showed that it has good heat resistance and acid resistance.In addition,the filler has good tolerance to divalent metal ions and better storage stability.