Purification, Characterization And Application Of Three Proteases From Different Resources

Proteases hydrolyze the peptide linkages in proteins, which are the important enzymes in industries. The wide distribution of proteases among animals, plants and microorganisms demonstrates that they are necessary for living organisms, and plays crucial physiological roles in quite diverse biological processes. In the present dissertation, the isolation, purification, characterization and application of proteases from three sources including bacteria, plant and fungi were investigated. The main conclusions are as follows:A comparative analysis on the distribution of protease activities among 90 plants, including fruits and vegetables, was performed.10 fruits and 14 vegetables showed protease activities above 10 U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit, broccoli, ginger, leek and red pepper at different pH values. These five plants may be promising candidates for novel protease exploitation according to the results of protease activities, SDS-PAEG, zymogram, protease pattern and peptide analysis. Protease activities, milk clotting activities and protein patterns of proteases from seven kiwifruit cultivars were analyzed, among which the actinidin from the cultivar Xuxiang exhibited the highest activity. Actinidin was purified using ammonium sulfate precipitation and ion-exchange chromatography. Its optimal pH and temperature were 3.5 and 40℃, respectively. It was stable within pH 3.5-6.0 and up to 40℃. Iodoacetamide strongly inhibited the protease activity of Actinidin, suggesting that it was a cysteine protease. Actinidin improved tenderness effectively on rabbit muscle at a lower concentration (0.25 mg/100g rabbit muscle). Meanwhile, hydrolysates from five plant-derived proteins by Actinidin showed highest peptide yield of 14.2% and ACE-inhibitory activity of 88.3%, indicating that it could be used as a promising protease for ACE-inhibitory peptides production.A protease producing bacterium CAU342A was isolated from soy sauce koji and was identified as Pseudomonas aeruginosa. The fermentation conditions for protease production by Pseudomonas aeruginosa CAU342A were optimized using the one-factor-at-a-time method and orthogonal test. The optimal fermentation conditions were obtained as follows (g/L):vinasse 30, yeast extract 15, Tween-80 0.5, NaCl 5, K2HPO4 7, KH2PO4 3, MnSO4 0.4, initial pH of 7.5, temperature of 30℃ and incubation of 72 h. Under the optimized fermentation conditions, the highest protease activity of 2653.5 U/mL was achieved. A protease PaproA was purified from the broth of Pseudomonas aeruginosa CAU342A using ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. The specific activity of PaproA was 8477.4 U/mg and the recovery yield was 9.8%. The enzyme was a monomer with molecular weight of 32.2 kDa on SDS-PAGE and 34.5 kDa by gel fitration. The optimal pH and temperature of PaproA were 9.0 and 55℃, respectively. It was stable in the pH range of 7.0-9.5 and under 60℃. The strong inhibition of EDTA on protease activity indicated that it was a metalloprotease. PaproA showed broad substrate specificity with strong hydrolysis ability towards casein, skimmed milk and protamine sulfate, but could not hydrolyze gelatin and collagen. PaproA exhibited great potential in the production of peptides with ACE inhibition activity and blended soy sauce.A novel protease gene (designated as RmproA) from Rhizomucor miehei CAU432 was successfully cloned and expressed in Pichia pastoris. The gene belongs to peptidase A1 family and has an open reading frame (ORF) of 1326 bp encoding 442 amino acids with no introns. The recombinant protease (RmproA) was secreted at high levels of 3480.4 U/mL by high cell density fermentation in a 5 L fermentor. RmproA was purified by QSFF ion-exchange chromatography and gel filtration chromatography with a specific activity of 773.3 U/mg. The purified enzyme was a monomer with molecular mass of 52.4 kDa on SDS-PAGE and was glycosylated. The optimal pH and temperature of RmproA were determined to be 5.5 and 55℃, respectively. It was stable within pH 5.0-8.0 and up to 50℃. Pepstatin A, an aspartic protease inhibitor, strongly inhibited the enzyme activity. RmproA displayed wide specificity towards casein, skimmed milk, azocasein and soy protein isolate, while could not hydrolyze protamine sulfate and collage. RmproA treated pork muscle showed lower shear force, indicating its application in meat tenderization.