Studies On Preparation Of Silver Carp Eggs Recombinant Cystatin And Its Application In The Inhibition Of Surimi Gel Weakening

In this study, the total RNA was extracted from silver carp eggs and the single-stranded cDNA was obtained by RT-PCR, then the silver carp eggs Cystatin (family II) about 333bp was amplified by PCR action with CystatinF: 5’-TAGGATCCACTGGGATTCCTGGAGGCCTTGTA-3’and Cystatin R: 5’-CCCTCGAGCATGCAGGTGTTTTCAGTGAC-3’as primers. After the clone of the recombinant plasmid Cystatin-pMD19-T, the positive colonies examined by PCR were confirmed by DNA sequencing further. The results showed that the amino acid sequence deduced from the DNA sequence contained the typical and conserved domain of Cystatin (family II), such as G(5)、QVAAG(49-53)、PW(98,99). Subsequently the prokaryotic expression vector Cystatin-pET-32a was constructed by the actions of double digestion with Bam HI and Xho I and ligation, and then it was transformed into competent E.coliDH5a, after detecting by PCR and double digestion, the prokaryoticthe expression vector is transferred to competent E.coliBL21 (DE3). Under the condition of lmmol/L IPTG and incubation 3h at 30℃, the fusion protein of trx-Cystatin was successfully expressed with the form of inclusion bodies. The inclusion bodies protein was collected after sonication and centrifuge of the deposited bacterial cells. After washing by 0.5mol/L, 1mol/L,5mol/L urea orderly, then the inclusion bodies was finally dissolved by 8mol/L urea. Then the soluble fusion protein trx-Cystatin was purified by nickel affinity chromatography. After hydrolysis by 1U/mg enterokinase at 37℃ for 16h, the sample was subjected to SP Sepharose Fast Flow cation exchange chromatography. Then a target protein of Cystatin activity was eluted with concentration of 0.2mol/L NaCl, and appear a single band about 15.8kD on SDS-PAGE.Further the inhibitory activity of recombinant silver carp Cystatin to papain、 cathepsinB、cathepsinL, pH stability and thermal stability, the type of inhibition and constant Ki were measured by the method of fluorescent synthetic peptide substrate. The results showed that the recombinant Cystatin can tolerate a wide pH range of 4.0 to 9.0, the activity was significantly decreased at pH 10.0-13.0; meantime, it was relatively stable at 4℃-60℃, the activity was significantly decreased above 60℃; the recombinant Cystatin was a competitive inhibitor and the inhibition constant Ki to papain is 0.0008nmol.Recombinant Cystatin was added to silver carp surimi to study its inhibition effect on surimi gel softening. The recombinant Cystatin was added into the surimi for experimental groups with the proportion of 30μg/g and 60μg/g and the ultrapure water of equal quantity was added into control group. The surimi gel breaking force of the group with Cystatin of 60μg/g and the control group were 330.66g±11.28g and 197.75g± 15.38g respectively, the experimental group was significantly increased 67.21%(p<0.01); And the surimi gel strength were 342.80g-cm±28.01 g-cm and 150.10g-cm±16.75g-cm respectively, the experimental group was significantly increased by 1.28 times, and the holding water rates were 91.63%±0.51% and 90.49%±0.56% respectively, the experimental group was increase by 1.25%(0.01<p<0.05) and the whiteness were 43.09 ±0.46 and 42.37±0.36 respectively, the experimental group increased by 1.69% (0.01<p<0.05). The indicators of 30μg/g surimi group were significantly(0.01<p<0.05) or highly significantly (p<0.01) higher than those in the control group, but a little less than 60μg/g surimi group with no significant difference(p>0.05) except the holding water rates. In addition, the hydrolysis of myosin heavy chain observed by SDS-PAGE, quantitative analysis showed that the hydrolysis of myosin heavy chain of experimental groups with 30μg/g and 60μg/g Cystatin reduced 15.79% and 17.89% respectively. Simultaneously, the analysis with scanning electron microscopy showed that the microstructure of surimi gel of experimental group looked smooth for the surface and with a compact structure and a well-arranged meshwork than that of the control group. The above results show that adding the silver carp recombinant Cystatin could inhibit the gel softening of silver carp surimi significantly.