Characterization of recombinant proteinase inhibitors in surimi application Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/fn107108g

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  • High proteolytic activity Pacific whiting muscle causes hydrolysis of myofibrillar protein and lowers surimi gel quality. Although food grade proteinase inhibitors can be used to prevent autolytic activity in surimi, their usage is limited due to their adverse effects on the organoleptic quality of surimi. Cystatins however can be used as a specific cysteine proteinase inhibitor without adverse effects. Recombinant cystatins were characterized for their inhibitory activity against cysteine proteinase and compared to egg white cystatin to develop a specific proteinase inhibitor to be used in Pacific whiting surimi. Soyacystatin expressed in E. Coil was purified with phenyl-Sepharose and DEAE 4.33 fold as a recombinant protein. Recombinant fish cystatin expressed in E. coil was also purified as a recombinant protein by affinity chromatography using Ni-NTA resin. Native egg white cystatin was purified 343 fold by using affinity chromatography on cm-papain-Sepharose. Fish cystatin, soyacystatin and egg white cystatin were tested for their inhibitory activity. The amount required to inhibit 50% activity of 2 [mu]g papain was 0.245 [mu]g and 0.455 [mu]g for soyacystatin and egg white cystatin, respectively. The amount of fish cystatin required to inhibit 90% activity of papain was 3.57 [mu]g while the amount of soyacystatin and egg white cystatin required to inhibit was 0.429 and 0.810 [mu]g, respectively. For the characterization study, the fish cystatin was not tested because of low recovery in the purification process. Also, the harsh chemical treatment and lengthy period required to renature the protein made fish cystatin unfeasible for surimi application. Egg white cystatin and soyacystatin were tested against 400 ng of purified cathepsin L from Pacific whiting fillet. The amount of soyacystatin and egg white cystatin required to inhibit 50% activity of cathepsin L were 18.77 ng and 32.14 ng, respectively. The complex formation between papain and both cystatins was demonstrated by isoelectric focusing gel. When papain and cystatins formed a complex, the pI of the complex was resolved in between pIs of cystatins and papain, resulting in changes in pI from those of cystatins and papain. Both cystatins showed high stability at the wide pH range (pH 4-10), and soyacystatin was more sensitive at high temperature than egg white cystatin. Soyacystatin lost 80% of its activity at 60°C by incubation for 30 min while egg white cystatin lost 70% of its activity by the same condition. Soyacystatin was tested for inhibition of autolytic activity in parasitized Pacific whiting fish fillet and Pacific whiting surimi. The results showed that 0.041% (w/w) soyacystatin inhibited almost 90% autolytic activity in Pacific whiting fish fillet. With Pacific whiting surimi, 75.57% autolytic activity was inhibited by 0.0052% (w/w) soyacystatin.
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