Fermentation Optimization And Purification Of Large Yellow Croaker Cystatin F In Genetic Engineering Pichia Pastoris

Surimi is one of the main aquatic products of deep processing.It can not only be used directly as raw material in the catering industry,but also is an important food-manufacturing raw material.However,gel softening occurs easily in the process due to the impact of protease,and influences the development of Surimi-product industry.Currently,improving rinsing process,updating heating mode,adding starch and non-muscle protein as well as other measures are used to reduce the influences of gel softening.However,the methods mentioned above are not effective and will bring bad influence on flavor and color,which makes their application very restricted.Cystatin F is a kind of cysteine protease inhibitor,which plays an important role in immune regulation.Preliminary studies in our laboratory have shown that the recombination large yellow croaker cystatin F(LycCysF)can not only effectively inhibit the activity of papain and recombinant large yellow croaker cathepsin B,L,S as well,but also effectively inhibit the cathepsin caused modori during the process of surimi processing,and enhance the gel strength,elasticity,hardness,chewiness,whiteness and water-holding capacity of surmi.Thus the recombinant LycCysF have a great prospect in surmi processing.A genetic engineering strain GS115/pPIC9K-Op2CysF has been obtaines in laboratory previously.In this study,the fermentation conditions of GS115/pPIC9K-Op2CysF were optimized.First,we studied the pH,methanol concentration,methanol induction time in shaking flask,and we found that its optimal inducing pH value is 4.8,the concentration of methanol in the culture should be less than 1%-2%and 72 h is the best induction time.On this basis,we used fermentor to produce Cystatin F in 5 L and 50 L fermentor.Eventually,on the basis of single factor experiments and small scale fermentations,we established the fermentation processes and parameters to produce Cystatin F in 1000L fermentor.The total cellular protein amount can reach 503 mg/L at the end of the fermentation,and the target protein accounted for 18.7%of the total cellular protein,reaching 94.1 mg/L,The establishment of fermentation process of GS115/pPIC9K-Op2CysF laid the foundation of scale production and further application of Cystatin FOn the basis of the established large-scale fermentation system,we further studied the large-scale purification method of cystatin F.We tried to purify the recombinant cystatin F by ion exchange chromatography,gel filtration chromatography,ammonium sulfate precipitation,affinity chromatography and some other protein purification methods.After fermentation completed,the fermentation broth were collected and pH value were adjusted to 7.4 with NaOH.Then the supernatant were obtained by centrifugation and concentrated using Vivaflow 50 tangential flow filters.The concerntated supernatant were subjected toSephadex G-25 for desalination,at last using Ni column for protein purification.The target protein recovery was 71.2%according this method for cystatin F purification.This method is easy to operate and enlarge.It has high recovery rate,which lays foundation for the further development and application of large-scale production of Cystatin F.This is the first time for recombinant large yellow croaker cystatin F to be appliedto surimi processing.In this study,a set of scientific methods to improve the quality of surmi product and control the protein degradation as well as the surmi modori was set up.