Production Of Genetically Engineered Rennet By Fermentation And Its Application

As the development of dairy industry, cheese production will boom in the coming years. Rennet is a necessary enzyme in cheese making process, which can induce coagulation of milk and add more flavors of cheese. With the soaring demand of cheese, the shortage of calf rennet has become a serious bottleneck to the further development of cheese industry. Researchers are seeking for the appropriate substitute for it. Although microbial rennet as an option has the advantages of low cost and short production cycle, its defections of potent proteolytic activity and high inactivation temperature will jeopardize the quality and quantity of cheese production. Hitherto, rennet used in cheese production in China was imported, so the cheese was also expensive. Currently recombinant chymosin is a favorable option. It is urgent to develop the recombinant rennet of our own intellectual property. The methylotrophic yeast, Pichia pastoris has proved to be an outstanding host for high level production of both secreted and intracellular proteins, featuring high security, high level of foreign proteins expression and growth to high cell densities.In this paper, factors affecting the rennet-production from the expression of a rennet gene of Mucor pusillus by recombinant Pichia pastoris were studied. The enzyme purification, properties and application in cheese manufacture were also researched. The main results as follows:(1) The growth curve showed that the yeast entered the stationary phase after incubation for 24h, total counts and viable yeast counts were observed after 216h, then started enter the decline phase after 240h. Analysis of enzymatic activity showed that rennet reached the maximum milk-clotting activity (300SU/mL) at 192h. Proteolytic activity, the ratio of milk-clotting activity and proteolytic activity (C/P) and total protein content were determined to be 10.75u/mL, 27.9, 0.189mg/mL, respectively. A strong band at about 47kDa was shown by SDS-PAGE.(2) The optimal growth conditions were obtained by single factor experiment: temperature 30℃, pH 5.2, rotating speed 280r/min, volume of medium 5% (12.5mL/ 250mL).(3) The optimal enzyme producing conditions were studied using Plackett-Burman method based on the single factor experiment. The crucial conditions were the volume of medium (A), methanol concentration (B) and pH (C) of the fermentation liquor. The regression equation was obtained. It was: Y=472.20-42.81A-8.03B+24.43C+ 55.34AB-16.99AC-47.57BC- 52.59A2-60.01 B2-131.34C2. The optimized parameters of these factors were 13.1%, 1.5% and 6.2, respectively, determined with response surface method. The highest milk-clotting activity (491.7SU/mL) was achieved on the set of parameters. The values predicted by the response surface method were proved to be correct and reliable in verification test.(4) The rennet was purified with ammonium sulfate(80%),ion-exchange chromatography using DEAE-52, and size-exclusion chromatography using Sephadex G-75, conferred 89.3-folds purification and 53.77% recovery of milk-clotting enzyme. SDS-PAGE revealed that after puriefication, the rennet produced by recombinant Pichia pastoris was of high purity.(5) The properties of rennet were studied: The optimal temperature and pH of the enzyme were 55℃and 5.4, respectively. The milk-clotting activity of the rennet was high under the acidic pHs, but low under the alkaline pHs. And stable at pH 6.0.(6) The clotting time decreased with the increasing of enzyme concentration. Total solid of skim milk prolonged the clotting time. The optimal concentration of CaCl2 was 0.05M. The curd properties, viscosity and texture were also investigated. The results showed the properties of milk coagulation induced by the two enzymes were similar.(7) Cheddar cheese made with recombinant rennet was similar to that made with calf rennet in sensory evaluation tests, physical and chemical characters and microbial tests. Although, the rennet secreted by recombinant Pichia pastoris has high proteolytic activity, but it did not bring about bitterness in cheese. Consequently, the application of recombinant rennet in cheese manufacture is good.