Expression Of Aspartic Proteases And Their Application In Soybean Protein And Shrimp Shell Hydrolysis

Aspartic protease is an important protease present in most organisms,such as plants,animals,bacteria,fungi and viruses.Since there were two highly conserved aspartic acids as catalytic residues,it is also called as aspartic protease.Its secondary structure is mainly composed of?-sheets,and tertiary structure is a highly symmetrical double-leaf structure.The catalytic site locates among the symmetrical cracks where peptide bonds are cleaved by activating water molecules.Aspartic proteins have wide applications,including in the leather industry,detergent addition,cheese processing,beverages and wines turbidity removing,and protease wastes hydrolysis?okara,shrimp shells,bacterial residues,mushroom residues,feathers,etc?.In this study,52 proteases genes were cloned from Candida tropicalis and Candida parapsilosis,then constructed into two recombinant expression vectors—inducible vector p ICh and constitutive vector p IC9BM-GAP,and the expression vectors integrated with the genome of Pichia pastoris GS115.It was successfully that constructed 47 recombinant protease expression strains,and two acidic proteases with high enzyme activity were selected from them.Among aspartic protease SAPP1 molecular weight is 47 k Da,showing maximum activity405.36 U/m L?inducible?and 113.98 U/m L?constitutive?at pH 3.0 and 50?,and it's relatively stable at pH 4.0-6.0 and below 50?.While the molecular weight of SAPT is 50 k Da,the optimal reaction conditions are pH 3.0,55?,the measured enzyme activity is 275.50 U/m L?inducible?and 202.39 U/m L?constitutive?,and it has good stability between pH 3.0-7.0 and below 50?.Using the two acidic proteases and commercial papain and alkaline protease hydrolyze soybean protein and shrimp shell,and it was preliminary explored hydrolysis effects.Under the optimal pH conditions,through SAPP1?pH 3.0?,SAPT?pH 3.0?,papain?pH 7.0?,alkaline protease?pH 9.0?hydrolysis,the recovery rate of soybean protein:22.92%,18.01%,13.19%,32.89%;the recovery rate shrimp shell protein:40.88%,39.79%,32.96%,44.23%.In comparison,alkaline protease is the best,SAPP1 is second,SAPT is third,papain is the worst.However,when the gastric environment is simulated at pH 3.0 in vitro,aspartic protease the soluble protein and amino acid concentration is 3-5 times higher than that of papain and alkaline protease.Apparently,SAPP1 and SAPT has more advantages in the gastric environment of animals.In addition,their soy protein and shrimp shells hydrolysates were antioxidant activity.The SAPP1,SAPT,papain and alkaline protease soybean hydrolysates that the DPPH free radical scavenge rates EC₅₀ were 2.21 mg/m L,1.04 mg/m L,1.77 mg/m L,6.87 mg/m L;and the EC₅₀ of shrimp shell hydrolysate were 0.37 mg/m L,0.39 mg/m L,0.77 mg/m L,0.56 mg/m L.Soy protein and shrimp shell hydrolysate antioxidant activity of aspartic protease SAPP1 and SAPT were significantly higher than that of papain and alkaline protease.In this research,two high activity aspartic proteases were heterologous expressed in Pichia pastoris,and preliminary hydrolysis experiments were carried out on soybean protein and shrimp shells.The hydrolysis effect is good and has biological activity.It has wide application prospects in the field of recycling of biological protein resources.At present,the main use of triangular shake flasks for fermentation is to use small fermentation tanks for fermentation to increase enzyme activity and enzyme production.At the same time,feed the grouper with hydrolysate as feed,and explore its application potential in feed additives.