Eukaryotic Expression And Purification Of Cathepsin B From Jian Carp And Its Relationship With Protein Oxidation And Texture Quality In Refrigerated Fish Slices

Cathepsins B（CTS B）is one of the most active lysosomal cysteine proteases in postmortem fish muscles.In order to investigate the relationship between CTS B and protein oxidation and the effect on physicochemical indexes and texture quality of fish slices during refrigeration,the eukaryotic cloning expression system was constructed and CTS B was prepared.Then,the relationship between the activity of endogenous CTS B and protein oxidation indexes of fish slices during refrigeration was explored,as well as the influence of exogenous CTS B on physicochemical indexes and texture quality of fish slices during refrigeration.The experimental results are as follows:（1）Eukaryotic expression and purification of recombinant CTS B in Jian carpIn this study,total RNA was extracted from the meat tissue of carp,and c DNA was obtained by reverse transcription.The recombinant expression vector p PIC9K-cts b was constructed from this template,and then the recombinant expression strain Pichia pastoris GS115-p PIC9K-cts b was constructed.The recombinant protein CTS B was expressed after methanol induction.The protein was purified by nickel ion affinity chromatography and showed a single band of 22.6 k Da on electrophoresis.（2）Endogenous CTS B of refrigerated carp meat slices and its correlation with protein oxidation indexesDuring 0-5 days of aerobic refrigeration at 4℃,the activity of endogenous CTS B of carp meat increased gradually.The activity of Ca²⁺-ATPase and total sulfhydryl groups decreased,while the surface hydrophobicity,ROS,disulfide bonds and carbonyl groups increased.The protein oxidation trend shown by the above protein oxidation indexes was basically consistent with the changes of CTS B,and there was a certain correlation.（3）Effects of recombinant protein CTS B on physicochemical indexes and texture quality of refrigerated carp meat slicesThe recombinant protein CTS B was evenly applied on the surface of carp meat slices in accordance with the ratio of 1.5 U/g fish（CTS B group）,and the same volume of sterile water was used as the negative control.The two groups were aerobic refrigerated at 4℃for0-5 d,and samples were taken at the same time point every day to determine the relevant indicators and for comparison.The results showed that the sensory score,Texture Profile Analysis（TPA）and shear force of CTS B group were significantly lower than those of control group（p<0.05）,while the SAP loss rate and p H of CTS B group were significantly higher than those of control group（p<0.05）.According to the results of SDS gel electrophoresis of myofibrillar protein and the observation of fish tissue sections,the myofibrillar fibers in CTS group B degraded more significantly at 4-5d,and the muscle fiber breakage in this group was more serious.On the 5th day,the intracellular myofibrillar fibers broke and dissolved in a large area.The application of exogenous CTS B accelerated the deterioration rate of fish quality.The above results provide relevant experimental basis and theoretical basis for further research on the mechanism of the change of quality of fish slices in cold storage.