Heterologous Expression Of Cathepsin B And Cathepsin L From Takifugu Rubripes And Properties Of Crude Enzyme Solution

The main edible of the fish is the muscles,and the liver will generally be discarded,resulting in waste.But for some fish,such as the liver of the Takifugu rubripes,is recognized as the most delicious parts.Takifugu rubripes have delicious taste,up to now,a series of umami peptides have been separated and identified from it,but its formation mechanism is not yet clear.Cathepsin B,L can degrade myofibrillar protein and troponin during muscle degradation to play an important role in the flavor formation.This paper provides some information for the further study on enzymatic characteristics and taste formation of cathepsin L in Takifugu rubripes.The study content in detail is as follows:(1)A total of 1482 bp and 1336 bp of Takifugu rubripes cathepsin B and L were obtained by searching for NCBI database through analysis sequence.RNA was extracted from muscle and liver,and then transcribed into cDNA as cloned template.PET-28a(+)as the expression vector and E.coli BL21 as engineering bacteria was respectively used to clone and express Cathepsin B,L.The recombinant cathepsin B,L was obtained with a size of 36 kDa,and its E.coli expression system is also successfully validated.(2)The crude enzyme solution of recombinant cathepsin B,L were purified in different ways,and it was found that the ammonium sulfate precipitation method could remove the other proteins.The enzymatic properties of the enzyme such as optimum temperature and Temperature stability and optimum pH and pH stability were measured by using Z-Arg-Arg-AMG,Z-Phe-Arg-AMC as the substrate of cathepsin B and L respectively.This paper provides some information for the further study on enzymatic characteristics and taste formation of cathepsin B,L in Takifugu rubripes.