The Genetic Recombination And Expression For Cystatin In Yeast And Its Application In Surimi

Cystatin inhibitors,also known as cystatin,are widely present in various organisms and are capable of inhibiting cysteine proteases containing cysteine(cySH)residues in the active center.The mechanism by which cysteine protease inhibitor F inhibits cysteine proteases is rarely reported compared to cysteine protease inhibitor C.In this work,the recombinant plasmid pPIC9K-CST7 was first constructed and then introduced into Pichia pastoris GS115.A methanol-induced expression and histidine-tag purification resulted in an active recombinant cysteine protease inhibitor F.The inhibitory activity of recombinant cystatin F was measured and the IC50 was calculated to be 4.78 ?M.The Lineweaver-Burk analysis was used to analyze the inhibition type.It was found that no significant change in Vmax was observed in the protease hydrolysis reaction with different concentrations of Cystatin F,and the Km value increased with the increase of inhibitor concentration.The inhibition type of cystatin F is competitive inhibition with protein substrate,and isothermal titration calorimetry shows its ability to bind to papain(Kd = 9.387×10-7M),showing recombinant cysteine according to stoichiometry.The ratio of acid protease inhibitor F to papain was 1:1.During the binding of Cystatin F and papain,the secondary structure of the ?-sheet was significantly increased,the percentage of random curl was reduced,and the fluorescence intensity was gradually reduced.Binding to the secondary structure of the active sites Cys25,Asp158,His159 in the papain crystal structure(PDB ID: 3LFY)and the fluorescent-emitting amino acid residues Tyr64,Trp177,cysteine protease inhibitor F and protein substrate Competitively binding to the papain active site results in significant inhibitory activity.To further verify the actual effect of expressing purified recombinant Cystatin F on enhancing the gel strength of surimi,recombinant Cystatin F was added to the surimi gel.The experimental results show that the recombinant cystatin F can significantly improve the gel strength,texture characteristics and water holding capacity of the surimi,but does not significantly affect the whiteness of the surimi.When the addition amount of recombinant cystatin was between 2.5‰10‰,the quality gradually increased with the increase of the addition amount,and the gel strength increased by 32%,and the recovery increased from 0.369 to 0.416,which increased by 12.7%.Water holding capacity increased from 63% to 67% and overall increased by 6.3%.When the amount of recombinant cystatin F was higher than 10‰,the inhibitory effect of cystatin F on cysteine protease was saturated,and there was no significant influence on the gel texture of the fish gill.This study provides a theoretical basis for the large-scale production of cysteine protease inhibitors and their use in surimi products.